okra


EVOLUTIONARY HOMOLOGS part 2/2

Mammalian RAD54 homologs

cDNAs encoding a human and a mouse homolog of RAD54 have been isolated. The human (hHR54) and mouse (mHR54) proteins are 48% identical to Rad54 and belong to the SNF2/SW12 family, which is characterized by amino-acid motifs found in DNA-dependent ATPases. The hHR54 gene maps to chromosome 1p32, and hHR54 protein is located in the nucleus. Levels of hHR54 mRNA increase in late G1 phase, as has been found for RAD54 mRNA. The level of mHR54 mRNA is elevated in organs of germ cell and lymphoid development and increased mHR54 expression correlates with the meiotic phase of spermatogenesis. hHR54 cDNA partially complements the methyl methanesulfonate-sensitive phenotype of S. cerevisiae rad54 delta cells. The tissue-specific expression of mHR54 is consistent with a role for the gene in recombination. The complementation experiments show that the DNA repair function of Rad54 is conserved from yeast to humans. These findings underscore the fundamental importance of DNA repair pathways: even though they are complex and involve multiple proteins, they seem to be functionally conserved throughout the eukaryotic kingdom (Kanaar, 1996).

A DNA fragments database search for sequences containing homology to known yeast DNA recombination and repair genes, yielded a cDNA fragment with high homology to RAD54. The complete cDNA sequence and the characterization of the genomic locus coding for the human homolog of the yeast RAD54 gene (hRAD54) are described. The yeast RAD54 belongs to the RAD52 epistasis group and appears to be involved in both DNA recombination and repair. The hRAD54 gene maps to chromosome 1p32 in a region of frequent loss of heterozygosity in breast tumors and encodes a protein of M(r) 93,000 that displays 52% identity to the yeast RAD54 protein. The hRAD54 protein sequence additionally contains all seven of the consensus segments of a superfamily of proteins with presumed or proven DNA helicase activity. Mutations in genes with consensus helicase homology have been found in cancer-prone syndromes such as xeroderma pigmentosum and Bloom syndrome as well as Werner's syndrome, in which patients age prematurely, and the X-linked mental retardation with alpha-thalassemia syndrome, ATR-X. The hRAD54 gene was examined in several breast tumors and breast tumor cell lines: although the gene region appears to be deleted in several tumors, at present no coding sequence mutations have been found (Rasio, 1997).

rad54 mutants of the yeast Saccharomyces cerevisiae are extremely X-ray sensitive and have decreased mitotic recombination frequencies because of a defect in double-strand break repair. A RAD54 homolog was disrupted in the chicken B cell line DT40, which undergoes immunoglobulin gene conversion and exhibits unusually high ratios of targeted to random integration after DNA transfection. Homozygous RAD54-/- mutant clones are highly X-ray sensitive, as compared to wildtype cells. The rate of immunoglobulin gene conversion is 6- to 8-fold reduced, and the frequency of targeted integration is at least two orders of magnitude decreased in the mutant clones. Reexpression of the RAD54 cDNA restores radiation resistance and targeted integration activity. The reported phenotype provides the first genetic evidence of a link between double-strand break repair and homologous recombination in vertebrate cells (Bezzubova, 1997).

Double-strand DNA break (DSB) repair by homologous recombination occurs through the RAD52 pathway in Saccharomyces cerevisiae. The phenotype of mouse RAD54-/- (mRAD54-/-) cells have been analyzed. Consistent with a DSB repair defect, these cells are sensitive to ionizing radiation, mitomycin C, and methyl methanesulfonate, but not to ultraviolet light. Gene targeting experiments demonstrate that homologous recombination in mRAD54-/- cells is reduced, when compared to wild-type cells. These results imply that, in addition to DNA end-joining mediated by DNA-dependent protein kinase, homologous recombination contributes to the repair of DSBs in mammalian cells. mRAD54-/- mice are shown to be viable and to exhibit apparently normal V(D)J and immunoglobulin class-switch recombination. Thus, mRAD54 is not required for the recombination processes that generate functional immunoglobulin and T cell receptor genes (Essers, 1997).

The cDNA for human protein HsRad54, which is a structural homolog of Saccharomyces cerevisiae recombination/repair protein Rad54, was cloned and expressed in Escherichia coli. As demonstrated by analysis in vitro and in vivo, HsRad54 protein interacts with human Rad51 recombinase. The interaction is mediated by the N-terminal domain of HsRad54 protein, which interacts with both free and DNA-bound HsRad51 protein (Golub, 1997).

Eukaryotic cells repair DNA double-strand breaks (DSBs) using at least two pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). Rad54 participates in the first recombinational repair pathway while Ku proteins are involved in NHEJ. To investigate the distinctive as well as redundant roles of these two repair pathways, an analysis was carried out of the mutants RAD54(-/-), KU70(-/-) and RAD54(-/-)/KU70(-/-), generated from the chicken B-cell line DT40. The NHEJ pathway plays a dominant role in repairing gamma-radiation-induced DSBs during G1-early S phase while recombinational repair is preferentially used in late S-G2 phase. RAD54(-/-)/KU70(-/-) cells are profoundly more sensitive to gamma-rays than either single mutant, indicating that the two repair pathways are complementary. Spontaneous chromosomal aberrations and cell death are observed in both RAD54(-/-) and RAD54(-/-)/KU70(-/-) cells, with RAD54(-/-)/KU70(-/-) cells exhibiting significantly higher levels of chromosomal aberrations than RAD54(-/-) cells. These observations provide the first genetic evidence that both repair pathways play a role in maintaining chromosomal DNA during the cell cycle (Takata, 1998).

Human Rad51 (hRad51) and Rad54 proteins are key members of the RAD52 group required for homologous recombination. hRad54 promotes transient separation of the strands in duplex DNA via its ATP hydrolysis-driven DNA supercoiling function. The ATPase, DNA supercoiling, and DNA strand opening activities of hRad54 are greatly stimulated through an interaction with hRad51. Importantly, hRad51 and hRad54 functionally cooperate in the homologous DNA pairing reaction that forms recombination DNA intermediates. These results should provide a biochemical model for dissecting the role of hRad51 and hRad54 in recombination reactions in human cells (Sigurdsson, 2002).

In eukaryotic cells, the repair of DNA double-strand breaks by homologous recombination requires a RecA-like recombinase, Rad51p, and a Swi2p/Snf2p-like ATPase, Rad54p. Yeast Rad51p and Rad54p support robust homologous pairing between single-stranded DNA and a chromatin donor. In contrast, bacterial RecA is incapable of catalyzing homologous pairing with a chromatin donor. Rad54p possesses many of the biochemical properties of bona fide ATP-dependent chromatin-remodeling enzymes, such as ySWI/SNF. Rad54p can enhance the accessibility of DNA within nucleosomal arrays, but it does not seem to disrupt nucleosome positioning. Taken together, these results indicate that Rad54p is a chromatin-remodeling enzyme that promotes homologous DNA pairing events within the context of chromatin (Jaskelioff, 2003).

In vivo and in vitro studies have suggested the following sequence of molecular events that lead to the recombinational repair of a DSB. First, the 5' ends of DNA that flank the break are resected by an exonuclease to create ssDNA tails. Next, Rad51p polymerizes onto these DNA tails to form a nucleoprotein filament that has the capability to search for a homologous duplex DNA molecule. After DNA homology has been located, the Rad51-ssDNA nucleoprotein filament catalyzes the formation of a heteroduplex DNA joint with the homolog. The process of DNA homology search and DNA joint molecule formation is called 'homologous DNA pairing and strand exchange'. Subsequent steps entail DNA synthesis to replace the missing information followed by resolution of DNA intermediates to yield two intact duplex DNA molecules (Jaskelioff, 2003).

The homologous DNA pairing activity of Rad51p is enhanced by Rad54p. Rad54p is a member of the Swi2p/Snf2p protein family that has DNA-stimulated ATPase activity and physically interacts with Rad51p. Because of its relatedness to the Swi2p/Snf2p family of ATPases, Rad54p may have chromatin remodeling activities in addition to its established role in facilitating Rad51p-mediated homologous pairing reactions. In this study it has been shown that Rad51p and Rad54p mediate robust D-loop formation with a chromatin donor, whereas the bacterial recombinase, RecA, can only function with naked DNA. Furthermore, the ATPase activity of Rad54p is essential for D-loop formation on chromatin and Rad54p can use the free energy from ATP hydrolysis to enhance the accessibility of nucleosomal DNA. Experiments are also presented to suggest that chromatin remodeling by Rad54p and yeast SWI/SNF involves DNA translocation (Jaskelioff, 2003).

These results suggest that Rad54p is an extremely versatile recombination protein that plays key roles in several steps of homologous recombination. Rad54p is required for optimal recruitment of Rad51p to a double strand break in vivo, and likewise Rad54p can promote formation of the presynaptic filament in vitro by helping Rad51p contend with the inhibitory effects of the ssDNA-binding protein replication protein A.2. Several studies over the past few years have also shown that the ATPase activity of Rad54p plays key roles subsequent to formation of the presynaptic filament. For instance, Rad54p is required for the Rad51p-nucleoprotein filament to form a heteroduplex joint DNA molecule, even when the homologous donor is naked DNA. In this case, it has been proposed that Rad54p might use the free energy from ATP hydrolysis to translocate along DNA, which facilitates the homology search process. This DNA-translocation model is fully consistent with findings that Rad54p can displace a DNA triplex and that the ATPase activity of Rad54p is proportional to DNA length. Rad54p also stimulates heteroduplex DNA extension of established joint molecules. Finally, Rad54p is required for Rad51p-dependent heteroduplex joint molecule formation with a chromatin donor. In this case, the results suggest that the ATPase activity of Rad54p is used to translocate the enzyme along the nucleosomal fiber, generating superhelical torsion, which leads to enhanced nucleosomal DNA accessibility. It seems likely that this chromatin remodeling activity of Rad54p might also facilitate additional steps after heteroduplex joint formation. Future studies are now poised to reconstitute the complete homologous recombinational repair reaction that fully mimics each step in the repair of chromosomal DNA double strand breaks in vivo (Jaskelioff, 2003).

The efficient and accurate repair of DNA double strand breaks (DSBs) is critical to cell survival, and defects in this process can lead to genome instability and cancers. In eukaryotes, the Rad52 group of proteins dictates the repair of DSBs by the error-free process of homologous recombination (HR). A critical step in eukaryotic HR is the formation of the initial Rad51-single-stranded DNA presynaptic nucleoprotein filament. This presynaptic filament participates in a homology search process that leads to the formation of a DNA joint molecule and recombinational repair of the DSB. The Rad54 protein functions as a mediator of Rad51 binding to single-stranded DNA; this activity does not require ATP hydrolysis. A novel Rad54-dependent chromatin remodeling event has been discovered that occurs in vivo during the DNA strand invasion step of HR. This ATP-dependent remodeling activity of Rad54 appears to control subsequent steps in the HR process (Wolner, 2005; full text of article).

RAD51 and RAD52 proteins: Upstream effectors of meiotic and mitotic repair

In the yeast Saccharomyces cerevisiae, mutations in either of the genes RAD51 or RAD52 result in severe defects in genetic recombination and the repair of double-strand DNA breaks. The RAD51 gene is a structural and functional homologue of the recA gene and the gene product participates in strand exchange and single-stranded-DNA-dependent ATP hydrolysis by means of nucleoprotein filament formation. These genes, and others of the RAD52 epistasis group (RAD50, RAD54, RAD55, RAD57, RAD59, MRE11 and XRS2), were first identified by their sensitivity to X-rays. They were subsequently shown to be required for spontaneous and induced mitotic recombination, meiotic recombination, and mating-type switching. Human homologs of RAD50, RAD51, RAD52, RAD54 and MRE11 have been identified. Targeted disruption of the murine RAD51 gene results in an embryonic lethal phenotype, indicating that Rad51 protein is required during cell proliferation. Biochemical studies have shown that human RAD51 encodes a protein of relative molecular mass 36,966 (hRad51) that promotes ATP-dependent homologous pairing and DNA strand exchange. As a structural and functional homolog of the RecA protein from Escherichia coli, hRad51 is thought to play a central role in recombination. Yeast Rad51 has been shown to interact with Rad52 protein, as does the human homolog. hRad52 is shown to stimulate homologous pairing by hRad51. The DNA-binding properties of hRad52 indicate that Rad52 is involved in an early stage of Rad51-mediated recombination (Benson, 1998).

Recently, human and rodent homologs of yeast repair genes Rad51 and Rad52 have been identified and proposed to play roles in DNA double-strand break (DSB) repair. In this study, cell cycle-dependent expression of human and rodent RAD51 and RAD52 proteins was monitored using two approaches. (1) Flow cytometric measurements of DNA content and immunofluorescence were used to determine the phase-specific levels of RAD51 and RAD52 protein expression in irradiated and control populations. The expression of both proteins is lowest in G0/G1, increases in S and reaches a maximum in G2/M. No difference is found in the whole-cell level of RAD51 or RAD52 protein expression between gamma-irradiated and control cell populations. (2) Cell cycle-dependent protein expression was confirmed by Western analysis of populations synchronized in G0, G1 and G2 phases. Analysis of V3, a hamster equivalent of SCID, indicates that the protein level increases of RAD51 and RAD52 from G0 to G1/S/G2 do not require DNA-PK (Chen, 1997).

Rad54 and DNA Ligase IV cooperate to maintain mammalian chromatid stability

Nonhomologous end joining (NHEJ) and homologous recombination (HR) represent the two major pathways of DNA double-strand break (DSB) repair in eukaryotic cells. NHEJ repairs DSBs by ligation of cognate broken ends irrespective of homologous flanking sequences, whereas HR repairs DSBs using an undamaged homologous template. Although both NHEJ and HR have been clearly implicated in the maintenance of genome stability, how these apparently independent and mechanistically distinct pathways are coordinated remains largely unexplored. To investigate the relationship between HR and NHEJ modes of DSB repair, cells doubly deficient for the NHEJ factor DNA Ligase IV (Lig4) and the HR factor Rad54 were generated. Lig4 and Rad54 cooperate to support cellular proliferation, repair spontaneous DSBs, and prevent chromosome and single chromatid aberrations. These findings demonstrate a role for NHEJ in the repair of DSBs that occur spontaneously during or after DNA replication, and reveal overlapping functions for NHEJ and Rad54-dependent HR in the repair of such DSBs (Mills, 2004).

The findings described here uncover a critical role for the HR protein Rad54 in cellular proliferation and the maintenance of genome stability within a setting of deficient NHEJ. Cells simultaneously deficient for both Rad54 and Lig4 fail to proliferate in culture, show a multiplicity of spontaneously occurring DSBs as evidenced by gamma-H2AX (a variant of histone H2A) focus formation, and exhibit high levels of spontaneous cytogenetic abnormalities. Notably, the occurrence of unresolved DSBs and the spontaneous genomic instability in Lig4-/- cells are markedly less, and of a different type, than in RL or Rad54 Lig4 p53 triply deficient (RLP) cells, indicating that simultaneous Rad54 and Lig4 deficiency produces a synthetic phenotype. Although a minor fraction of gamma-H2AX-positive RL cells undergo apoptosis, most RL cells do not express common apoptotic makers, and thus appear to acquire gamma-H2AX foci as a result of spontaneously occurring DSBs (Mills, 2004).

Cells deficient for both Rad54 and Lig4 incur a very high proportion of single-chromatid breaks and gaps, relative to either deficiency alone, thus demonstrating a defect in postreplication DSB repair. Since DNA repair pathway usage varies with cell cycle progression, these defects may arise, in part, because cells in the G2 phase of the cell cycle exhibit a relatively greater dependence on Rad54 for maintenance of genome stability. Although an important role for Rad54 in postreplication DNA repair is not unexpected and may be unmasked by cell cycle delay in G2, an overlapping role for NHEJ in this phase of the cell cycle has been more speculative. One recent report of DNA-PKcs versus XRCC3 in the repair of IR-induced DSBs in CHO cells provides a suggestion that NHEJ can play a role in all phases of the cell cycle. It has also been proposed that NHEJ can, in some cases, resolve DSBs arising from stalled replication forks, although this has remained controversial. The current study demonstrates a role for Lig4, and by extension NHEJ, in the repair of DSBs that arise spontaneously during postreplication phases of the cell cycle, although the source of such breaks remains to be determined (Mills, 2004).

The observation that both Lig4 and Rad54 play roles in maintaining genome stability in G2 demonstrate that NHEJ and HR pathways can overlap during (post)replication phases of the cell cycle. This is further supported by recent reports concerning the repair of IR or aphidicolin-induced breaks: reports have suggested that NHEJ may also participate in the repair of a subset of these exogenously induced post-G1 breaks. The slightly higher overall rate of genomic instability in Lig4, versus Rad54, single mutant cells indicates that Lig4-dependent DSB repair is favored over Rad54-dependent repair, even in postreplication phases of the cell cycle and suggests two possible models. The first model indicates that Rad54- and Lig4-dependent pathways, although possibly remaining mechanistically separate, may also overlap in the repair of a subset of DSBs arising during or after DNA replication. Such a functional interrelationship would result in a synergistic effect on chromosome or chromatid instability when both pathways are deficient. In addition, Lig4- and Rad54-dependent pathways may directly interact in the repair of some DSBs, possibly by a coupled NHEJ-HR mechanism, although this remains speculative. A second model holds that Rad54 and Lig4 can both function during postreplication, but remain functionally distinct, with each addressing a subset of overall DSBs. By this model, double deficiency would result in an additive but not synthetic phenotype. This study has shown clear evidence that loss of Rad54 and Lig4 produces, in addition to a synergistic increase in instability, a synthetic shift toward chromatid breaks, relative to either single mutant. Thus, the findings described here support the first model and indicate an interplay between Rad54 and Lig4 in the repair of postreplication DSBs (Mills, 2004).

Werner syndrome protein participates in a complex with RAD51, RAD54, RAD54B and ATR in response to ICL-induced replication arrest

Werner syndrome (WS) is a rare genetic disorder characterized by genomic instability caused by defects in the WRN gene encoding a member of the human RecQ helicase family. RecQ helicases are involved in several DNA metabolic pathways including homologous recombination (HR) processes during repair of stalled replication forks. Following introduction of interstrand DNA crosslinks (ICL), WRN relocated from nucleoli to arrested replication forks in the nucleoplasm where it interacted with the HR protein RAD52. In this study, fluorescence resonance energy transfer (FRET) and immune-precipitation experiments were used to demonstrate that WRN participates in a multiprotein complex including RAD51, RAD54, RAD54B and ATR in cells where replication has been arrested by ICL. The WRN-RAD51 and WRN-RAD54B direct interaction was verified in vitro. These data support a role for WRN also in the recombination step of ICL repair (Otterlei, 2006).

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