The Interactive Fly
Zygotically transcribed genes

DNA Replication enzymes and protein cofactors

How does DNA replication occur?

Chromatin state marks cell-type- and gender-specific replication of the Drosophila genome

Genetic organization of interphase chromosome bands and interbands in Drosophila melanogaster

Histone H4K20 tri-methylation at late-firing origins ensures timely heterochromatin replication



DNA polymerases and subunits

Origin recognition complex


Mini-chromosome maintenance family


DNA replication factor A complex - a single stranded DNA-binding protein complex



Other proteins

anti-silencing factor 1
histone chaperone that assists in chromatin assembly and remodeling during DNA replication, transcription activation, and gene silencing

cdc6
AAA-superfamily ATP helicase involved in initiation of DNA replication - involved in the formation of the prereplicative complex -
checkpoint protein involved in controlling the G2/M transition

Disc proliferation abnormal
An MCM4 homolog - component of licensing factor

double parked
conserved protein required for DNA replicaton

DNA ligase I
Joins Okazaki fragments

DNA ligase II
Functions in repair (?)

DP transcription factor
transcription factor - obligate dimerization partner of E2f1 and E2f2 - required for normal cell proliferation,
optimal DNA synthesis, and efficient G2/M progression

geminin
limits DNA replication by preventing Mcm loading onto chromatin - interacts with Double parked (Drosophila cdt1)

p53
Transcription factor functioning in DNA repair and apoptosis

Proliferating cell nuclear antigen (PCNA) (common alternative name: Mutagen-sensitive 209)
Polymerase-delta/epsilon processivity factor

RNase H1
Involved in Okazaki fragment maturation

Topoisomerase 1
Unlinks parental strands - involved in replication

Topoisomerase 2
Unlinks parental strands and progeny duplexes


How does DNA replication take place?


Why should a developmental biologist be interested in DNA replication? There are at least three reasons. (1) For a given origin of replication, there is a link between gene expression and timing of DNA replication, and understanding the basis of this link is important. (2) The mechanism of gene replication is by necessity involved with the restructuring of chromatin and the regulatory implications of that event. (3) There are fail-safe mechanisms to ensure that each origin of replication fires only once per cell cycle, and these mechanisms involve an interaction of cyclins with licensing factor, a chromatin component. Thus, in the future there will be a developing understanding of the relationship between cell cycle, DNA replication, chromatin components, and changes in gene expression.

The first signal for initiation of replication involves replication licensing factor (RLF), which 'licenses' replication origins by putting them into an initiation-competent state. The second signal, S-phase promoting factor, induces licensed origins to initiate, and in doing so removes the license. RLF of Xenopus can be separated into two essential components, RLF-M and RLF-B, both of which are required for licensing. RLF-M, a fraction containing members of the minichromosome maintenance family, associates with chromatin prior to replication but is removed during replication. Drosophila MCM2 and MCM4 homologs have been identified (See disc proliferation abnormal for information about both of these). RLF-M's reassociation with chromatin requires passage through mitosis. RLF-M requires RLF-B, an as yet uncharacterized fraction, for binding RLF-M to DNA. Apparently RLF binds to origins of replication, but the basis for this binding has not yet been characterized (Chong, 1996 and references).

The focus of all replication forks is the helicase, which catalyzes the transition from double- to single-stranded DNA. In eukaryotes, the identification of the enzyme that acts at chromosomal replication forks awaits further investigation, but the SV40 T antigen fulfills the helicase function in SV40 replication. The origin of replication is selected and identified in yeast by the origin recognition complex, of which one Drosophila homolog (Orc2) has been identified. Identification of other components of the ORC and unraveling the nature of DNA sequences at the origin are currently very active subjects of research.

The replication process is semidiscontinuous. Proteins comprising the replication machine act in concert to unwind the parental strands and carry out the simultaneous synthesis of the two progeny strands. Both progeny strands are synthesized in the 5' to 3' direction, but since parental DNA strands are antiparallel, two distinct mechanisms of DNA synthesis are required. One of the two progeny strands (the leading strand) is synthesized continuously in the direction of fork movement. The other (the lagging strand) is synthesized discontinuously in the direction opposite to fork movement. Discontinuous DNA synthesis on the lagging-strand templates involves the related synthesis of oligoribonucleotide primers, which are then elongated into short DNA chains (Okazaki fragments). Following their synthesis, Okazaki fragements are processed to remove the RNA primers and joined together to form an interrupted progeny strand (Brush, 1996).

As the replication fork advances, a helix-destabilizing protein is required to maintain the single-stranded DNA structure that serves as template for RNA priming and DNA synthesis. In eukaryotes, Replication protein A performs this function. This phosphoprotein consists of three subunits.

DNA synthesis is initiated by the bifunctional pol-alpha:primase complex, a heterotetrameric phosphoprotein. The primase activity resides in the smallest subunit and is tightly associated with the next largest, which is thought to tether the primase to the catalytic subunit. The remaining subunit has no known catalytic function, but it may contribute to recruitment of pol-alpha:primase to the replication fork. The main function of pol-alpha:primase is to serve as a priming enzyme. The primase catalyzes the synthesis of complementary oligoribonucleotides, which are then extended a short distance by the polymerase activity. The pol-alpha:primase serves exclusively to initiate DNA synthesis on the lagging strand, and dissociation from the DNA provides a primer terminus for assembly of the PCNA/pol-delta complex, which serves to extend the RNA/DNA primers originally synthesized by pol-alpha:primase.

The heterodimeric DNA polymerase-delta is involved in the elongation stage of DNA replication, acting both on the leading and lagging strands. Unlike pol-alpha:primase, the polymerase-delta does not act alone but requires the action of two auxiliary factors, the multisubunit replication factor C (RF-C) which binds to the primer terminus of the lagging strand immediately after RNA/DNA primer synthesis has been completed by pol-alpha:primase, allowing the subsequent assembly of a functional pol-delta complex. Once bound the primer-template junction, RF-C loads PCNA onto the DNA in an energy-dependent process. PCNA then functions as a processivity factor, acting as a clamp to prevent sliding of polymerase in the reverse direction. RNase H1 acts with another enzyme to remove the RNA primer used to initiate the lagging strand, while DNA ligase I joins the nacent DNA fragments to complete the synthesis of the lagging strand.

It is not clear how pol-alpha:primase synthesized DNA is proofread, as this enzyme has no exonuclease activity. the catalytic subunit of polymerase-delta contains a 3' to 5' exonuclease activity, functioning in proofreading, which allows high-fidelity DNA synthesis (Brush, 1996).

Chromatin state marks cell-type- and gender-specific replication of the Drosophila genome

Duplication of eukaryotic genomes during S phase is coordinated in space and time. In order to identify zones of initiation and cell-type- as well as gender-specific plasticity of DNA replication, replication timing, histone acetylation, and transcription was profiled throughout the Drosophila genome. Two waves of replication initiation were identified with many distinct zones firing in early-S phase, along with multiple, less defined peaks at the end of S phase, suggesting that initiation becomes more promiscuous in late-S phase. A comparison of different cell types revealed widespread plasticity of replication timing on autosomes. Most occur in large regions, but only half coincide with local differences in transcription. In contrast to confined autosomal differences, a global shift in replication timing occurs throughout the single male X chromosome. Unlike in females, the dosage-compensated X chromosome replicates almost exclusively early. This difference occurs at sites that are not transcriptionally hyperactivated, but show increased acetylation of Lys 16 of histone H4 (H4K16ac). This suggests a transcription-independent, yet chromosome-wide process related to chromatin. Importantly, H4K16ac is also enriched at initiation zones as well as early replicating regions on autosomes during S phase. Together, this study reveals novel organizational principles of DNA replication of the Drosophila genome and suggests that H4K16ac is more closely correlated with replication timing than is transcription (Schwaiger, 2009).

The high resolution of these replication timing profiles allowed zones of replication initiation to be identified throughout S phase, that were confirmed in combination with measuring small nascent strand abundance. This revealed that sites of early initiation are rather distinct, which manifests in the timing profile as single peaks or a few peaks clustered together in early-S phase, followed by long stretches of the replication timing profile without changes in slope. Late initiation zones often reside in close proximity to other late initiation zones. The feature of distinct peaks of early initiation in Drosophila is very distinct from mammalian genomes (Hiratani, 2008), where many more sites of initiation of similar timing are clustered together, resulting in large regions up to several megabase pairs of early replication timing (Schwaiger, 2009).

Interestingly, the frequency of initiation appears discontinuous with high rates in early-S, a reduced frequency in mid-S, and again increased appearance of initiation sites in late-S phase. The high frequency and proximity of late-firing initiation zones suggest that late regions are replicated by many proximal late-firing origins of replication. This finding is particularly interesting in light of a recent report that suggested the absence of a checkpoint to control for the completion of DNA replication before mitosis (Torres-Rosell, 2007). This would in turn require a mechanism that mediates rapid replication of unreplicated regions in late-S phase, which could be achieved by a promiscuous activation of many proximal origins. Interestingly, replicative stress that reduces replication fork progression leads to a decrease in inter-origin distance through activation of normally dormant origins. It is conceivable that a similar situation is encountered in late replicating regions (Schwaiger, 2009).

Since the previously reported correlation between replication timing and transcription in Drosophila was not absolute, the percentage of the genome that replicates in a tissue-specific fashion remained to be tested quantitatively. For example, the general correlation could be driven by housekeeping genes that are active in most cells, resulting in a uniform replication timing program. This study showed that dynamic replication timing differs significantly between two Drosophila cell types, affecting at least 20% of autosomal DNA. It was also shown by two different methodologies that this plasticity of DNA replication coincides with transcription differences in only half of all cases (Schwaiger, 2009).

Early replication was shown previously to correlate with transcription levels over 180 kb, leading to the suggestion that replication timing integrates transcription over large regions. Consistent with this model, it was found that dynamic replication timing often occurs in large (~100 kb) regions encompassing many genes. Interestingly, genes with related function often cluster together in the Drosophila genome, and such clusters tend to be similarly 100 kb in size. In mammalian genomes, this clustering appears functionally related to chromatin structure, suggesting that widespread open chromatin at developmentally regulated multigene loci could lead to early replication or vice versa. This, in turn, might increase the potential of gene expression over large regions as in the case of genes important for wing disc development in Cl8 cells, where early replication could render the locus poised for activation (Schwaiger, 2009).

Localized differences in gene expression of a fraction of genes in a large region might also account for replication timing differences. Indeed, some, but not all, genes in differentially replicating regions are strongly differentially expressed between the two cell types. Thus, while gene expression could account for much of the observed changes on autosomes, a considerable fraction does not display transcriptional changes. It seems unlikely that the analysis missed such changes since noncoding transcription was measured as well as RNA polymerase abundance (Schwaiger, 2009).

The relation between replication timing and chromatin structure has been controversial. Transcription itself involves an opening of chromatin structure, and thus early replication could in many situations be downstream from transcriptional activation. However, previous work using injected plasmids suggested a role for early replication in mediating increased levels of histone acetylation. This led to a model in which replication timing mediates an open chromatin structure required for transcription. This suggestion is compatible with the genome-wide analysis, where a preferential location of H4K16ac was observed not only to active genes, but also to early replicating regions that are not transcribed. It is possible that early replication and elevated H4K16ac at inactive genes will result in a more open chromatin confirmation compared with late replicating inactive genes. This might render them more responsive to downstream activating cues, and thus replication timing could modulate the sensitivity to activators. This process could also function in maintenance of an active state through cell division. Importantly, however, this mechanism does not override the parallel process of transcription-coupled acetylation, as late replicating genes that are actively transcribed are still hyperacetylated (Schwaiger, 2009).

Interestingly, a strong abundance of H4K16ac was observed at sites of initiation during S phase. Several single-gene studies have suggested a positive function of histone acteylation for origin activity. Other reports, however, did not support this model. Recent maps of human replication initiation suggest that early origins are marked by H3K9/K14 acetylation (Lucas, 2007). However, no genome-wide correlation between active chromatin marks and early origin firing was observed in S. cerevisiae, where specific sequences function as origins of replication. This study identified a preferential localization of H4K16ac to initiation zones throughout the Drosophila genome compatible with a function of acetylation. In this study, focus was placed on acetylation of H4K16 because this residue has been functionally linked to higher-order chromatin compaction and chromatin opening on the dosage-compensated X in Drosophila (Schwaiger, 2009).

It has been proposed that origins of replication lie frequently between promoters of active genes, which would make transcription and replication fork progression co-oriented. Furthermore, transcription and replication are thought to be coordinated in the nucleus to be spatially and temporally separated. It thus seems plausible that the enrichment of H4K16ac in initiation zones reflects location between highly acetylated, active promoters. According to this model, proximity to active promoters would result in an open chromatin confirmation through increased H4K16ac, which in turn enhances origin firing (Schwaiger, 2009).

Importantly, however, enrichment for H4K16ac was observed at initiation zones that are not proximal to active genes, arguing against a simple process that is solely transcription-coupled. Open chromatin structure, reflected and potentially even mediated by H4K16ac, could make DNA more accessible for efficient initiation of DNA replication and thus provide a sequence-independent component that could contribute to origin localization and activity. While these are testable models, they do require a fine-mapping of actual origins at a resolution higher than the current detection of zones of initiation at the level of several kilobases (Schwaiger, 2009).

This analysis reveals the almost complete absence of late replication on the single X chromosome in male Drosophila cells. About 90% of female late replicating regions on the X replicate early in males, while autosomes show no advanced replication. Such chromosome-wide advance in replication timing has not been observed previously. In mammals, transcriptional inactivation of one of the female X chromosomes correlates with its late replication, reflecting the efficient silencing of this chromosome and increased chromatin compaction. In contrast, dosage compensation in flies involves the twofold up-regulation of genes already active in females and an open chromatin state mediated by H4K16ac. Interestingly, this study showed that advanced replication of the dosage-compensated X occurs mostly outside of transcriptionally activated regions and thus is unlikely to be accounted for by transcriptional changes. Importantly, the local increases in H4K16ac, which are detected throughout the male X chromosome, can be directly related to this loss of late replication. Reduction of the responsible Histone-Acetyltransferase Mof leads to a block in cell division, making it difficult to test this model. Notably, slightly delayed replication of the X chromosome was detected in the few cells that were in S phase in the knockdown population. While this is compatible with a model that Mof-mediated H4K16 acetylation advances replication of intergenic regions on the male X chromosome, the predominant effect on the cell cycle precluded further analysis (Schwaiger, 2009).

This suggests a transcription-independent, chromatin-dependent process, which leads to early replication chromosome-wide. While this likely reflects a different chromatin compaction, it is tempting to speculate that it also reflects a particular nuclear organization as the dosage-compensated X chromosome has been shown to associate directly with nuclear pores (Schwaiger, 2009).

Together these findings provide new principles of the replication timing program of the Drosophila genome and its dynamics relative to histone acetylation and transcription. The data further support a model in which open chromatin structure is a general feature of early replication timing and could potentially even advance replication of entire chromosomes (Schwaiger, 2009).

Genetic organization of interphase chromosome bands and interbands in Drosophila melanogaster

Drosophila melanogaster polytene chromosomes display specific banding pattern; the underlying genetic organization of this pattern has remained elusive for many years. This paper analyzed 32 cytology-mapped polytene chromosome interbands. Molecular locations of these interbands was estimated, their molecular and genetic organization was described and it was demonstrated that polytene chromosome interbands contain the 5' ends of housekeeping genes. As a rule, interbands display preferential 'head-to-head' orientation of genes. They are enriched for 'broad' class promoters characteristic of housekeeping genes and associate with open chromatin proteins and Origin Recognition Complex (ORC) components. In two regions, 10A and 100B, coding sequences of genes whose 5'-ends reside in interbands map to constantly loosely compacted, early-replicating, so-called 'grey' bands. Comparison of expression patterns of genes mapping to late-replicating dense bands vs genes whose promoter regions map to interbands shows that the former are generally tissue-specific, whereas the latter are represented by ubiquitously active genes. Analysis of RNA-seq data (modENCODE-FlyBase) indicates that transcripts from interband-mapping genes are present in most tissues and cell lines studied, across most developmental stages and upon various treatment conditions. A special algorithm was developed to computationally process protein localization data generated by the modENCODE project; it was shown that Drosophila genome has about 5700 sites that demonstrate all the features shared by the interbands cytologically mapped to date (Zhimulev, 2014. PubMed ID: 25072930).

DNA copy-number control through inhibition of replication fork progression

Proper control of DNA replication is essential to ensure faithful transmission of genetic material and prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict cell-cycle regulation. Importantly, DNA replication is highly influenced by developmental cues. In Drosophila, specific regions of the genome are repressed for DNA replication during differentiation by the SNF2 domain-containing protein Suppressor of Under-Replication (SuUR) through an unknown mechanism. This study demonstrates that SuUR is recruited to active replication forks and mediates the repression of DNA replication by directly inhibiting replication fork progression instead of functioning as a replication fork barrier. Mass spectrometry identification of SUUR-associated proteins identified the replicative helicase member CDC45 as a SUUR-associated protein, supporting a role for SUUR directly at replication forks. These results reveal that control of eukaryotic DNA copy number can occur through the inhibition of replication fork progression (Nordman, 2014: PubMed).

Histone H4K20 tri-methylation at late-firing origins ensures timely heterochromatin replication

Among other targets, the protein lysine methyltransferase PR-Set7 (see Drosophila SET domain containing 7) induces histone H4 lysine 20 monomethylation (H4K20me1), which is the substrate for further methylation by the Suv4-20h methyltransferase. Although these enzymes have been implicated in control of replication origins, the specific contribution of H4K20 methylation to DNA replication remains unclear. This study shows that H4K20 mutation in mammalian cells, unlike in Drosophila, partially impairs S-phase progression and protects from DNA re-replication induced by stabilization of PR-Set7. Using Epstein-Barr virus-derived episomes, it was further demonstrated that conversion of H4K20me1 to higher H4K20me2/3 states by Suv4-20h is not sufficient to define an efficient origin per se, but rather serves as an enhancer for MCM2-7 helicase (see Drosophila MCM5) loading and replication activation at defined origins. Consistent with this, it was found that Suv4-20h-mediated H4K20 tri-methylation (H4K20me3) is required to sustain the licensing and activity of a subset of ORCA/LRWD1-associated origins, which ensure proper replication timing of late-replicating heterochromatin domains. Altogether, these results reveal Suv4-20h-mediated H4K20 tri-methylation as a critical determinant in the selection of active replication initiation sites in heterochromatin regions of mammalian genomes (Brustel, 2017).

list of proteins involved in DNA replication


References

Brush, G. S. and Kelly, T. J. (1996). Mechanisms for replicating DNA. In "DNA replication in eukaryotic cells". Ed. M. L. DePamphilis. pp 1-43 Cold Spring Harbor Laboratory Press, Plainview, NY.

Brustel, J., Kirstein, N., Izard, F., Grimaud, C., Prorok, P., Cayrou, C., Schotta, G., Abdelsamie, A. F., Dejardin, J., Mechali, M., Baldacci, G., Sardet, C., Cadoret, J. C., Schepers, A. and Julien, E. (2017). Histone H4K20 tri-methylation at late-firing origins ensures timely heterochromatin replication. EMBO J 36(18): 2726-2741. PubMed ID: 28778956

Chong, J. P. J., Thömmes, P and Blow, J. J. (1996). The role of MCM/P1 proteins in licensing of DNA replication. Trends in Biochem. Sci. 21: 102-106. Medline abstract: 8882583

Hiratani, I., et al. (2008). Global reorganization of replication domains during embryonic stem cell differentiation. PLoS Biol. 6: e245. PubMed Citation: 18842067

Lucas, I., et al. (2007). High-throughput mapping of origins of replication in human cells. EMBO Rep. 8: 770-777. PubMed Citation: 17668008

Nordman, J. T., Kozhevnikova, E. N., Verrijzer, C. P., Pindyurin, A. V., Andreyeva, E. N., Shloma, V. V., Zhimulev, I. F. and Orr-Weaver, T. L. (2014). DNA copy-number control through inhibition of replication fork progression. Cell Rep 9: 841-849. PubMed ID: 25437540

Schwaiger, M., et al. (2009). Chromatin state marks cell-type- and gender-specific replication of the Drosophila genome. Genes Dev. 23(5): 589-601. PubMed Citation: 19270159

Torres-Rosell, J., et al. (2007). Anaphase onset before complete DNA replication with intact checkpoint responses. Science 315: 1411-1415. PubMed Citation: 17347440

Zhimulev, I. F., Zykova, T. Y., Goncharov, F. P., Khoroshko, V. A., Demakova, O. V., Semeshin, V. F., Pokholkova, G. V., Boldyreva, L. V., Demidova, D. S., Babenko, V. N., Demakov, S. A. and Belyaeva, E. S. (2014). Genetic organization of interphase chromosome bands and interbands in Drosophila melanogaster. PLoS One 9: e101631. PubMed ID: 25072930

date revised: 25 October 2017

Zygotically transcribed genes

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