grapes
Checkpoints maintain the order of cell-cycle events. At G2/M, a checkpoint blocks mitosis in response to
damaged or unreplicated DNA. There are significant differences in the checkpoint responses to damaged DNA
and unreplicated DNA, although many of the same genes are involved in both responses. To identify new genes
that function specifically in the DNA replication checkpoint pathway, a search was carried out for high-copy suppressors of
overproducer of Cdc25p [OPcdc25(+)], which lacks a DNA replication checkpoint. Two classes of suppressors
were isolated. One class includes a new gene encoding a putative DEAD box helicase, suppressor of uncontrolled
mitosis [sum3(+)]. This gene negatively regulates the cell-cycle response to stress when overexpressed and
restores the checkpoint response by a mechanism that is independent of Cdc2p tyrosine phosphorylation. The
second class includes chk1(+) and the two Schizosaccharomyces pombe 14-3-3 genes, rad24(+) and rad25(+),
which appear to suppress the checkpoint defect by inhibiting Cdc25p. rad24Delta mutants are
defective in the checkpoint response to the DNA replication inhibitor hydroxyurea at 37 degrees:
cds1Delta rad24Delta mutants, like cds1Delta chk1Delta mutants, are entirely checkpoint deficient at 29 degrees.
These results suggest that chk1(+) and rad24(+) may function redundantly with cds1(+) in the checkpoint
response to unreplicated DNA (Forbes, 1999).
Fission yeast p56(chk1) kinase is known to be involved in the DNA damage checkpoint but not to be
required for cell cycle arrest following exposure to the DNA replication inhibitor hydroxyurea (HU).
For this reason, p56(chk1) is not considered necessary for the DNA replication checkpoint, which
acts through the inhibitory phosphorylation of p34(cdc2) kinase activity. Two chk1 alleles have been isolated in a search for
Schizosaccharomyces pombe mutants that abolish the S phase cell cycle arrest of a thermosensitive
DNA polymerase delta strain at 37 degrees C. These alleles are
proficient in terms of the DNA damage checkpoint, but they induce mitotic catastrophe in several S phase
thermosensitive mutants. The mitotic catastrophe correlates with a decreased level of
tyrosine phosphorylation of p34(cdc2). The deletion of chk1 and the chk1
alleles abolish the cell cycle arrest and induce mitotic catastrophe in cells exposed to HU, if the cells
are grown at 37 degrees C. These findings suggest that chk1 is important for the maintenance of the
DNA replication checkpoint in S phase thermosensitive mutants and that the p56(chk1) kinase must
possess a novel function that prevents premature activation of p34(cdc2) kinase under conditions of
impaired DNA replication at 37 degrees C (Francesconi, 1997).
The role of fission yeast Cut5/Rad4 in genome maintenance is considered to be unique because it is
required for three distinct cell processes: replication, replication checkpoint, and normal UV sensitivity. However, it is unknown just how Cut5 protein is linked to other checkpoint proteins, and what
part it plays in replication and UV sensitivity. Cut5 interacts with a novel checkpoint protein, Crb2; this interaction is needed for normal genome maintenance. The carboxyl terminus of Crb2 resembles yeast Rad9 and human 53BP1 and BRCA1. Crb2 is required for checkpoint arrests induced by irradiation and
polymerase mutations, but is not required for those induced by inhibited nucleotide supply. After
UV damage, Crb2 is transiently modified, probably phosphorylated, with
phosphorylation similarly timed in Chk1 kinase, which is reported to restrain Cdc2 activation.
Crb2 modification requires other damage-sensing checkpoint proteins but not Chk1,
suggesting that Crb2 acts upstream of Chk1. The modified Crb2 exists as a
slowly sedimenting form, whereas Crb2 in undamaged cells is in a rapidly sedimenting
structure. Cut5 and Crb2 interact with Chk1 in a two-hybrid system. Moderate overexpression of Chk1 suppresses the phenotypes of cut5 and crb2
mutants. Cut5, Crb2, and Chk1 thus may form a checkpoint sensor-transmitter
pathway to arrest the cell cycle (Saka, 1997).
The G2-M-phase transition is controlled by cell-cycle checkpoint pathways that
inhibit mitosis if previous events are incomplete or if the DNA is damaged. Genetic analyses in yeast
have defined two related, but distinct, pathways that prevent mitosis--one which acts when S phase
is inhibited, and one which acts when the DNA is damaged. In the fission yeast Schizosaccharomyces
pombe, many of the gene products involved have been identified. Six 'radiation checkpoint' (rad) gene
products are required for both the S-M and DNA-damage checkpoints, whereas Chk1, a putative
protein kinase, is required only for the DNA-damage checkpoint and not for the S-M checkpoint
following the inhibition of DNA synthesis. A third mitotic
control checkpoint pathway has been defined in fission yeast, which prevents mitosis when passage through 'start' (the
commitment point in G1) is compromised. In cycling cells arrested at start, mitosis is prevented by a
Chk1-dependent pathway. In the absence of Chk1, G1 cells attempt an abortive mitosis with a 1C
DNA content without entering S phase. Similar results are seen in the absence of Rad17, a typical
example of a rad gene product. Genetic dissection of checkpoints in logarithmically
growing fission yeast has identified a pathway that couples mitosis to correct passage through start.
This pathway is related to the DNA-structure checkpoints, which ensure that mitosis is dependent on
the completion of replication and the integrity of the DNA. It is proposed that all three mitotic control
checkpoints monitor distinct DNA or protein structures at different stages in the cell cycle (Carr, 1995).
The dependence of cell-cycle progression on the integrity of the genome has been
described as checkpoint control. A number of mutants of the fission yeast
Schizosaccharomyces pombe, selected for their sensitivity to DNA damage caused by
radiation (rad mutants) or to the DNA synthesis inhibitor hydroxyurea (hus mutants)
have been classified as checkpoint mutants because they fail to arrest the cell cycle in
response to DNA damage or incompletely replicated DNA. Coupling control of the cell
cycle to the
checkpoint pathways that monitor DNA repair and replication is essential. In a search for components that interact with the cell-cycle
regulatory kinase p34cdc2, a novel fission yeast protein kinase homolog has been identified which is involved in cell-cycle arrest when DNA damage has occurred or when unligated DNA is present. The gene encoding this protein has been called chk1 for checkpoint kinase. Multiple copies of chk1 partially rescue the ultraviolet sensitivity of rad1-1, a mutant deficient in checkpoint control. The identification of a gene involved in checkpoint control as one which rescues a cdc2 mutant, links the rad1-dependent DNA-damage-sensing pathway and p34cdc2 activity (Walworth, 1993).
To investigate mechanisms that ensure the dependency relationships between cell
cycle events and checkpoints that prevent progression through the
cell cycle after DNA damage, mutants have been isolated defective in the checkpoint
and feedback control pathways. Eleven new loci have been isolated and characterized that define distinct classes of mutants defective in one or more of the
checkpoint and feedback control pathways. Two mutants, rad26.T12 and rad27.T15,
were selected for molecular analysis. The null allele of the rad26 gene (rad26.d)
shares the phenotype reported for the "checkpoint rad" mutants rad1, rad3, rad9,
rad17, and hus1, which are all defective in the radiation checkpoint and in the feedback
controls that ensure the order of cell cycle events. The null allele of the rad27 gene
(rad27.d) defines a new class of Schizosaccharomyces pombe mutants. The rad27 gene, also known as chk1, is a complementing gene that codes for a putative protein kinase, which is required for cell cycle
arrest after DNA damage, but not for the feedback control that links mitosis to the
completion of prior DNA synthesis. These properties are similar to those of the rad9
gene of Saccharomyces cerevisiae. A comparative analysis of the radiation responses
in rad26.d, rad26.T12, and rad27.d cells has revealed the existence of two separable
responses to DNA damage controlled by the "checkpoint rad" genes. The first, G2
arrest, is defective in rad27.d and rad26.d but is unaffected in rad26.T12 cells. The
second response is not associated with G2 arrest after DNA damage and is defective
in rad26.d and rad26.T12, but not rad27.d cells. A study of the radiation sensitivity of
these mutants through the cell cycle suggests that this second response is associated
with S phase and that the checkpoint rad mutants, in addition to an inability to arrest
mitosis after radiation, are defective in an S phase radiation checkpoint (al-Khodairy, 1994).
The protein kinase Chk1 is required for cell cycle arrest in response to DNA damage. The 14-3-3 proteins Rad24 and Rad25
physically interact with Chk1 in fission yeast. Association of Chk1 with 14-3-3 proteins is stimulated in response to DNA damage. DNA damage
results in phosphorylation of Chk1 and the 14-3-3 proteins bind preferentially to the phosphorylated form. Genetic analysis has independently
implicated both Rad24 and Rad25 in the DNA-damage checkpoint pathway. It is suggested that DNA damage-dependent association of
phosphorylated Chk1 with 14-3-3 proteins mediates an important step along the DNA-damage checkpoint pathway, perhaps by directing Chk1 to
a particular substrate or to a particular location within the cell. An additional role for 14-3-3 proteins in the DNA-damage checkpoint has been
suggested based on the observation that human Chk1 can phosphorylate Cdc25C in vitro creating a 14-3-3 binding site. These results suggest that
in fission yeast the interaction between the 14-3-3 proteins and Cdc25 does not require Chk1 function and is unaffected by DNA damage, in
sharp contrast to the interaction between the 14-3-3 proteins and Chk1 (Chen, 1999).
The protein kinase Chk1 is required in the fission yeast Schizosaccharomyces pombe for delaying cell cycle progression in response to DNA damage. Chk1 becomes phosphorylated when DNA is damaged by a variety of agents, including the anti-tumor drug camptothecin. To further characterize the behavior of Chk1 in response to DNA damage, PCR-based mutagenesis of the chk1 gene coupled with in vivo gap repair was used to generate mutant alleles. Of 44 chk1 mutants recovered, six encode full-length proteins that confer a DNA damage-sensitive phenotype. All of the alleles render cells checkpoint-defective, but confer subtle differences in sensitivity to camptothecin or UV light. Mutant alleles were sequenced and served to identify regions of the protein that are critical for checkpoint function (Wan, 2001).
Acquisition of lineage-specific cell cycle duration is a central feature of metazoan development. The mechanisms by which this is achieved during early embryogenesis are poorly understood. In the nematode Caenorhabditis elegans, differential cell cycle duration is apparent starting at the two-cell stage, when the larger anterior blastomere AB divides before the smaller posterior blastomere P1. How anterior-posterior (A-P) polarity cues control this asynchrony remains to be elucidated. Early C. elegans embryos possess a hitherto unrecognized DNA replication checkpoint that relies on the PI-3-like kinase atl-1 and the kinase chk-1. Preferential activation of this checkpoint in the P1 blastomere contributes to asynchrony of cell division in two-cell-stage wild-type embryos. Furthermore, preferential checkpoint activation is largely abrogated in embryos that undergo equal first cleavage following inactivation of Galpha signaling. These findings establish that differential checkpoint activation contributes to acquisition of distinct cell cycle duration in two-cell-stage C. elegans embryos and suggest a novel mechanism coupling asymmetric division to acquisition of distinct cell cycle duration during development (Brauchle, 2003).
This work reveals that the DNA replication checkpoint is functional in one- and two-cell-stage C. elegans embryos. Since entry into mitosis is merely delayed, even in hydroxyurea-treated embryos, this checkpoint is less potent at preventing cell cycle progression than that acting in somatic cells, where entry into mitosis is blocked following inhibition of DNA replication. Nevertheless, these findings are in contrast to observations in embryos of Drosophila, Xenopus, or Zebrafish, where aphidicolin or hydroxyurea do not affect cell cycle progression prior to the midblastula transition. Interestingly, the DNA replication checkpoint is also functional in early embryos of fucus and sea urchin; this finding indicates that the DNA replication checkpoint may contribute to modulation of cell cycle duration during early development of a number of organisms (Brauchle, 2003).
The DNA replication checkpoint in early C. elegans embryos relies on two core components of the evolutionarily conserved checkpoint signal transduction cascade: ATR-related atl-1 and chk-1. ATR is also the PI-3-like kinase seemingly required for the DNA replication checkpoint in vertebrate somatic cells, since human fibroblasts overexpressing a kinase-inactive ATR protein are hypersensitive to aphidicolin and hydroxyurea. Moreover, Xenopus egg extracts supplemented with sperm nuclei and treated with aphidicolin no longer arrest following immunodepletion of ATR. Similarly, Chk1 appears to be the main downstream effector of ATR in vertebrates. ATR phosphorylates Chk1 in Xenopus egg extracts, and this phosphorylation is essential for Chk1 to prevent entry into mitosis following DNA replication checkpoint activation. Furthermore, ATR/mei-41 and Chk1/grapes are essential for the DNA replication checkpoint in Drosophila. These observations indicate that ATR and Chk1 play a conserved role in the DNA replication checkpoint across metazoan evolution (Brauchle, 2003).
It has become increasingly clear that the DNA replication checkpoint can be utilized in the absence of defective DNA replication to modulate cell cycle progression. For instance, careful timing has revealed that ATR/mei-41 and CHK-1/grapes are required to lengthen S phases at the midblastula transition in Drosophila embryos. Moreover, immunodepletion of ATR or Chk1 from Xenopus egg extracts results in premature entry into mitosis. The current results mirror these observations and demonstrate for the first time that differential modulation of constitutive signaling can contribute to acquisition of distinct cell cycle duration during early development (Brauchle, 2003).
These findings suggest that A-P polarity regulates timing of cell division in wild-type two-cell-stage C. elegans embryos through two distinct mechanisms: (1) polarity cues govern 60% of the time difference through an atl-1/chk-1-independent mechanism that remains to be identified; (2) polarity cues result in preferential activation of the atl-1/chk-1-dependent DNA replication checkpoint in the smaller blastomere, P1, and account for the remaining 40% time difference. Why should there be two mechanisms to ensure acquisition of lineage-specific cell cycle duration? Interestingly, despite having a 40% reduction in the time difference between AB and P1, most atl-1/chk-1(RNAi) embryos give rise to viable adults, though they lack a germline. Perhaps the existence of two partially redundant mechanisms provides a means to ensure robust acquisition of distinct cell cycle duration during early embryonic development (Brauchle, 2003).
An attractive hypothesis is that preferential checkpoint activation in P1 results from the unequal first cleavage of wild-type embryos, since preferential activation is largely abrogated in embryos that undergo equal first cleavage following inactivation of Gα signaling. In apparent contradiction with this working model, par-4 mutant embryos undergo unequal first cleavage, yet exhibit near synchronous division at the two-cell stage. However, probably as a result of incomplete or delayed cytokinesis, cytoplasm often flows from AB into P1 in par-4 mutant embryos; this could alter cell cycle progression in the two blastomeres. Compatible with this view, daughter nuclei confined to a common cytoplasm following lack of cytokinesis at the one-cell stage undergo synchronous nuclear envelope breakdown. Furthermore, the apparent paradox posed by par-4 mutant embryos may be resolved if the impact of cell size on cell cycle progression can be detected in full only when polarity cues are intact. Future work, including experiments in which the first cleavage can be equalized without interfering with polarity cues or Gα signaling, will help elucidate the mechanisms by which A-P polarity ensures differential checkpoint activation during early development (Brauchle, 2003).
To gain insight into the function and organization of proteins assembled on the DNA in response to genotoxic insult, the phosphorylation of the Schizosaccharomyces pombe PCNA-like checkpoint protein Rad9 was investigated. C-terminal T412/S423 phosphorylation of Rad9 by Rad3ATR occurs in S phase without replication stress. Rad3ATR and Tel1ATM phosphorylate these same residues, plus additional ones, in response to DNA damage. In S phase and after damage, only Rad9 phosphorylated on T412/S423, but not unphosphorylated Rad9, associates with a two-BRCT-domain region of the essential Rad4TOPBP1 protein. Rad9-Rad4TOPBP1 interaction is required to activate the Chk1 damage checkpoint but not the Cds1 replication checkpoint. When the Rad9-T412/S423 are phosphorylated, Rad4TOPBP1 coprecipitates with Rad3ATR, suggesting that phosphorylation coordinates formation of an active checkpoint complex (Furuya, 2004).
Most of the proteins involved in the DNA damage and DNA replication checkpoint have been identified, and the majority are highly conserved through evolution. Many features of the checkpoint pathways remain unexplained, including their apparent complexity and the fact that many of the same proteins participate in both the DNA damage and DNA replication responses. The data presented in this study begin to uncover the molecular organization of the checkpoint proteins following their recruitment to sites of DNA damage or collapsed DNA replication forks. It is suggested that one of the reasons for the apparent complexity of the system is because it allows cells to distinguish between similar biochemical consequences of DNA damage (such as ssDNA-RPA complexes) that occurs in distinct circumstances (such as induced damage in G2 and collapsed replication forks). It is important to make these distinctions because different signaling responses to the cell cycle and the DNA repair apparatus will be appropriate in each case. The data suggest that the Rad3ATR-dependent phosphorylation of Rad9 promotes association between Rad9 and Rad4TOPBP1 through phospho-specific BRCT-domain interactions during unperturbed S phase, and that this helps cells distinguish collapsed forks from DNA damage in G2 cells. It is intriguing that a phospho-specific BRCT-mediated interaction between BRCA1 and BACH1 in human cells is promoted by cyclin-dependent kinase activity against BACH1 in G2 and also by the G2 checkpoint. Together, these observations suggest that a combination of phosphorylation events can orchestrate the organization of the checkpoint apparatus before it is activated and that, upon activation, the consequent phospho-specific protein interactions dictate the downstream consequences of this activation (Furuya, 2004).
Arrest of the cell cycle at the G2 checkpoint, induced by DNA damage, requires inhibitory
phosphorylation of the kinase Cdc2 in both fission yeast and human cells. The kinase Wee1 (Drosophila homolog: wee) and the
phosphatase Cdc25, which regulate Cdc2 phosphorylation, were evaluated as targets of Chk1, a kinase
essential for the checkpoint. Fission yeast cdc2-3w Deltacdc25 cells, which express activated Cdc2
and lack Cdc25, are responsive to Wee1 but insensitive to Chk1 and irradiation. Expression of large
amounts of Chk1 produces the same phenotype as does loss of the cdc25 gene in cdc2-3w cells. Cdc25
associates with Chk1 in vivo and is phosphorylated when copurified in Chk1 complexes. These
findings identify Cdc25, but not Wee1, as a target of the DNA damage checkpoint (Furnari, 1997).
The G2 DNA damage checkpoint ensures maintenance of cell viability by delaying
progression into mitosis in cells that have suffered genomic damage. It is controlled
by a number of proteins which are hypothesized to transduce signals through cell cycle
regulators to delay activation of p34cdc2. Studies in mammalian cells have correlated
induction of inhibitory tyrosine 15 (Y15) phosphorylation on p34cdc2 with the response
to DNA damage. However, genetic studies in fission yeast have suggested that the
major Y15 kinase, p107wee1, is not required for the cell cycle delay in response to
DNA damage, although it is required for survival after irradiation. Thus, the target of
the checkpoint, and hence the mechanism of cell cycle delay, has remained unknown. This paper shows that Y15 phosphorylation is maintained in checkpoint-arrested fission yeast cells. Wee1 is required for cell cycle arrest induced by up-regulation of chk1, an essential component of this checkpoint. p107wee1 is
hyperphosphorylated in cells delayed by chk1 overexpression or UV irradiation, and p56chk1 can phosphorylate p107wee1 directly in vitro. These observations suggest that in response to DNA damage, p107wee1 is phosphorylated by p56chk1 in vivo, and this results in maintenance of Y15 phosphorylation and hence G2 delay. In the absence of wee1, other Y15 kinases, such as p66mik1, may partially substitute for
p107wee1 to induce cell cycle delay, but this wee1-independent delay is insufficient to
maintain full viability. This study establishes a link between a G2 DNA damage
checkpoint function and a core cell cycle regulator (O'Connell, 1997).
A common cellular response to DNA damage is cell cycle arrest. This checkpoint
control has been the subject of intensive genetic investigation, but the biochemical
mechanism that prevents mitosis following DNA damage is unknown. In Schizosaccharomyces pombe, as well as vertebrates, the timing of mitosis under normal circumstances is determined by the balance of kinases and phosphatases that regulate inhibitory phosphorylation of Cdc2. In S. pombe, the phosphorylation occurs
on tyrosine-15. This method of mitotic control is also used in S. pombe to couple
mitosis with completion of DNA replication, but the role of Cdc2 tyrosine
phosphorylation in the Chk1 kinase-mediated DNA damage checkpoint has remained
uncertain. The G2 DNA damage checkpoint arrest in S. pombe depends on the inhibitory tyrosine phosphorylation of Cdc2 carried out by the Wee1 and Mik1 kinases. The rate of Cdc2
tyrosine dephosphorylation is reduced by irradiation. This result implicates regulation of
Cdc2 tyrosine dephosphorylation, mainly carried out by the Cdc25 tyrosine
phosphatase, as an important part of the mechanism by which the DNA damage
checkpoint induces Cdc2 inhibition and G2 arrest (Rhind, 1997).
Fission yeast cells expressing the human gene encoding the cyclin-dependent kinase inhibitor protein
p21Cip1 are severely compromised for cell cycle progress. The degree of cell cycle inhibition is
related to the level of p21Cip1 expression. Inhibited cells have a 2C DNA content and are judged by
cytology and pulsed field gel electrophoresis to be in the G2 phase of the cell cycle. p21Cip1
accumulates in the nucleus and is associated with p34cdc2 and PCNA. Thus, p21Cip1 interacts with
the same targets in fission yeast as in mammalian cells. Elimination of p34cdc2 binding by mutation
within the cyclin-dependent kinase binding domain of p21Cip1 exaggerates the cell cycle delay
phenotype. By contrast, elimination of PCNA binding by mutation within the PCNA-binding domain
completely abolishes the cell cycle inhibitory effects. Yeast cells expressing wild-type p21Cip1 and the
mutant form that is unable to bind p34cdc2 show enhanced sensitivity to UV. Cell cycle inhibition by
p21Cip1 is largely abolished by deletion of the chk1+ gene, which monitors radiation damage, and is
considerably enhanced in cells deleted for the rad3+ gene, which monitors both DNA damage and the
completion of DNA synthesis. Overexpression of PCNA also results in cell cycle arrest in G2 and
this phenotype is also abolished by deletion of chk1+ and enhanced in cells deleted for rad3+. These
results formally establish a link between PCNA and the products of the rad3+ and chk1+ checkpoint
genes (Tournier, 1997).
Chk1 is a kinase crucial for genomic integrity and an effector of ATR (ATM and Rad3-realated) in DNA damage response. This study shows that Chk1 regulates the DNA damage-induced ubiquitination of proliferating cell nuclear antigen (PCNA), which facilitates the continuous replication of damaged DNA. Surprisingly, this Chk1 function requires the DNA replication protein Claspin but not ATR. Claspin, which is stabilized by Chk1, regulates the binding of the ubiquitin ligase Rad18 to chromatin. Timeless, a Claspin-associating protein, is also required for efficient PCNA ubiquitination. Thus, Chk1 and the Claspin-Timeless module of replication forks not only participate in ATR signaling, but also protect stressed forks independently of ATR (Yang, 2008).
Chk1 kinase, a DNA damage/replication G2 checkpoint kinase, has recently been shown to
phosphorylate and inhibit Cdc25C, a Cdc2 Tyr-15 phosphatase, thereby directly linking the G2
checkpoint to negative regulation of Cdc2. Immature Xenopus oocytes are arrested naturally at the
first meiotic prophase (prophase I) or the late G2 phase, with sustained Cdc2 Tyr-15 phosphorylation.
A Xenopus homolog of Chk1 has been cloned, its developmental expression determined, and
its possible role in prophase I arrest of oocytes examined. Xenopus Chk1 protein is expressed at
approximately constant levels throughout oocyte maturation and early embryogenesis. Overexpression
of wild-type Chk1 in oocytes prevents the release from prophase I arrest by progesterone. Conversely,
specific inhibition of endogenous Chk1 either by overexpression of a dominant-negative Chk1 mutant or
by injection of a neutralizing anti-Chk1 antibody facilitates prophase I release by progesterone.
Moreover, when ectopically expressed in oocytes, a Chk1-nonphosphorylatable Cdc25C mutant alone
can induce prophase I release much more efficiently than wild-type Cdc25C; if endogenous Chk1
function is inhibited, however, even wild-type Cdc25C can induce the release very efficiently. These
results suggest strongly that Chk1 is involved in physiological prophase I arrest of Xenopus oocytes via
the direct phosphorylation and inhibition of Cdc25C. Chk1 might function
either as a G2 checkpoint kinase or as an ordinary cell cycle regulator in prophase-I-arrested oocytes (Nakajo, 1999).
The checkpoint kinase Xchk1 becomes phosphorylated in Xenopus egg extracts in response to DNA replication blocks or
UV-damaged DNA. Xchk1 is also required for the cell cycle delay that is induced by unreplicated or UV-damaged DNA. Xatr, the Xenopus homolog of ATR (Drosophila homolog: meiotic 41) was removed from egg extracts by immunodepletion. In Xatr-depleted extracts,
the checkpoint-associated phosphorylation of Xchk1 is abolished, and the cell cycle delay induced by replication blocks is
strongly compromised. Xatr from egg extracts phosphorylates recombinant Xchk1 in vitro, but not a mutant form of Xchk1
(Xchk1-4AQ) containing nonphosphorylatable residues in its four conserved SQ/TQ motifs. Recombinant human ATR, but not a kinase-inactive mutant,
phosphorylated the same sites in Xchk1. Furthermore, the Xchk1-4AQ mutant was found to be defective in mediating a checkpoint response in egg extracts.
These findings suggest that Xchk1 is a functionally important target of Xatr during a checkpoint response to unreplicated or UV-damaged DNA (Guo, 2000).
Chk1, a nuclear DNA damage/replication G2 checkpoint kinase, phosphorylates Cdc25 and causes its nuclear exclusion in
yeast and mammalian cells, thereby arresting the cell at the G2 phase until DNA repair/replication is completed. Chk1 is
also involved, at least in part, in the natural G2 arrest of immature Xenopus oocytes, but it is unknown how Chk1 inhibits
Cdc25 function and undergoes regulation during oocyte maturation. By using enucleated oocytes, it has been shown that Chk1
inhibits Cdc25 function in the cytoplasm of G2-arrested oocytes and that Cdc25 is activated exclusively in the cytoplasm
of maturing oocytes. Moreover, Chk1 activity is not appreciably altered during maturation, being maintained
at basal levels; the C-terminal truncation mutants of Chk1 have very high kinase activities, strong abilities to inhibit
maturation, and altered subcellular localization in oocytes. These results suggest that the Chk1/Cdc25 pathway is involved cytoplasmically in G2 arrest of Xenopus oocytes, but moderately and independent of the
G2 checkpoint, and that the C-terminal region of Chk1 negatively regulates its kinase activity and also determines its
subcellular localization. Based on these results, Chk1 (with the basal activity) may function
as an ordinary regulator of Cdc25 in oocytes (and in other cell types) and Chk1 might be hyperactivated in response to
the G2 checkpoint via its dramatic conformational change (Oe, 2001).
To explore how Chk1 kinase activity is maintained at
basal levels throughout oocyte maturation, a series of Chk1 C-terminal truncation mutants was expressed in oocytes. Chk1 mutants with truncations of the C-terminal 15 to 60 amino acids, particularly those of 40 and 60 amino acids, undergo hyperautophosphorylation, possess markedly enhanced kinase activity (up to a 25-fold activity of wild-type Chk1, and strongly inhibited oocyte
maturation. These remarkable features were not observed with Chk1 mutants with a truncation of the C-terminal 5 or 10 amino acids (as well as with wild-type
Chk1). Thus, strikingly, removal of amino acids 11-60 from
the C-terminus causes hyperactivation of Chk1, suggesting
that this C-terminal region, which is relatively well conserved, negatively acts on Chk1 kinase activity. (This negative regulation probably accounts for the
relatively low basal activity of endogenous Chk1 during
maturation.) A recent study has shown that, upon
crystal structure analysis, the N-terminal catalytic domain
(~260 amino acids) of recombinant human Chk1 has an
open kinase conformation and that the C-terminal regulatory
domain (~220 amino acids) negatively impacts Chk1
kinase activity in vitro (Chen, 2000). Clearly, the negatively impacting region resides mainly in the C-terminal one-fourth of the regulatory domain and
is also active in vivo. How then might the C-terminal
region negatively act on Chk1 kinase activity in vivo? In
principle, the C-terminal region could bind some transacting
factor(s) that negatively regulates Chk1 kinase activity.
Reportedly, however, the negative effect of the
C-terminal regulatory domain observed in vitro is seen with
'purified' recombinant Chk1 (Chen, 2000). Thus, it
seems more likely that the C-terminal region negatively
regulates Chk1 activity by directly interacting with the
kinase domain or by acting as an autoinhibitory domain. In G2-arrested oocytes, while wild-type Chk1 and Chk1 mutants with a truncation of the C-terminal 5 or 10 amino
acids localize to both the nucleus and the cytoplasm (but
being concentrated in the former),
all the other mutants (D15- to D60-Chk1) localize exclusively
to the cytoplasm. This finding indicates that
the C-terminal region -- at least a region surrounding amino
acid 15 from the C-terminus -- is required for normal
nuclear localization of Chk1. This C-terminal region apparently
contains stretches of sequence rich in basic amino
acids and a test protein fused to the C-terminal region can localize to the nucleus. Thus, the C-terminal region of Chk1 may contain a nuclear targeting signal(s) that is analogous to the classical nuclear localization signal. If so, Chk1 might be
imported to the nucleus by the importin-alpha/beta heterodimer (Oe, 2001).
Wee1 inactivates the Cdc2-cyclin B complex during interphase by phosphorylating Cdc2 on Tyr-15. The activity of Wee1 is highly regulated during the cell cycle. In frog egg extracts Xenopus Wee1 (Xwee1) is present in a hypophosphorylated, active form during interphase and undergoes down-regulation by extensive phosphorylation at M-phase. Xwee1 is also regulated by association with 14-3-3 proteins. In partcular, both Xenopus 14-3-3epsilon and 14-3-3zeta are found associated with His6-GST-Xwee1 in egg extracts. Binding of 14-3-3 to Xwee1 occurs during interphase, but not M-phase, and requires phosphorylation of Xwee1 on Ser-549. A mutant of Xwee1 (S549A) that cannot bind 14-3-3 is substantially less active than wild-type Xwee1 in its ability to phosphorylate Cdc2. This mutation also affects the intranuclear distribution of Xwee1. In cell-free kinase assays, Xchk1 phosphorylates Xwee1 on Ser-549. The results of experiments in which Xwee1, Xchk1, or both were immunodepleted from Xenopus egg extracts suggest that these two enzymes are involved in a common pathway in the DNA replication checkpoint response. Replacement of endogenous Xwee1 with recombinant Xwee1-S549A in egg extracts attenuates the cell cycle delay induced by addition of excess recombinant Xchk1. Taken together, these results suggest that Xchk1 and 14-3-3 proteins act together as positive regulators of Xwee1 (Lee, 2001).
In Xenopus embryos, cell cycle elongation and degradation of Cdc25A (a Cdk2 Tyr15 phosphatase) occur naturally at the midblastula transition (MBT), at which time a physiological DNA replication checkpoint is thought to be activated by the exponentially increased nucleo-cytoplasmic ratio. The checkpoint kinase Chk1, but not Cds1 (Drosophila homolog: loki), is activated transiently at the MBT in a maternal/zygotic gene product-regulated manner and is essential for cell cycle elongation and Cdc25A degradation at this transition. A constitutively active form of Chk1 can phosphorylate Cdc25A in vitro and can target it rapidly for degradation in pre-MBT embryos. Intriguingly, for this degradation, however, Cdc25A also requires a prior Chk1-independent phosphorylation at Ser73. Ectopically expressed human Cdc25A can be degraded in the same way as Xenopus Cdc25A. Finally, Cdc25A degradation at the MBT is a prerequisite for cell viability at later stages. Thus, the physiological replication checkpoint is activated transiently at the MBT by developmental cues, and activated Chk1, only together with an unknown kinase, targets Cdc25A for degradation to ensure later development (Shimuta, 2002).
Thus Chk1, but not Cds1, is essential for cell cycle elongation or the physiological DNA replication checkpoint at the MBT and for cell viability shortly after the MBT in Xenopus. Chk1 is also essential for normal cell proliferation at the MBT in Drosophila and at the blastocyst stage in mice. In these organisms, however, it is not known whether Chk1 undergoes any regulation at the relevant stages of development. The results in Xenopus do show, however, that Chk1 undergoes ATR-mediated phosphorylation (and hence activation) weakly and transiently at the MBT or, more accurately, during the maternal/zygotic transition. This result, which is the first direct demonstration of a physiological Chk1 activation in metazoan development, would imply that replication checkpoint signaling in early embryos is induced, rather than constitutive, and intrinsically weak. Notably, however, very low basal levels of Chk1 modification was consistently observed even in post-MZT embryos, which could imply that constitutive replication checkpoint signaling occurs at low basal levels in normal cells. At present, it is not known whether such basal signaling is essential for later development, although, in Drosophila, ATR or Chk1 has been shown to be non-essential for post-embryonic development (Shimuta, 2002).
Prior to the midblastula transition (MBT), Xenopus laevis embryos do not engage cell cycle checkpoints, although overexpression of the kinase XChk1 arrests cell divisions. At the MBT, XChk1 transiently activates and promotes cell cycle lengthening. In this study, endogenous XChk1 was inhibited by the expression of dominant-negative XChk1 (DN-XChk1). Development appeared normal until the early gastrula stage, when cells lost attachments and chromatin condensed. TUNEL and caspase assays indicate these embryos die by apoptosis during gastrulation. Embryos with unreplicated DNA likewise die by apoptosis. Embryos expressing DN-XChk1 proceed through additional rapid rounds of DNA replication but initiate zygotic transcription on schedule. Therefore, XChk1 is essential in the early Xenopus embryo for cell cycle remodeling and for survival after the MBT (Carter, 2003).
The checkpoint mediator protein Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing aphidicolin-induced DNA replication blocks. During this checkpoint response, Claspin becomes phosphorylated on threonine 906 (T906), which creates a docking site for Plx1, the Xenopus Polo-like kinase. This interaction promotes the phosphorylation of Claspin on a nearby serine (S934) by Plx1. After a prolonged interphase arrest, aphidicolin-treated egg extracts typically undergo adaptation and enter into mitosis despite the presence of incompletely replicated DNA. In this process, Claspin dissociates from chromatin, and Chk1 undergoes inactivation. By contrast, aphidicolin-treated extracts containing mutants of Claspin with alanine substitutions at positions 906 or 934 (T906A or S934A) are unable to undergo adaptation. Under such adaptation-defective conditions, Claspin accumulates on chromatin at high levels, and Chk1 does not decrease in activity. These results indicate that the Plx1-dependent inactivation of Claspin results in termination of a DNA replication checkpoint response (Yoo, 2004).
Checkpoint pathways prevent cell-cycle progression in the event of
DNA lesions. Checkpoints are well defined in mitosis, where lesions can be the result of extrinsic damage, and they are critical in meiosis, where DNA breaks are a programmed step in meiotic recombination. In mitotic yeast cells, the Chk1 protein couples DNA repair to the cell-cycle machinery. The Atm and Atr proteins (see Drosophila mei-41) are mitotic cell-cycle proteins that also associate with chromatin during meiotic prophase I. The
genetic and regulatory interaction between Atm and mammalian Chk1 appears to be
important for integrating DNA-damage repair with cell-cycle arrest.
Structural homologs of yeast Chk1 have been identified in human and mouse. Chk1(Hu/Mo) has protein kinase activity and is expressed in the testis. Chk1 accumulates in late zygotene and pachytene spermatocytes and is present along synapsed meiotic chromosomes. Chk1 localizes along the unsynapsed axes of X and Y chromosomes in pachytene spermatocytes. The association of Chk1 with meiotic chromosomes and levels of Chk1 protein depend on a functional Atm gene product, but Chk1 is not
dependent on p53 for meiosis I functions. Mapping of CHK1 to human
chromosomes indicates that the gene is located at 11q22-23, a region marked by
frequent deletions and loss of heterozygosity in human tumors. The Atm-dependent presence of Chk1 in mouse cells and along meiotic chromosomes, and the late pachynema co-localization of Atr and Chk1 on the unsynapsed axes of the paired X and Y chromosomes, suggest that Chk1 acts as an integrator for Atm and
Atr signals and may be involved in monitoring the processing of meiotic recombination.
Mapping of the CHK1 gene to a region of frequent loss of heterozygosity
in human tumors at 11q22-23 indicates that the CHK1 gene is a candidate tumor
suppressor gene (Flaggs, 1997).
In response to DNA damage, mammalian cells prevent cell cycle progression through the control of
critical cell cycle regulators. A human gene has been identified that encodes the protein Chk1, a homolog of
the Schizosaccharomyces pombe Chk1 protein kinase, which is required for the DNA damage
checkpoint. Human Chk1 protein is modified in response to DNA damage. In vitro Chk1 binds to
and phosphorylates the dual-specificity protein phosphatases Cdc25A, Cdc25B, and Cdc25C, which
control cell cycle transitions by dephosphorylating cyclin-dependent kinases. Chk1 phosphorylates
Cdc25C on serine-216. Serine-216
phosphorylation creates a binding site for 14-3-3 protein and inhibits function of the phosphatase. These
results suggest a model whereby in response to DNA damage, Chk1 phosphorylates and inhibits
Cdc25C, thus preventing activation of the Cdc2-cyclin B complex and mitotic entry (Sanchez, 1997).
Human Cdc25C is a dual-specificity protein phosphatase that controls entry into
mitosis by dephosphorylating the protein kinase Cdc2. Throughout interphase, but not
in mitosis, Cdc25C is phosphorylated on serine-216 and binds to members of the
highly conserved and ubiquitously expressed family of 14-3-3 proteins (see Drosophila Leonardo). A mutation
preventing phosphorylation of serine-216 abrogates 14-3-3 binding. Conditional
overexpression of this mutant perturbs mitotic timing and allows cells to escape the
G2 checkpoint arrest induced by either unreplicated DNA or radiation-induced
damage. Chk1, a fission yeast kinase involved in the DNA damage checkpoint
response, phosphorylates Cdc25C in vitro on serine-216. These results indicate that
serine-216 phosphorylation and 14-3-3 binding negatively regulate Cdc25C and identify
Cdc25C as a potential target of checkpoint control in human cells (Peng, 1997).
Checkpoints maintain the order and fidelity of events of the cell cycle by blocking mitosis in response to
unreplicated or damaged DNA. In most species this is accomplished by preventing activation of the
cell-division kinase Cdc2, which regulates entry into mitosis. The Chk1 kinase, an effector of the
DNA-damage checkpoint, phosphorylates Cdc25, an activator of Cdc2. Phosphorylation of Cdc25
promotes its binding to 14-3-3 proteins, preventing it from activating Cdc2. It is proposed that a
similar pathway is required for mitotic arrest in the presence of unreplicated DNA (that is, in the
replication checkpoint) in fission yeast. It is shown by mutagenesis that Chk1 functions redundantly with
the kinase Cds1 at the replication checkpoint and that both kinases phosphorylate Cdc25 on the same
sites, which include serine residues at positions 99, 192 and 359. Mutation of these residues reduces
binding of 14-3-3 proteins to Cdc25 in vitro and disrupts the replication checkpoint in vivo. It is concluded
that both Cds1 and Chk1 regulate the binding of Cdc25 to 14-3-3 proteins as part of the checkpoint
response to unreplicated DNA (Zeng, 1998).
The recent discovery of checkpoint kinases has suggested the conservation of checkpoint mechanisms between yeast and mammals.
In yeast, the protein kinase Chk1 is thought to mediate signaling associated with the DNA damage checkpoint of the cell cycle.
However, the function of Chk1 in mammals has remained unknown. Targeted disruption of Chk1 in mice has shown that Chk1-/- embryos exhibit gross morphologic abnormalities in nuclei as early as the blastocyst stage. In culture, Chk1-/- blastocysts show a severe defect in outgrowth of the inner cell mass and undergo apoptosis. DNA replication block and DNA damage
fail to arrest the cell cycle before initiation of mitosis in Chk1-/- embryos. These results may indicate that Chk1 is indispensable for cell
proliferation and survival through maintaining the G2 checkpoint in mammals (Takai, 2000).
Checkpoint genes cause cell cycle arrest when DNA is damaged or DNA replication
is blocked. Although a human homolog of Chk1 (hChk1) has recently been reported
to be involved in the DNA damage checkpoint through phosphorylation of Cdc25A,
B, and C, it is not known at which phase(s) of the cell cycle hChk1 functions
and how hChk1 causes cell cycle arrest in response to DNA damage. In the present
study, it is demonstrated that, in normal human fibroblasts (MJ90), hChk1 is
expressed specifically at the S to M phase of the cell cycle at both the RNA and
protein levels and that it is localized to the nucleus at this time. hChk1
activity, as determined by phosphorylation of Cdc25C, is readily detected at the
S to M phase of the cell cycle, and DNA damage induced by UV or ionizing
radiation does not enhance the expression of hChk1 or its activity. Furthermore,
hChk1 exists in an active form at the S to M phase in fibroblasts derived from
patients with ataxia telangiectasia (AT): these patients lack the functional AT mutated
(ATM) gene product, suggesting that hChk1 expression is independent of
functional ATM. Taken together with the findings that phosphorylation of Cdc25C
on serine 216 is increased at the S to M phase, it is suggested that at this
particular phase of the cell cycle, even in the absence of DNA damage, hChk1
phosphorylates Cdc25C on serine 216: this modification is considered to be a prerequisite
for the G2/M checkpoint. Thus, hChk1 may play an important role in keeping
Cdc25C prepared for responding to DNA damage by phosphorylating its serine
residue at 216 during the S to M phase (Kaneko, 1999).
Chk1, an evolutionarily conserved protein kinase, has been implicated in cell cycle checkpoint control in lower eukaryotes. By gene
disruption, it has been shown that CHK1 deficiency results in a severe proliferation defect and death in embryonic stem (ES) cells, and
peri-implantation embryonic lethality in mice. Through analysis of a conditional CHK1-deficient cell line, it has been demonstrated that ES
cells lacking Chk1 have a defective G2/M DNA damage checkpoint in response to gamma-irradiation (IR). CHK1
heterozygosity modestly enhances the tumorigenesis phenotype of WNT-1 transgenic mice. In human cells, Chk1 is phosphorylated on serine
345 (S345) in response to UV, IR, and hydroxyurea (HU). Overexpression of wild-type Atr enhances, whereas overexpression of the kinase-defective mutant
Atr inhibits S345 phosphorylation of Chk1 induced by UV treatment. Taken together, these data indicate that Chk1 plays an essential role in the mammalian
DNA damage checkpoint, embryonic development, and tumor suppression, and that Atr regulates Chk1 (Liu, 2000).
In human cells, Chk1 is phosphorylated in response to DNA damage. Because SQ sites are known substrates of Atm and Atr, rabbit polyclonal antibodies were raised to peptides containing phosphorylated serine in the conserved SQ
sites in human Chk1 protein. Only the anti-phospho-S345 (anti-p-S345) antibodies produce a signal specific for the phospho-antigen-peptide. Chk1 can be
immunoprecipitated with anti-Chk1 or anti-p-S345 antibodies from lysates prepared from 293T cells that have been either untreated or treated with hydroxyurea
(HU), UV, or IR. Although the anti-Chk1 antibodies bring down equivalent amounts of Chk1 proteins from all cell lysates, the anti-p-S345 antibodies
immunoprecipitate Chk1 proteins only from HU-, UV-, or IR-treated cells, but not from untreated cells. This experiment suggests that in human cells
Chk1 is phosphorylated on S345 in response to DNA damage or replication blocks.
Because ATR and CHK1 disruptions both lead to peri-implantation embryonic lethality in mice, it was asked whether Atr regulates Chk1 in response to DNA
damage. By transient transfection experiments, it has been shown that overexpression of wild-type Atr, but not the kinase-defective mutant Atr, increases the
phosphorylation of co-transfected Chk1 on S345 in response to UV. Furthermore, overexpression of the kinase-defective mutant Atr in an inducible
cell line inhibits the UV-induced S345 phosphorylation of endogenous Chk1. These results suggest that Atr functions
upstream of Chk1 in the mammalian DNA damage response pathway and is a major regulator of Chk1 phosphorylation after DNA damage (Liu, 2000).
Atm responds primarily to DNA-damaging agents that cause double-stranded
breaks, such as IR, and Atm phosphorylates Chk2. Atm and Chk2 together phosphorylate Brca1 and p53, and activate cellular responses including G1 arrest. In contrast, Atr responds primarily to agents like UV and HU that can potentially interfere with DNA replication, and Atr phosphorylates Chk1. It is possible
that Atr and Chk1 phosphorylate Brca1, p53 in addition to Cdc25C, and
activate cellular responses including G2 arrest. Furthermore, the two pathways have significant overlap and often cooperate with each other to ensure prompt
and efficient repair of DNA damage and to maintain genomic integrity. When one pathway is genetically compromised, they can also function redundantly,
although probably to a lesser extent and with different kinetics. For example, the rapid p53 stabilization in response to IR is greatly reduced in ATM mutant cells, but it does occur much later, which is probably due to Atr. Similar
observations have been made for Chk2 phosphorylation in response to IR. This may reflect the fact that in addition to
tailoring the cellular response to different types of DNA damage, cells have many responses that are commonly required toward different types of genotoxic
stress (Liu, 2000 and references therein).
Chk1 is an evolutionarily conserved protein kinase that regulates cell cycle progression in response to checkpoint activation. Agents that block DNA replication or cause certain forms of DNA damage induce the phosphorylation of human Chk1. The phosphorylated form of Chk1 possesses higher intrinsic protein kinase activity and elutes more quickly on gel filtration columns. Serines 317 and 345 were identified as sites of phosphorylation in vivo, and ATR (the ATM- and Rad3-related protein kinase) phosphorylates both of these sites in vitro. Furthermore, phosphorylation of Chk1 on serines 317 and 345 in vivo is ATR dependent. Mutants of Chk1 containing alanine in place of serines 317 and 345 are poorly activated in response to replication blocks or genotoxic stress in vivo, are poorly phosphorylated by ATR in vitro, and are not found in faster-eluting fractions by gel filtration. These findings demonstrate that the activation of Chk1 in response to replication blocks and certain forms of genotoxic stress involves phosphorylation of serines 317 and 345. In addition, this study implicates ATR as a direct upstream activator of Chk1 in human cells (Zhao, 2001).
The breast cancer tumor-suppressor gene, BRCA1, encodes a protein with a BRCT domain -- a motif that is found in many proteins that are implicated in DNA damage response and in genome stability. Phosphorylation of BRCA1 by the DNA
damage-response proteins ATM, ATR and hCds1/Chk2 changes in response to DNA
damage and at replication-block checkpoints. Although cells that lack BRCA1 have an abnormal response to DNA damage, the exact role of BRCA1 in this process has remained unclear. BRCA1 is shown in this study to be essential for activating the Chk1 kinase that regulates DNA damage-induced G2/M arrest. Thus, BRCA1 controls the expression, phosphorylation and cellular localization of Cdc25C and Cdc2/cyclin
B kinase-proteins that are crucial for the G2/M transition. BRCA1
regulates the expression of both Wee1 kinase, an inhibitor of Cdc2/cyclin B
kinase, and the 14-3-3 family of proteins that sequesters phosphorylated Cdc25C and Cdc2/cyclin B kinase in the cytoplasm. It is concluded that BRCA1 regulates key effectors that control the G2/M checkpoint and is therefore involved in regulating the onset of mitosis (Yarden, 2002).
The role of the PI 3-kinase cascade in regulation of cell growth is well established. PKB (protein kinase B) is a key downstream effector of the PI 3-kinase pathway and is best known for its antiapoptotic effects and the role it plays in initiation of S phase. PKB activity is high in the G2/M phase of the cell cycle in epithelial cells. Inhibition of the PI 3-kinase pathway in MDCK cells induces apoptosis at the G2/M transition, prevents activation of cyclin B-associated kinase, and prohibits entry of the surviving cells into mitosis. All of these consequences of the inhibition of PI 3-kinase are relieved by expression of a constitutively active form of PKB (caPKB), indicating that PKB plays a role in regulation of the G2/M phase. Inhibition of PI 3-kinase results in activation of Chk1, whereas constitutively active PKB inhibits the ability of Chk1 to become activated in response to treatment with hydroxyurea. Preliminary data show that PKB phosphorylates the Chk1 polypeptide in vitro on serine 280. These results not only implicate PKB activity in transition through the G2/M stage of the cell cycle, but they also suggest the existence of crosstalk between the PI 3-kinase pathway and the key regulators of the DNA damage checkpoint machinery (Shtivelman, 2002).
The human Cdc25A phosphatase plays a pivotal role at the G1/S transition by activating cyclinE and A/Cdk2 complexes through dephosphorylation. In response to ionizing radiation (IR), Cdc25A is phosphorylated by both Chk1 and Chk2 on Ser-123. This in turn leads to ubiquitylation and rapid degradation of Cdc25A by the proteasome resulting in cell cycle arrest. In response to UV-irradiation, Cdc25A is phosphorylated at a different serine residue, Ser-75. Significantly, Cdc25A mutants carrying alanine instead of either Ser-75 or Ser-123 demonstrate that only Ser-75 mediates protein stabilization in response to UV-induced DNA damage. As a consequence, cyclinE/Cdk2 kinase activity is high. Furthermore, Cdc25A is phosphorylated by Chk1 on Ser-75 in vitro and the same site is also phosphorylated in vivo. Taken together, these data strongly suggest that phosphorylation of Cdc25A on Ser-75 by Chk1 and its subsequent degradation is required to delay cell cycle progression in response to UV-induced DNA lesions (Hassepass, 2003).
The checkpoint kinases Chk1 and Chk2 are central to the induction of cell cycle
arrest, DNA repair, and apoptosis as elements in the DNA-damage checkpoint.
In several human tumor cell lines, Chk1 and Chk2 control the
induction of the p53 related transcription factor p73 in response to DNA damage.
Multiple experimental systems were used to show that interference with or
augmentation of Chk1 or Chk2 signaling strongly impacts p73 accumulation.
Furthermore, Chk1 and Chk2 control p73 mRNA accumulation after DNA damage.
E2F1 directs p73 expression in the presence and absence
of DNA damage. Chk1 and Chk2, in turn, are vital to E2F1 stabilization and
activity after genotoxic stress. Thus, Chk1, Chk2, E2F1, and p73 function in a
pathway mediating p53-independent cell death produced by cytotoxic drugs. Since
p53 is often obviated through mutation as a cellular port for anticancer
intervention, this pathway controlling p53 autonomous pro-apoptotic signaling is
of potential therapeutic importance (Urist, 2004).
GRASP65, a structural protein of the Golgi apparatus, has been linked
to the sensing of Golgi structure and the integration of this information with
the control of mitotic entry in the form of a Golgi checkpoint.
Cdk1-cyclin B is the major kinase phosphorylating GRASP65 in mitosis, and
phosphorylated GRASP65 interacts with the polo box domain of the polo-like
kinase Plk1. GRASP65 is phosphorylated in its C-terminal domain at four
consensus sites by Cdk1-cyclin B, and mutation of these residues to alanine
essentially abolishes both mitotic phosphorylation and Plk1 binding. Expression
of the wild-type GRASP65 C-terminus but not the phosphorylation defective mutant
in normal rat kidney cells causes a delay but not the block in mitotic entry
expected if this were a true cell cycle checkpoint. These findings identify a
Plk1-dependent signalling mechanism potentially linking Golgi structure and cell
cycle control, but suggest that this may not be a cell cycle checkpoint in the
classical sense (Preisinger, 2005 ).
The Timeless protein is essential for circadian rhythm in Drosophila. The Timeless orthologue in mice is essential for viability and appears to be required for the maintenance of a robust circadian rhythm as well. The human Timeless protein interacts with both the circadian clock protein cryptochrome 2 and with the cell cycle checkpoint proteins Chk1 and the ATR-ATRIP complex and plays an important role in the DNA damage checkpoint response. Down-regulation of Timeless in human cells seriously compromises replication and intra-S checkpoints, indicating an intimate connection between the circadian cycle and the DNA damage checkpoints that is in part mediated by the Timeless protein (Unsal-Kacmaz, 2005).
The human homologue of the C. elegans biological clock protein CLK-2 (HCLK2) associates with the S-phase checkpoint components ATR, ATRIP, claspin and Chk1. Consistent with a critical role in the S-phase checkpoint, HCLK2-depleted cells accumulate spontaneous DNA damage in S-phase, exhibit radio-resistant DNA synthesis, are impaired for damage-induced monoubiquitination of FANCD2 and fail to recruit FANCD2 and Rad51 (critical components of the Fanconi anaemia and homologous recombination pathways, respectively) to sites of replication stress. Although Thr 68 phosphorylation of the checkpoint effector kinase Chk2 remains intact in the absence of HCLK2, claspin phosphorylation and degradation of the checkpoint phosphatase Cdc25A are compromised following replication stress as a result of accelerated Chk1 degradation. ATR phosphorylation is known to both activate Chk1 and target it for proteolytic degradation, and depleting ATR or mutation of Chk1 at Ser 345 restored Chk1 protein levels in HCLK2-depleted cells. It is concluded that HCLK2 promotes activation of the S-phase checkpoint and downstream repair responses by preventing unscheduled Chk1 degradation by the proteasome (Collins, 2007).
The ATR-dependent DNA damage response pathway can respond to a diverse group of
lesions as well as inhibitors of DNA replication. Using the Xenopus egg extract
system, it has been shown that lesions induced by UV irradiation and cis-platinum cause
the functional uncoupling of MCM helicase and DNA polymerase activities, an
event previously shown for aphidicolin. Inhibition of uncoupling during
elongation with inhibitors of MCM7 or Cdc45, a putative helicase cofactor,
results in abrogation of Chk1 phosphorylation, indicating that uncoupling is
necessary for activation of the checkpoint. However, uncoupling is not
sufficient for checkpoint activation, and DNA synthesis by Polalpha is also required.
Finally, using plasmids of varying size, it has been demonstrated that all of the unwound
DNA generated at a stalled replication fork can contribute to the level of Chk1
phosphorylation, suggesting that uncoupling amplifies checkpoint signaling at
each individual replication fork. Taken together, these observations indicate
that functional uncoupling of MCM helicase and DNA polymerase activities occurs
in response to multiple forms of DNA damage and that there is a general
mechanism for generation of the checkpoint-activating signal following DNA
damage (Byun, 2005).
One critical component of the DNA damage response pathway is the ATR-ATRIP
complex. ATR is a phosphatidylinositol kinase-related protein kinase that is
thought to function as both a sensor and transducer in the DNA damage response.
ATR, and its associated protein ATRIP, respond to a broad spectrum of genotoxic
agents that includes ultraviolet light, topoisomerase inhibitors,
alkylating agents, and cis-platinum, as well as chemicals that disrupt
replication, such as aphidicolin and hydroxyurea (HU). Following DNA damage, ATR
phosphorylates and activates the checkpoint kinase Chk1.
In higher eukaryotes, the phosphorylation of Chk1 also requires the
activities of the Rad9-Rad1-Hus1 (RHR, aka 9-1-1) complex and Claspin.
The RHR complex is a PCNA-related complex that is loaded on to
primed DNA in vitro and is
recruited to sites of DNA damage in vivo.
Claspin was initially identified as a protein that binds the activated form of
Chk1, and it has been shown to bind chromatin throughout S phase (Byun, 2005 and references therein).
Recruitment of ATR and Rad1 to chromatin
and activation of Chk1 requires DNA replication in Xenopus egg extracts following several
types of DNA damage. Studies in mammalian cells also indicate that ATR binds
UV-damaged chromatin in S phase but not G1 phase. In
addition, other studies show that a replication fork must be established in
Saccharomyces cerevisiae for checkpoint activation induced by methylmethane
sulfonate (MMS). Taken together, these observations
suggest that one or more replication-dependent events are needed to generate the
signal that ATR recognizes for many types of DNA damage (Byun, 2005 and references therein).
Although the exact nature of the biochemical signal(s) responsible for
activating the ATR pathway and the replication-dependent steps necessary for its
formation are still unclear, evidence from a number of different systems
supports a central role for replication protein A (RPA)-coated single-stranded
DNA (ssDNA) in the response. In yeast, certain RPA mutants exhibit a checkpoint
defect and also adapt more rapidly to DNA damage.
In addition, knock-down of the ssDNA-binding protein
RPA results in a significant loss of both Chk1 phosphorylation and ATR foci
formation following DNA damage in mammalian cells. In
Xenopus egg extracts, RPA is also required for the recruitment of ATR to
chromatin following treatment with aphidicolin or etoposide
and for the recruitment of ATR to poly(dA)70 ssDNA.
Importantly, in vitro experiments have shown that RPA is
sufficient for the binding of ATRIP to ssDNA and that RPA
also facilitates the association of the RHR complex with DNA (Byun, 2005 and references therein).
Interestingly, the amount of ssDNA appears to increase following genotoxic
stress, as RPA accumulates on chromatin in Xenopus extracts and mammalian cells
treated with UV, MMS, HU, or aphidicolin. Moreover, in
budding yeast, increased amounts of ssDNA have been observed by electron
microscopy following HU treatment. In the case of DNA damage,
the mechanism by which this ssDNA accumulates is not known, nor is it clear if
ssDNA accumulation is required for checkpoint activation. In principle, a number
of DNA repair (e.g., nucleotide excision repair, base excision repair) and
recombination processes could lead to the generation of ssDNA following DNA
damage at several points in the cell cycle. Alternatively, during DNA
replication, ssDNA could be formed if DNA polymerases are slowed by lesions and
the replicative helicase continues to unwind DNA. Indeed, uncoupling of helicase
and polymerase activities has been observed in the presence of
aphidicolin, and recent studies have shown that this aphidicolin-induced uncoupling is dependent on the MCM helicase (Byun, 2005 and references therein).
This uses a cell-free extract system derived from Xenopus eggs
to examine the mechanism by which ssDNA accumulates
following DNA damage. The appearance and disappearance of a
highly unwound form of plasmid DNA that accumulates following aphidicolin
treatment correlates with the phosphorylation of Chk1
on Ser 344 (S344). Importantly, this hyperunwound form of DNA is also observed
upon replication of plasmid DNA damaged with either UV or cis-platinum. This
suggests that DNA damage induces uncoupling of helicase and polymerase
activities and that these lesions, as well as aphidicolin, may generate a common
checkpoint-activating DNA structure. Moreover, while stalling the replication
fork with aphidicolin results in a robust checkpoint response, it is found that
aphidicolin elicits no checkpoint when the MCM DNA helicase is inactivated.
Using plasmids of varying sizes, it is also shown that functional uncoupling of DNA
unwinding and DNA synthesis during S phase may serve to amplify the level of
Chk1 phosphorylation that can be achieved at each individual replication fork.
Finally, it is demonstrated that although DNA unwinding is necessary for checkpoint
activation, it is not sufficient and additional DNA synthesis by Pol is
needed. Taken together, these results suggest that functional uncoupling of
helicase and polymerase activities is necessary to convert DNA lesions and
chemical inhibitors of DNA replication into the signal(s) that activate the
ATR-dependent checkpoint (Byun, 2005).
The ATM (ataxia-telangiectasia mutated) and ATR (ataxia-telangiectasia and
Rad3-related) kinases respond to DNA damage by phosphorylating cellular target
proteins that activate DNA repair pathways and cell cycle checkpoints in order
to maintain genomic integrity. The oncogenic p53-induced
serine/threonine phosphatase PPM1D (or Wip1: Drosophila homolog Protein phosphatase 2C) dephosphorylates two ATM/ATR
targets, Chk1 and p53. PPM1D binds Chk1 and dephosphorylates the ATR-targeted
phospho-Ser 345, leading to decreased Chk1 kinase activity. PPM1D also
dephosphorylates p53 at phospho-Ser 15. PPM1D dephosphorylations are correlated
with reduced cellular intra-S and G2/M checkpoint activity in response to DNA
damage induced by ultraviolet and ionizing radiation. Thus, a primary function
of PPM1D may be to reverse the p53 and Chk1-induced DNA damage and cell cycle
checkpoint responses and return the cell to a homeostatic state following
completion of DNA repair. These homeostatic functions may be partially
responsible for the oncogenic effects of PPM1D when it is amplified and
overexpressed in human tumors (Lu, 2005).
Claspin (potential Drosophila homolog: CG32251) is required for the phosphorylation and activation of the Chk1 protein kinase by ATR during DNA replication and in response to DNA damage. This checkpoint pathway plays a critical role in the resistance of cells to genotoxic stress. Human Claspin is cleaved by caspase-7 during the initiation of apoptosis. In cells, induction of DNA damage by etoposide at first produced rapid phosphorylation of Chk1 at a site targeted by ATR. Subsequently, etoposide causes activation of caspase-7, cleavage of Claspin, and dephosphorylation of Chk1. In apoptotic cell extracts, Claspin is cleaved by caspase-7 at a single aspartate residue into a large N-terminal fragment and a smaller C-terminal fragment that each contain different functional domains. The large N-terminal fragment was heavily phosphorylated in a human cell-free system in response to double-stranded DNA oligonucleotides, and this fragment retained Chk1 binding activity. In contrast, the smaller C-terminal fragment did not bind Chk1, but did associate with DNA and inhibited the DNA-dependent phosphorylation of Chk1 associated with its activation. These results indicate that cleavage of Claspin by caspase-7 inactivates the Chk1 signaling pathway. This mechanism may regulate the balance between cell cycle arrest and induction of apoptosis during the response to genotoxic stress (Clarke, 2005).
The E2F transcription factor family is known to play a key role in the timely expression of genes required for cell cycle progression and proliferation, but only a few E2F target genes have been identified. The possibility that E2F regulators play a broader role was suggested by identifying additional genes bound by E2F in living human cells. A protocol was developed to identify genomic binding sites for DNA-binding factors in
mammalian cells that combines immunoprecipitation of cross-linked protein-DNA complexes with DNA microarray analysis. Among ~1200 genes expressed during cell cycle entry, it was found that the promoters of
127 are bound by the E2F4 transcription factor in primary fibroblasts. A subset of these targets is also bound by E2F1. Most previously identified target genes known to have roles in DNA replication and cell cycle control and represented on the microarray were confirmed by this analysis. A remarkable cadre of genes was identifed with no previous connection to E2F regulation, including genes
that encode components of the DNA damage checkpoint and repair pathways, as well as factors involved in chromatin
assembly/condensation, chromosome segregation, and the mitotic spindle checkpoint. These data indicate that E2F directly links cell cycle
progression with the coordinate regulation of genes essential for both the synthesis of DNA as well as its surveillance (Ren, 2002).
One of the most surprising findings was the identification of a cluster of genes involved in several different checkpoints. Two genes involved in the DNA damage checkpoint, p53 and Chk1, were identifed. p53 is induced in response to DNA damage and acts to enforce a cell cycle block in
G1 phase. The identification of p53
as an E2F target was unanticipated, because the p53 promoter
lacks a recognizable E2F consensus site. This finding
may be explained by the indirect recruitment of E2F by additional
promoter-bound factors. E2F has been shown previously to indirectly
increase levels of p53 through activation of the
p14ARF gene, a component of the
p14ARF-Mdm2 stabilization pathway. These results suggest that
E2F may also directly control p53 expression levels. This
finding is also intriguing in light of previous reports implicating an essential role for both p53 and the pRB family in the G1 DNA
damage arrest checkpoint. The mechanisms underlying the pRB requirement for this G1 block are not known, although a role for
E2F-responsive genes has been postulated. A
second checkpoint gene, Chk1, was also identified in the E2F
location analysis. CHK1 is required for the G2 DNA damage
(and perhaps an S phase) checkpoint. Interestingly, pRB is required for Chk1 down-regulation and resumption of G2 after DNA damage, suggesting that E2F could be involved in Chk1 gene expression (Ren, 2002).
The ARF tumour suppressor (p14ARF in humans, p19ARF in mice) is a central component of the cellular defence against
oncogene activation. The expression of ARF, which shares a genetic locus with the p16INK4a tumour suppressor,
is regulated by the action of transcription factors such as members of the E2F family.
ARF can bind to and inhibit the Hdm2 protein (Mdm2 in mice), which functions as an
inhibitor and E3 ubiquitin ligase for the p53 transcription factor. In addition to activating p53 through binding Mdm2, ARF
possesses other functions, including an ability to repress the transcriptional
activity of the antiapoptotic RelA(p65) NF-kappaB subunit.
ARF induces the ATR- and Chk1-dependent phosphorylation of the RelA
transactivation domain at threonine 505, a site required for ARF-dependent
repression of RelA transcriptional activity. Consistent with this effect, ATR
and Chk1 are required for ARF-induced sensitivity to tumour necrosis factor-alpha induced cell death. Significantly, ATR activity is also required for
ARF-induced p53 activity and inhibition of proliferation. ARF achieves these
effects by activating ATR and Chk1. Furthermore, ATR and its scaffold protein
BRCA1, but not Chk1, relocalise to specific nucleolar sites. These results
reveal novel functions for ARF, ATR and Chk1 together with a new pathway
regulating RelA NF-kappaB function. Moreover, this pathway provides a mechanism
through which ARF can remodel the cellular response to an oncogenic challenge
and execute its function as a tumour suppressor (Rocha, 2005).
The spindle checkpoint delays anaphase onset in cells with mitotic spindle defects. Chk1, a component of the DNA damage and replication checkpoints, protects vertebrate cells against spontaneous chromosome missegregation and is required to sustain anaphase delay when spindle function is disrupted by taxol, but not when microtubules are completely depolymerized by nocodazole. Spindle checkpoint failure in Chk1-deficient cells correlates with decreased Aurora-B kinase activity and impaired phosphorylation and kinetochore localization of BubR1. Furthermore, Chk1 phosphorylates Aurora-B and enhances its catalytic activity in vitro. It is proposed that Chk1 augments spindle checkpoint signaling and is required for optimal regulation of Aurora-B and BubR1 when kinetochores produce a weakened signal. In addition, Chk1-deficient cells exhibit increased resistance to taxol. These results suggest a mechanism through which Chk1 could protect against tumorigenesis through its role in spindle checkpoint signaling (Zachos, 2007).
Evasion of DNA damage-induced cell death, via mutation of the p53 tumor suppressor or overexpression of prosurvival Bcl-2 family proteins, is a key step toward malignant transformation and therapeutic resistance. Depletion or acute inhibition of checkpoint kinase 1 (Chk1) is sufficient to restore γ-radiation-induced apoptosis in p53 mutant zebrafish embryos. Surprisingly, caspase-3 is not activated prior to DNA fragmentation, in contrast to classical intrinsic or extrinsic apoptosis. Rather, an alternative apoptotic program is engaged that cell autonomously requires atm (ataxia telangiectasia mutated), atr (ATM and Rad3-related) and caspase-2, and is not affected by p53 loss or overexpression of bcl-2/xl. Similarly, Chk1 inhibitor-treated human tumor cells hyperactivate ATM, ATR, and caspase-2 after γ-radiation and trigger a caspase-2-dependent apoptotic program that bypasses p53 deficiency and excess Bcl-2. The evolutionarily conserved 'Chk1-suppressed' pathway defines a novel apoptotic process, whose responsiveness to Chk1 inhibitors and insensitivity to p53 and BCL2 alterations have important implications for cancer therapy (Sidi, 2008).
The mitotic regulators Wee1 and Cdk1 can inactivate each other through inhibitory phosphorylations. This double-negative feedback loop is part of a bistable trigger that makes the transition into mitosis abrupt and decisive. To generate a bistable response, some component of a double-negative feedback loop must exhibit an ultrasensitive response to its upstream regulator. This study experimentally demonstrates that Wee1 exhibits a highly ultrasensitive response to Cdk1. Several mechanisms can, in principle, give rise to ultrasensitivity, including zero-order effects, multisite phosphorylation, and competition mechanisms. It was found that the ultrasensitivity in the inactivation of Wee1 arises mainly through two competition mechanisms: competition between two sets of phosphorylation sites in Wee1 and between Wee1 and other high-affinity Cdk1 targets. Based on these findings, it was possible to reconstitute a highly ultrasensitive Wee1 response with purified components. Competition provides a simple way of generating the equivalent of a highly cooperative allosteric response (Kim, 2007).
The mitotic regulator cyclin B-Cdk1 is controlled by a system of two double-negative feedback loops and a positive feedback loop. Active Cdk1 brings about the inactivation of the nuclear kinase Wee1 and the cytoplasmic, membrane-associated kinase Myt1, which, when active, can inactivate Cdk1 through phosphorylation of Thr 14 and/or Tyr 15. In addition, active Cdk1 brings about the activation of Cdc25, which can then dephosphorylate the sites phosphorylated by Wee1 and Myt1 (Kim, 2007 and references therein).
Under the proper circumstances, positive and double-negative feedback loops can exhibit bistability. This means that the system can adopt either of two alternative steady states in response to a constant stimulus and can toggle between these states in response to small changes in stimulus. Recent experimental work has demonstrated that the Cdk1/Wee1/Myt1/Cdc25 system is, in fact, bistable and can toggle between a stable interphase state, with Cdk1 and Cdc25 inactive and Wee1 and Myt1 active, and a stable mitotic state with Cdk1 and Cdc25 active and Wee1 and Myt1 inactive (Kim, 2007 and references therein).
A bistable signaling system must include a positive feedback loop, a double-negative feedback loop, or the equivalent, although sometimes this feedback loop may be difficult to appreciate. However, the presence of positive or double-negative feedback does not guarantee that a system will be bistable; the shapes of the steady-state stimulus/response curves for the individual legs of the loop are important as well. It is also important that some component of the loop exhibit a sigmoidal, ultrasensitive steady-state response -- that is, a nonlinear response resembling that of a cooperative enzyme -- rather than a hyperbolic, Michaelian response. For the particular case of a two-component positive or double-negative feedback, it is easy to show that simple Michaelian response functions do not support bistability. This is also true for an arbitrarily large positive feedback loop, provided that the components of the loop constitute a strongly monotone system. Given that the Cdk1/Wee1/Myt1/Cdc25 system is bistable, it seemed plausible that some component of the system would exhibit a highly ultrasensitive steady-state response to its upstream regulator (Kim, 2007 and references therein).
Ultrasensitivity is not only important for allowing positive feedback loops to generate bistable responses, but also for effective signal propagation down cascades. With Michaelian steady-state responses, an n-fold change in stimulus always yields a less than n-fold change in response. After several levels in a signaling cascade, the loss in signal contrast can be severe. Ultrasensitivity can help restore the original contrast and can also amplify it, converting graded inputs into more abrupt and switch-like outputs. In addition, ultrasensitivity is required for generating oscillations in negative feedback loops of certain lengths. Thus, ultrasensitivity may be important in a wide range of signaling contexts (Kim, 2007 and references therein).
Several examples of ultrasensitive responses have been documented experimentally. The earliest were the phosphorylation of phosphorylase and isocitrate dehydrogenase in vitro, where the observed sigmoidal responses could be largely attributed to zero-order ultrasensitivity, a phenomenon that occurs when the kinase and/or phosphatase that regulate the steady-state level of substrate phosphorylation are operating near saturation. More recent examples have included the activation of MEK and p42 MAPK by Mos in Xenopus oocyte extracts, AMP kinase activation in INS-1 cells, and JNK activation in several cell types. Thus, ultrasensitivity is a recurring motif in cell signaling (Kim, 2007 and references therein).
A variety of plausible mechanisms have been proposed to account for the ultrasensitivity observed in these systems. For example, the ultrasensitive response of MEK to Mos may be generated by competition between CK2β and MEK for access to Mos, and the response of p42 MAPK to MEK may be generated by nonprocessive multisite phosphorylation. However, these postulated mechanisms remain largely untested (Kim, 2007 and references therein).
This study examined whether the inactivation of Wee1 by Cdk1 in Xenopus egg extracts is Michaelian or ultrasensitive. It was found that the inactivation of Wee1, as assessed by the steady-state phosphorylation of one critical residue (Thr 150), is highly ultrasensitive, with an apparent Hill coefficient of 3.5. Several plausible mechanisms were tested to account for the observed ultrasensitivity. It was found that some of the ultrasensitivity is intrinsic to the core Wee1/cyclin B-Cdk1/Wee1 phosphatase system, with the main mechanism for intrinsic ultrasensitivity being competition between two sets of phosphorylation sites in Wee1 for access to Cdk1. In addition, much of the observed ultrasensitivity is extrinsic to the core system. The main mechanism of extrinsic ultrasensitivity appears to be competition between Wee1 and other high-affinity substrates for access to Cdk1. As proof of principle for the idea of competition as a source of ultrasensitivity, the highly ultrasensitive, switch-like inhibition of Wee1 by Cdk1 was reconstituted in vitro with purified recombinant components (Kim, 2007).
When cells are arrested in S phase, a subset of p53 target genes fails to be strongly induced despite the presence of high levels of p53. When DNA replication is inhibited, reduced p21 mRNA accumulation is correlated with a marked reduction in transcription elongation. This study shows that ablation of the protein kinase Chk1 rescues the p21 transcription elongation defect when cells are blocked in S phase, as measured by increases in both p21 mRNA levels and the presence of the elongating form of RNA polymerase II (RNAPII) toward the 3' end of the p21 gene. Recruitment of specific elongation and 3' processing factors (DSIF, CstF-64, and CPSF-100) is also restored. While additional components of the RNAPII transcriptional machinery, such as TFIIB and CDK7, are recruited more extensively to the p21 locus after DNA damage than after replication stress, their recruitment is not enhanced by ablation of Chk1. Significantly, ablating Chk2, a kinase closely related in substrate specificity to Chk1, does not rescue p21 mRNA levels during S-phase arrest. Thus, Chk1 has a direct and selective role in the elongation block to p21 observed during S-phase arrest. These findings demonstrate for the first time a link between the replication checkpoint mediated by ATR/Chk1 and the transcription elongation/3' processing machinery (Beckerman, 2009).
This study establishes a previously unknown link between the polyadenylation machinery and a DNA damage effector kinase. What is perhaps most striking about the results is that several proteins (DSIF, CPSF-100, and CstF-64) show no difference in recruitment to the TATA region of the p21 promoter after HU, but show a progressive loss in occupancy as the p21 gene is traversed by RNAPII. This is most pronounced at or downstream from the poly(A) site. DSIF, CPSF-100, and CstF-64 have all been shown to associate with RNAPII along the gene, and treatment of cells with DRB, which inhibits RNAPII phosphorylation at Ser2, decreases the association of CstF and CPSF subunits with distal regions of p21. Therefore, it cannot be ruled out, at this point, that the decreased recruitment of these processing factors at the 3' end of p21, and their subsequent rescue by Chk1 inhibition, does not simply reflect the decrease in Ser2-phosphorylated RNAPII under these conditions. However, a more interesting hypothesis is that these polyadenylation factors are regulated by Chk1 in such a way as to affect the efficiency of transcription elongation. Several reports have shown that polyadenylation factors impact transcription, especially elongation and termination, and several SR proteins have been shown to stimulate transcription elongation. Intriguingly, preliminary data indicate that Chk1 is recruited to the TATA region of p21 after HU treatment. This provides further support for a direct role for Chk1 in regulating transcription elongation at this locus. Future studies will investigated whether Chk1 directly phosphorylates CstF and CPSF subunits and if so, whether this affects transcription elongation (Beckerman, 2009).
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