Post-replicative Gaps Research Articles

Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Post-replicative Gap Repair

Invivo, replication forks proceed beyond replication-blocking lesions by way of downstream repriming, generating daughter strand gaps that are subsequently processed by post-replicative repair pathways such as homologous recombination and translesion synthesis (TLS). The way these gaps are filled during TLS is presently unknown. The structure of gap repair synthesis was assessed by sequencing large collections of single DNA molecules that underwent specific TLS events invivo. The higher error frequency of specialized relative to replicative polymerases allowed us to visualize gap-filling events athigh resolution. Unexpectedly, the data reveal that a specialized polymerase, Pol V, synthesizes stretches of DNA both upstream and downstream of a site-specific DNA lesion. Pol V-mediated untargeted mutations are thus spread over several hundred nucleotides, strongly eliciting genetic instability on either side of a given lesion. Consequently, post-replicative gap repair may be a source of untargeted mutations critical for gene diversification in adaptation and evolution.

The role of HERC2 and RNF8 ubiquitin E3 ligases in the promotion of translesion DNA synthesis in the chicken DT40 cell line.

The replicative DNA polymerases are generally blocked by template DNA damage. The resulting replication arrest can be released by one of two post-replication repair (PRR) pathways, translesion DNA synthesis (TLS) and template switching by homologous recombination (HR). The HERC2 ubiquitin ligase plays a role in homologous recombination by facilitating the assembly of the Ubc13 ubiquitin-conjugating enzyme with the RNF8 ubiquitin ligase. To explore the role of HERC2 and RNF8 in PRR, we examined immunoglobulin diversification in chicken DT40 cells deficient in HERC2 and RNF8. Unexpectedly, the HERC2(-/-) and RNF8(-/-) cells and HERC2(-/-)/RNF8(-/-) double mutant cells exhibit a significant reduction in the rate of immunoglobulin (Ig) hypermutation, compared to wild-type cells. Further, the HERC2(-/-) and RNF8(-/-) mutants exhibit defective maintenance of replication fork progression immediately after exposure to UV while retaining proficient post-replicative gap filling. These mutants are both proficient in mono-ubiquitination of PCNA. Taken together, these results suggest that HERC2 and RNF8 promote TLS past abasic sites and UV-lesions at or very close to stalled replication forks.

When mismatch repair met translesion synthesis

When mismatch repair met translesion synthesis

From egg to gastrula: how the cell cycle is remodeled during the Drosophila mid-blastula transition.

Many, if not most, embryos begin development with extremely short cell cycles that exhibit unusually rapid DNA replication and no gap phases. The commitment to the cell cycle in the early embryo appears to preclude many other cellular processes that only emerge as the cell cycle slows just prior to gastrulation at a major embryonic transition known as the mid-blastula transition (MBT). As reviewed here, genetic and molecular studies in Drosophila have identified changes that extend S phase and introduce a post-replicative gap phase, G2, to slow the cell cycle. Although many mysteries remain about the upstream regulators of these changes, we review the core mechanisms of the change in cell cycle regulation and discuss advances in our understanding of how these might be timed and triggered. Finally, we consider how the elements of this program may be conserved or changed in other organisms.

NuA4 Initiates Dynamic Histone H4 Acetylation to Promote High-Fidelity Sister Chromatid Recombination at Postreplication Gaps

CAG/CTG trinucleotide repeats are unstable, fragile sequences that strongly position nucleosomes, but little is known about chromatin modifications required to prevent genomic instability at these or other structure-forming sequences. We discovered that regulated histone H4 acetylation is required to maintain CAG repeat stability and promote gap-induced sister chromatid recombination. CAG expansions in the absence of H4 HATs NuA4 and Hat1 and HDACs Sir2, Hos2, and Hst1 depended on Rad52, Rad57, and Rad5 and were therefore arising through homology-mediated postreplication repair (PRR) events. H4K12 and H4K16 acetylation were required to prevent Rad5-dependent CAG repeat expansions, and H4K16 acetylation was enriched at CAG repeats during S phase. Genetic experiments placed the RSC chromatin remodeler in the same PRR pathway, and Rsc2 recruitment was coincident with H4K16 acetylation. Here we have utilized a repetitive DNA sequence that induces endogenous DNA damage to identify histone modifications that regulate recombination efficiency and fidelity during postreplication gap repair.

Regulatory Role of Ubiquitin in Eukaryotic DNA Translesion Synthesis

Although often associated with proteasome-mediated protein degradation, ubiquitin plays essential nondegradative roles in a myriad of cellular processes, including chromatin dynamics, membrane trafficking, innate immunity, and DNA damage response. The recent progress in understanding DNA translesion synthesis (TLS), an important branch of DNA damage response, has largely been stimulated by the finding that ubiquitination of an essential nuclear protein, proliferating cell nuclear antigen (PCNA), controls precisely how eukaryotic cells respond to DNA damage. Despite the remarkable activity of the TLS polymerases in synthesizing past the damaged nucleotides, they are intrinsically error-prone on the normal DNA template. Therefore, a stringent regulation of the TLS polymerases is essential for the faithful replication of the DNA genome. Here we review the structure and function of the Y-family TLS polymerases and their interactions with ubiquitin and monoubiquitinated PCNA (Ub-PCNA). Driven by the need for monoubiquitinated PCNA in a sufficient quantity and purity, researchers developed both chemical and enzymatic methods for PCNA monoubiquitination, which have propelled our understanding of the structure of Ub-PCNA by X-ray crystallography and small-angle X-ray scattering. Together with studies using a reconstituted polymerase switching assay, these investigations revealed a surprising conformational flexibility of ubiquitin as a modifier on PCNA. Although the molecular details of TLS in cells still need to be deciphered, two working models, polymerase switching and postreplicative gap filling, have been proposed and tested in both in vitro and cellular systems. Evidence for both models is discussed herein. Compared to PCNA monoubiquitination, polyubiquitination of PCNA in DNA damage response is much less well understood and will be the subject of a future investigation. Given the close connection of DNA damage response and anticancer therapy, an in-depth understanding of the eukaryotic translesion synthesis and its regulation by ubiquitin will likely provide new opportunities for therapeutic intervention.

Analysis of CPD Ultraviolet Lesion Bypass in Chicken DT40 Cells: Polymerase η and PCNA Ubiquitylation Play Identical Roles

Translesion synthesis (TLS) provides a mechanism of copying damaged templates during DNA replication. This potentially mutagenic process may operate either at the replication fork or at post-replicative gaps. We used the example of T-T cyclobutane pyrimidine dimer (CPD) bypass to determine the influence of polymerase recruitment via PCNA ubiquitylation versus the REV1 protein on the efficiency and mutagenic outcome of TLS. Using mutant chicken DT40 cell lines we show that, on this numerically most important UV lesion, defects in polymerase η or in PCNA ubiquitylation similarly result in the long-term failure of lesion bypass with persistent strand gaps opposite the lesion, and the elevation of mutations amongst successful TLS events. Our data suggest that PCNA ubiquitylation promotes CPD bypass mainly by recruiting polymerase η, resulting in the majority of CPD lesions bypassed in an error-free manner. In contrast, we find that polymerase ζ is responsible for the majority of CPD-dependent mutations, but has no essential function in the completion of bypass. These findings point to a hierarchy of access of the different TLS polymerases to the lesion, suggesting a temporal order of their recruitment. The similarity of REV1 and REV3 mutant phenotypes confirms that the involvement of polymerase ζ in TLS is largely determined by its recruitment to DNA by REV1. Our data demonstrate the influence of the TLS polymerase recruitment mechanism on the success and accuracy of bypass.

CHK1 activity is required for continuous replication fork elongation but not stabilization of post-replicative gaps after UV irradiation

Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. The checkpoint kinase, CHK1 has been shown to stabilize replication forks following hydroxyurea treatment. Therefore, we wanted to test if the increased UV sensitivity caused by the unspecific kinase inhibitor caffeine—inhibiting ATM and ATR amongst other kinases—is explained by inability to activate the CHK1 kinase to stabilize replicative structures. For this, we used cells deficient in polymerase η (Polη), a translesion synthesis polymerase capable of properly bypassing the UV-induced cis–syn TT pyrimidine dimer, which blocks replication. These cells accumulate gaps behind progressing replication forks after UV exposure. We demonstrate that both caffeine and CHK1 inhibition, equally retards continuous replication fork elongation after UV treatment. Interestingly, we found more pronounced UV-sensitization by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of replicative structures after caffeine treatment, but not after CHK1 inhibition, in UV-irradiated cells. This demonstrates that CHK1 activity is not required for stabilization of gaps induced during replication of UV-damaged DNA. These data suggest that elongation and stabilization of replicative structures at UV-induced DNA damage are distinct mechanisms, and that CHK1 is only involved in replication elongation.

High Fidelity Required

Molecular Biology Covalent modifications of histones in nucleosomes can determine characteristic gene expression states for specific cells and tissues. These marks are thought to be maintained through cell division and DNA replication by the close coordination of histone eviction from the parental DNA with the transfer of the “parental” histones onto the nascent daughter DNA strands. The modifications on these recycled histones are then copied to new nucleosomes added to counter the twofold dilution on the daughter DNA. The fidelity of histone transfer depends on its close coordination with DNA replication. Sarkies et al. find that the normally silent [rho]-globin gene, which contains G quadruplex (G4) sequences that disrupt DNA replication, becomes activated in chicken cells lacking the specialized DNA polymerase REV1. REV1 helps the DNA replication machinery bypass G4 DNA. In the absence of REV1, post-replicative gaps are suggested to form in the vicinity of the G4 DNA, which are filled in later, thereby uncoupling replication and parental histone transfer. In the absence of the coupled transfer, new histones are used to populate the repaired gaps. New histones bear activating marks—not the repressive marks of the [rho]-globin parental nucleosomes—resulting in [rho]-globin gene activation. Many of the genes activated in cells lacking REV1 also have G4 consensus sequences close to their promoters, suggesting that histone recycling is critical for maintaining epigenetic states. Mol. Cell 40 , 1 (2010).

The Werner's Syndrome protein collaborates with REV1 to promote replication fork progression on damaged DNA

DNA damage tolerance pathways facilitate the bypass of DNA lesions encountered during replication. These pathways can be mechanistically divided into recombinational damage avoidance and translesion synthesis, in which the lesion is directly bypassed by specialised DNA polymerases. We have recently shown distinct genetic dependencies for lesion bypass at and behind the replication fork in the avian cell line DT40, bypass at the fork requiring REV1 and bypass at post-replicative gaps requiring PCNA ubiquitination by RAD18. The WRN helicase/exonuclease, which is mutated in the progeroid and cancer predisposition disorder Werner's Syndrome, has previously been implicated in a RAD18-dependent DNA damage tolerance pathway. However, WRN has also been shown to be required to maintain normal replication fork progression on a damaged DNA template, a defect reminiscent of REV1-deficient cells. Here we use the avian cell line DT40 to demonstrate that WRN assists REV1-dependent translesion synthesis at the replication fork and that PCNA ubiquitination-dependent post-replicative lesion bypass provides an important backup mechanism for damage tolerance in the absence of WRN protein.

Postreplication gaps at UV lesions are signals for checkpoint activation

Exposure of eukaryotic cells to UV light induces a checkpoint response that delays cell-cycle progression after cells enter S phase. It has been hypothesized that this checkpoint response provides time for repair by signaling the presence of structures generated when the replication fork encounters UV-induced DNA damage. To gain insight into the nature of the signaling structures, we used time-lapse microscopy to determine the effects of deficiencies in translesion DNA polymerases on the checkpoint response of the fission yeast Schizosaccharomyces pombe. We found that disruption of the genes encoding translesion DNA polymerases Polkappa and Poleta significantly prolonged the checkpoint response, indicating that the substrates of these enzymes are signals for checkpoint activation. Surprisingly, we found no evidence that the translesion polymerases Rev1 and Polzeta repair structures that are recognized by the checkpoint despite their role in maintaining viability after UV irradiation. Quantitative flow cytometry revealed that cells lacking translesion polymerases replicate UV-damaged DNA at the same rate at WT cells, indicating that the enhanced checkpoint response of cells lacking Polkappa and Poleta is not the result of stalled replication forks. These observations support a model in which postreplication DNA gaps with unrepaired UV lesions in the template strand act both as substrates for translesion polymerases and as signals for checkpoint activation.

Contributions of Nucleotide Excision Repair, DNA Polymerase η, and Homologous Recombination to Replication of UV-irradiated Herpes Simplex Virus Type 1

The effects of UV irradiation on herpes simplex virus type 1 (HSV-1) gene expression and DNA replication were examined in cell lines containing mutations inactivating the XPA gene product required for nucleotide-excision repair, the DNA polymerase eta responsible for translesion synthesis, or the Cockayne syndrome A and B (CSA and CSB) gene products required for transcription-coupled nucleotide excision repair. In the absence of XPA and CSA and CSB gene products, virus replication was reduced 10(6)-, 400-, and 100-fold, respectively. In DNA polymerase eta mutant cells HSV-1 plaque efficiency was reduced 10(4)-fold. Furthermore, DNA polymerase eta was strictly required for virus replication at low multiplicities of infection but dispensable at high multiplicities of infection. Knock down of Rad 51, Rad 52, and Rad 54 levels by RNA interference reduced replication of UV-irradiated HSV-1 150-, 100-, and 50-fold, respectively. We find that transcription-coupled repair efficiently supports expression of immediate early and early genes from UV-irradiated HSV-1 DNA. In contrast, the progression of the replication fork appears to be impaired, causing a severe reduction of late gene expression. Since the HSV-1 replisome does not make use of proliferating cell nuclear antigen, we attribute the replication defect to an inability to perform proliferating cell nuclear antigen-dependent translesion synthesis by polymerase switching at the fork. Instead, DNA polymerase eta may act during postreplication gap filling. Homologous recombination, finally, might restore the physical and genetic integrity of the virus chromosome.

Separate Domains of Rev1 Mediate Two Modes of DNA Damage Bypass in Mammalian Cells

The Y family DNA polymerase Rev1 has been proposed to play a regulatory role in the replication of damaged templates. To elucidate the mechanism by which Rev1 promotes DNA damage bypass, we have analyzed the progression of replication on UV light-damaged DNA in mouse embryonic fibroblasts that contain a defined deletion in the N-terminal BRCT domain of Rev1 or that are deficient for Rev1. We provide evidence that Rev1 plays a coordinating role in two modes of DNA damage bypass, i.e., an early and a late pathway. The cells carrying the deletion in the BRCT domain are deficient for the early pathway, reflecting a role of the BRCT domain of Rev1 in mutagenic translesion synthesis. Rev1-deficient cells display a defect in both modes of DNA damage bypass. Despite the persistent defect in the late replicational bypass of fork-blocking (6-4)pyrimidine-pyrimidone photoproducts, overall replication is not strongly affected by Rev1 deficiency. This results in almost completely replicated templates that contain gaps encompassing the photoproducts. These gaps are inducers of DNA damage signaling leading to an irreversible G(2) arrest. Our results corroborate a model in which Rev1-mediated DNA damage bypass at postreplicative gaps quenches irreversible DNA damage responses.

PCNA Ubiquitination and REV1 Define Temporally Distinct Mechanisms for Controlling Translesion Synthesis in the Avian Cell Line DT40

Translesion synthesis (TLS) is a potentially mutagenic method of bypassing DNA damage encountered during replication that requires the recruitment of specialized DNA polymerases to stalled replication forks or postreplicative gaps. Current models suggest that TLS is activated by monoubiquitination of the DNA sliding clamp PCNA. However, in higher organisms, fully effective TLS also requires a noncatalytic function of the Y family polymerase REV1. Using the genetically tractable chicken cell line DT40, we show that TLS at stalled replication forks requires that both the translesion polymerase-interaction domain and ubiquitin-binding domain in the C terminus of REV1 are intact. Surprisingly, however, PCNA ubiquitination is not required to maintain normal fork progression on damaged DNA. Conversely, PCNA ubiquitination is essential for filling postreplicative gaps. Thus, PCNA ubiquitination and REV1 play distinct roles in the coordination of DNA damage bypass that are temporally separated relative to replication fork arrest.

Exploring the roles of Mus81-Eme1/Mms4 at perturbed replication forks

Cells of all living organisms have evolved complex mechanisms that serve to stabilise, repair and restart stalled, blocked and broken replication forks. The heterodimeric Mus81-Eme1/Mms4 structure-specific endonuclease appears to play an important role(s) in homologous recombination-mediated processing of such perturbed forks. This enzyme has been implicated in the cleavage of stalled and blocked replication forks to initiate recombination, as well as in the processing of recombination intermediates that result from repairing damaged forks. In this review we assess the biochemical and genetic evidence for the mitotic role of Mus81-Eme1/Mms4 at replication forks and in repairing post-replication DNA damage. Mus81 appears to act when replication is impeded by genotoxins or by impairment of the replication machinery, or when arrested replication forks are not adequately protected. We discuss how its action is regulated by the S-phase cell cycle checkpoint, depending on the nature of the stalled or damaged fork. We also present a new way in which Mus81 may limit crossing over during the repair of post-replication gaps, and explore Mus81's interplay with other components of the recombination machinery, including the RecQ helicases that also play important roles in processing replication and recombination intermediates.

Cleavage of Holliday Junctions by the Escherichia coli RuvABC Complex

The Escherichia coli RuvABC proteins process recombination intermediates during genetic recombination and recombinational repair. Although early biochemical studies indicated distinct RuvAB-mediated branch migration and RuvC-mediated Holliday junction resolution reactions, more recent studies have shown that the three proteins act together as a "resolvasome" complex. In this work we have used recombination intermediates made by RecA to determine whether the RuvAB proteins affect the sequence specificity of the RuvC resolvase. We find that RuvAB proteins do not alter significantly the site specificity of RuvC-dependent cleavage, although under certain conditions, they do affect the efficiency of cleavage at particular sites. The presence of RecA also influences cleavage at some sites. We also show that the RuvAB proteins act upon transient strand exchange intermediates made using substrates that have the opposite polarity of those preferred by RecA. Together, our results allow us to develop further a model for the recombinational repair of DNA lesions that lead to the formation of post-replication gaps during DNA replication. The novel features of this model are as follows: (i) the RuvABC resolvasome recognizes joints made by RecA; (ii) resolution by RuvABC occurs at specific sites containing the RuvC consensus cleavage sequence 5'-(A/T)TT downward arrow(G/C)-3'; and (iii) Holliday junction resolution often occurs close to the initiating gap without significant heteroduplex DNA formation.

Is RecF a DNA replication protein?

Maintenance of genetic integrity is of foremost importance to the cell. Thus, cells are well equipped with a number of repair mechanisms for different types of DNA damage. All DNA polymerases are very sensitive to pyrimidine dimers, a major DNA lesion of UV irradiation. Inhibition of DNA replication by dimers, if not recovered, causes cell death. Escherichia coli cells have at least five different ways to deal with dimers (1). Dimers can be directly reversed by a reaction (photoreactivation) catalyzed by photolyase or can be removed by UvrABC-catalyzed excision repair. The remaining dimers can be tolerated by the replication machinery in different mechanisms. A modified DNA polymerase is able to replicate past noninstructive dimers by a mechanism termed translesion replication (1). Major mechanisms of dimer tolerance, however, involve two recombinational repair activities. The first of the two is RecF-dependent postreplication gap repair. It was demonstrated that DNA replication stalls at a dimer but can restart downstream of the dimer (2, 3). Thus, newly synthesized DNA after UV irradiation contains single-strand (ss) gaps opposite dimers (2). These gaps are subsequently filled by a mechanism involving homologous recombination (4). The second recombinational repair activity is RecBCD-dependent double-strand break (DSB) repair. It is known that a significant fraction of UV-induced lesions is converted to DSBs (5), which can be repaired by a mechanism that requires homologous recombination functions (6). A crucial aspect of recombinational repair is the extent to which the repair process involves DNA replication. Recently, RecBCD-dependent DSB repair was shown to require extensive semiconservative DNA replication that initiates at a D-loop, a recombination intermediate (6). Now, the paper by Courcelle et al. (7) in this issue of the Proceedings suggests that RecF protein, previously identified as a recombination protein, may assist replisome reassembly after UV irradiation. The proposal opens the door to the possibility that RecF-dependent postreplication gap repair also might involve extensive DNA replication.

Mating-type suppression of the DNA-repair defect of the yeast rad6 delta mutation requires the activity of genes in the RAD52 epistasis group.

The RAD6 gene of Saccharomyces cerevisiae is required for post-replication repair of UV-damaged DNA, UV mutagenesis, and sporulation. Here, we show that the radiation sensitivity of a MATa rad6 delta strain can be suppressed by the MAT alpha 2 gene carried on a multicopy plasmid. The a1-alpha 2 suppression is specific to the RAD6 pathway, as mutations in genes required for nucleotide excision repair or for recombinational repair do not show such mating-type suppression. The a1-alpha 2 suppression of the rad6 delta mutation requires the activity of the RAD52 group of genes, suggesting that suppression occurs by channelling of post-replication gaps present in the rad6 delta mutant into the RAD52 recombinational repair pathway. The a1-alpha 2 repressor could mediate this suppression via an enhancement in the expression, or the activity, of recombination genes.

Yeast DNA repair protein RAD5 that promotes instability of simple repetitive sequences is a DNA-dependent ATPase.

The RAD5 gene of Saccharomyces cerevisiae functions in postreplication repair of ultraviolet damaged DNA, and interestingly, it also has a role in increasing the instability of simple repetitive sequences in the genome. In contrast to DNA mismatch repair genes which function in maintaining constant length of repeat sequences, RAD5 promotes alterations in the length of repeat sequences. In this work, we purify the RAD5 protein to near homogeneity from yeast cells and show that it is a single-stranded DNA-dependent ATPase. The possible roles of RAD5 ATPase in postreplication gap filling and in increasing the incidence of length alterations of repeat sequences are discussed.

Effects of Post-treatment Incubation on Recombinogenesis in Incision-proficient and Incision-deficient Strains of Saccharomyces cerevisiae: I. Recombinogenesis after UV- and .GAMMA.-Ray-irradiation.

After UV-irradiation of G1 phase cells, most gene conversion and crossing-over took place in cells of incision-proficient strains without post-irradiation incubation. In contrast, incision-deficient cells markedly induced both of these recombinational events only after irradiated cells were incubated for several hours in liquid growth medium before selection. These results indicate that when G1 cells are irradiated with UV recombinational events are initiated during that G1 phase in incision-proficient strains; whereas, they are not initiated in irradiated incision-deficient strains before the cells reach the S/G2 phase. Experimental gamma-ray results also suggest that post-irradiation incubation is not required for incision-proficient and -deficient strains to initiate mitotic recombinational events. Taken together, these results show that the formation of incision nicks and of post-replication gaps in DNA appears to be necessary for the initiation of recombinational events after UV treatment; whereas, it is not required after gamma-irradiation.