Fate of DNA replication fork encountering a single DNA lesion during oriC plasmid DNA replication in vitro

BACKGROUND: The inhibition of DNA replication fork progression by DNA lesions can lead to cell death or genome instability. However, little is known about how such DNA lesions affect the concurrent synthesis of leading- and lagging-strand DNA catalysed by the protein machinery used in chromosomal replication. Using a system of semi-bidirectional DNA replication of an oriC plasmid that employs purified replicative enzymes and a replication-terminating protein of Escherichia coli, we examined the dynamics of the replication fork when it encounters a single abasic DNA lesion on the template DNA. RESULTS: A DNA lesion located on the lagging strand completely blocked the synthesis of the Okazaki fragment extending toward the lesion site, but did not affect the progression of the replication fork or leading-strand DNA synthesis. In contrast, a DNA lesion on the leading strand stalled the replication fork in conjunction with strongly inhibiting leading-strand synthesis. However, about two-thirds of the replication forks encountering this lesion maintained lagging-strand synthesis for about 1 kb beyond the lesion site, and the velocity with which the replication fork progressed seemed to be significantly reduced. CONCLUSIONS: The blocking DNA lesion affects DNA replication differently depending on which strand, leading or lagging, contains the lesion.     

Long inverted repeat transiently stalls DNA replication by forming hairpin structures on both leading and lagging strands

Long inverted repeats (LIRs), often found in eukaryotic genomes, are unstable in Escherichia coli where they are recognized by the SbcCD (the bacterial Mre11/Rad50 homologue), an endonuclease/exonuclease capable of cleaving hairpin DNA. It has long been postulated that LIRs form hairpin structures exclusively on the lagging‐strand template during DNA replication, and SbcCD cleaves these hairpin‐containing lagging strands to generate DNA double‐strand breaks. Using a reconstituted oriC plasmid DNA replication system, we have examined how a replication fork behaves when it meets a LIR on DNA. We have shown that leading‐strand synthesis stalls transiently within the upstream half of the LIR. Pausing of lagging‐strand synthesis at the LIR was not clearly observed, but the pattern of priming sites for Okazaki fragment synthesis was altered within the downstream half of the LIR. We have found that the LIR on a replicating plasmid was cleaved by SbcCD with almost equal frequency on both the leading‐ and lagging‐strand templates. These data strongly suggest that the LIR is readily converted to a cruciform DNA, before the arrival of the fork, creating SbcCD‐sensitive hairpin structures on both leading and lagging strands. We propose a model for the replication‐dependent extrusion of LIRs to form cruciform structures that transiently impede replication fork movement.     

Role of the Escherichia coli RecQ DNA helicase in SOS signaling and genome stabilization at stalled replication forks

The RecQ protein family is a highly conserved group of DNA helicases that play roles in maintaining genomic stability. In this study, we present biochemical and genetic evidence that Escherichia coli RecQ processes stalled replication forks and participates in SOS signaling. Cells that carry dnaE486, a mutation in the DNA polymerase III alpha-catalytic subunit, induce an RecA-dependent SOS response and become highly filamented at the semirestrictive temperature (38 degrees C). An recQ mutation suppresses the induction of SOS response and the filamentation in the dnaE486 mutant at 38 degrees C, causing appearance of a high proportion of anucleate cells. In vitro, RecQ binds and unwinds forked DNA substrates with a gap on the leading strand more efficiently than those with a gap on the lagging strand or Holliday junction DNA. RecQ unwinds the template duplex ahead of the fork, and then the lagging strand is unwound. Consequently, this process generates a single-stranded DNA (ssDNA) gap on the lagging strand adjacent to a replication fork. These results suggest that RecQ functions to generate an initiating signal that can recruit RecA for SOS induction and recombination at stalled replication forks, which are required for the cell cycle checkpoint and resumption of DNA replication.     

Role of DNA damage-induced replication checkpoint in promoting lesion bypass by translesion synthesis in yeast

Unrepaired DNA lesions in the template strand block the replication fork. In yeast, Mec1 protein kinase-mediated replication checkpoint prevents the breakdown of replication forks and maintains viability in DNA-damaged cells going through the S phase. By ensuring that the replisome does not dissociate from the fork stalled at the lesion site, the replication checkpoint presumably coordinates the action of lesion bypass processes with the replisome. However, it has remained unclear as to which of the lesion bypass processes—translesion synthesis (TLS) and/or template switching—depend on the activation of the replication checkpoint. Here we determine if the Mec1 kinase and the subunits of the checkpoint clamp and the clamp loader are required for TLS. We show that proficient TLS can occur in the absence of these checkpoint proteins in nucleotide excision repair (NER)-proficient cells; however, in the absence of NER, checkpoint protein-mediated Rev1 phosphorylation contributes to increasing the proficiency of DNA polymerase ζ-dependent TLS.     

A single (6-4) photoproduct inhibits plasmid DNA replication in xeroderma pigmentosum variant cell extracts

The human skin cancer-prone disease xeroderma pigmentosum variant (XPV) results from a mutation in the human RAD30 gene, which encodes the lesion bypass DNA polymerase eta. XPV cells are characterized by delayed completion of DNA replication and increased mutagenesis following UV-irradiation. Using extracts of an XPV lymphoblast cell line (GM2449C) that has a truncating mutation in the RAD30 gene, we investigated the effect of a (6-4) photoproduct and a cyclobutane pyrimidine dimer (CPD), at a unique -TT- site on either the leading or lagging strand, on plasmid DNA replication. Compared to normal cell extracts, XPV cell extracts have a reduced capacity to carry out complete replication of DNA containing either a (6-4) photoproduct or a CPD on the leading strand, whereas there is little difference between the two cell extracts in replication of DNA containing a lesion on the lagging strand. Inhibition of replication in the presence of a (6-4) photoproduct is attributed to arrest of nascent DNA strand synthesis at the lesion site; in XPV cell extracts, the proportion of arrested products is increased compared to that of normal cell extracts. These results are consistent with a requirement for functional DNA polymerase eta in the replication of a double-stranded plasmid containing either a (6-4) photoproduct or a CPD, on the leading but not the lagging strand.     

Human POT1 is required for efficient telomere C-rich strand replication in the absence of WRN

Mechanisms of telomere replication remain poorly defined. It has been suggested that G-rich telomeric strand replication by lagging mechanisms requires, in a stochastic way, the WRN protein. Here we show that this requirement is more systematic than previously thought. Our data are compatible with a situation in which, in the absence of WRN, DNA synthesis at replication forks is uncoupled, thus allowing replication to continue on the C strand, while single G strands accumulate. We also show that in cells in which both WRN and POT1 are limiting, both G- and C-rich telomeric strands shorten, suggesting a complete replication block. Under this particular condition, expression of a fragment spanning the two POT1-OB (oligonucleotide-binding) fold domains is able to restore C (but not G) strand replication, suggesting that binding of POT1 to the lagging strand allows DNA synthesis uncoupling in the absence of WRN. Furthermore, in vitro experiments indicate that purified POT1 has a higher affinity for the telomeric G-rich strand than purified RPA. We propose a model in which the relative enrichments of POT1 versus RPA on the telomeric lagging strand allows or does not allow uncoupling of DNA synthesis at the replication fork. Our study reveals an unanticipated role for hPOT1 during telomere replication.     

Papillomavirus E1 Proteins: Form,Function, and Features

The E1 proteins are the essential origin recognition proteins for papillomavirus (PV) replication. E1 proteins bind to specific DNA elements in the viral origin of replication and assemble into hexameric helicases with the aid of a second viral protein, E2. The resultant helicase complex initiates origin DNA unwinding to provide the template for subsequent syntheses of progeny DNA. In addition to ATP-dependent helicase activity, E1 proteins interact with and recruit several host cell replication proteins to viral origin, including DNA polymerase and RPA. This review will compare the basic structures and features of the human (HPV) and bovine (BPV1) papillomaviruses with an emphasis on mechanisms of replication function.     

Inhibition of simian virus 40 DNA replication by ultraviolet light

The effects of ultraviolet light (uv) upon SV40 DNA synthesis in monkey cells were examined to determine whether replication forks were halted upon encountering lesions in the DNA, or alternatively whether lesions were rapidly bypassed. Ultraviolet light inhibits elongation of nascent DNA strands; the extent of incorporation of [3H]deoxythymidine ( [3H]dT) into DNA decreases with increasing uv fluence. Inhibition begins within minutes of irradiation, and becomes more pronounced with increasing time after irradiation. The synthesis of form I (covalently closed) molecules is inhibited even more severely than is total incorporation: post-uv incorporation is predominantly into replication intermediates. In contrast to previous reports, we find that replication intermediates labeled after uv resemble those in unirradiated cells, and contain covalently closed parental strands. DNA strands made after uv are approximately the size of parental DNA which has been cleaved at pyrimidine dimers by a uv endonuclease, indicating that they do not extend past dimers. The hypothesis that replication forks are halted upon encountering pyrimidine dimers in the template strand is consistent with these data.     

Analysis of mutations induced by replication of UV-damaged plasmid DNA in hela cell extracts

We have used an SV40-based shuttle vector, pZ189, to investigate the capacity of HeLa cell extracts to reproduce the in vivo process of mutation fixation. We showed previously that when UV-irradiated pZ189 is replicated in these extracts, bypass of UV photoproducts occurs, resulting in base substitution mutations in the supF gene of the vector. Here we report the DNA sequence characterization of a collection of 60 of these UV-induced mutants. Most of the mutations observed are single or tandem double base substitutions at dipyrimidine sites; of these, approximately 90% are G:C→A:T transitions. Mutations are observed predominantly at a few sites, in particular at positions 155 and 156 in the supF sequence. No dramatic differences in the mutation spectrum were observed when the orientation of the supF gene was reversed with respect to the SV40 origin of replication, suggesting that mutation fixation occurs similarly on both the leading and the lagging strands for DNA replication. Generally, the mutational hot spots observed in vitro are at the same sites as those observed when UV-irradiated pZ189 was passaged in human or monkey cells in culture. Thus, it appears that the replication and mutagenesis of UV-damaged templates in HeLa cell extracts accurately reflects these processes in the intact cell. © 1995 Wiley-Liss, Inc.     

ATR phosphorylates SMARCAL1 to prevent replication fork collapse

The DNA damage response kinase ataxia telangiectasia and Rad3-related (ATR) coordinates much of the cellular response to replication stress. The exact mechanisms by which ATR regulates DNA synthesis in conditions of replication stress are largely unknown, but this activity is critical for the viability and proliferation of cancer cells, making ATR a potential therapeutic target. Here we use selective ATR inhibitors to demonstrate that acute inhibition of ATR kinase activity yields rapid cell lethality, disrupts the timing of replication initiation, slows replication elongation, and induces fork collapse. We define the mechanism of this fork collapse, which includes SLX4-dependent cleavage yielding double-strand breaks and CtIP-dependent resection generating excess single-stranded template and nascent DNA strands. Our data suggest that the DNA substrates of these nucleases are generated at least in part by the SMARCAL1 DNA translocase. Properly regulated SMARCAL1 promotes stalled fork repair and restart; however, unregulated SMARCAL1 contributes to fork collapse when ATR is inactivated in both mammalian and Xenopus systems. ATR phosphorylates SMARCAL1 on S652, thereby limiting its fork regression activities and preventing aberrant fork processing. Thus, phosphorylation of SMARCAL1 is one mechanism by which ATR prevents fork collapse, promotes the completion of DNA replication, and maintains genome integrity.     

ZRANB3 is a structure-specific ATP-dependent endonuclease involved in replication stress response

To efficiently duplicate their genomic content, cells must overcome DNA lesions that interfere with processive DNA replication. These lesions may be removed and repaired, rather than just tolerated, to allow continuity of DNA replication on an undamaged DNA template. However, it is unclear how this is achieved at a molecular level. Here we identify a new replication-associated factor, ZRANB3 (zinc finger, RAN-binding domain containing 3), and propose its role in the repair of replication-blocking lesions. ZRANB3 has a unique structure-specific endonuclease activity, which is coupled to ATP hydrolysis. It cleaves branched DNA structures with unusual polarity, generating an accessible 3'-OH group in the template of the leading strand. Furthermore, ZRANB3 localizes to DNA replication sites and interacts with the components of the replication machinery. It is recruited to damaged replication forks via multiple mechanisms, which involve interactions with PCNA, K63-polyubiquitin chains, and branched DNA structures. Collectively, our data support a role for ZRANB3 in the replication stress response and suggest new insights into how DNA repair is coordinated with DNA replication to maintain genome stability.     

(6-4) Photoproducts and not cyclobutane pyrimidine dimers are the main UV-induced mutagenic lesions in Chinese hamster cells

A partial revertant (RH1–26) of the UV-sensitive Chinese hamster V79 cell mutant V-H1 (complementation group 2) was isolated and characterized. It was used to analyze the mutagenic potency of the 2 major UV-induced lesions, cyclobutane pyrimidine dimers and (6-4) photoproducts. Both V-H1 and RH1–26 did not repair pyrimidine dimers measured in the genome overall as well as in the active hprt gene. Repair of (6-4) photoproducts from the genome overall was slower in V-H1 than in wild-type V79 cells, but was restored to normal in RH1–26. Although V-H1 cells have a 7-fold enhanced mutagenicity, RH1–26 cells, despite the absence of pyrimidine dimer repair, have a slightly lower level of UV-induced mutagenesis than observed in wild-type V79 cells. The molecular nature of hprt mutations and the DNA-strand specificity were similar in V79 and RH1–26 cells but different from that of V-H1 cells. Since in RH1–26 as well as in V79 cells most hprt mutations were induced by lesions in the non-transcribed DNA strand, in contrast to the transcribed DNA strand in V-H1, the observed mutation-strand bias suggests that normally (6-4) photoproducts are preferentially repaired in the transcribed DNA strand. The dramatic influence of the impaired (6-4) photoproduct repair in V-H1 on UV-induced mutability and the molecular nature of hprt mutations indicate that the (6-4) photoproduct is the main UV-induced mutagenic lesion.     

Short CCG repeat in huntingtin gene is an obstacle for replicative DNA polymerases,potentially hampering progression of replication fork

Trinucleotide repeats (TNRs) are highly unstable in genomes, and their expansions are linked to human disorders. DNA replication is reported to be involved in TNR instability, but the current models are insufficient in explaining TNR expansion is induced during replication. Here, we investigated replication fork progression across huntingtin (HTT)‐gene‐derived fragments using an Escherichia coli oriC plasmid DNA replication system. We found most of the forks to travel smoothly across the HTT fragments even when the fragments had a pathological length of CAG/CTG repeats (approximately 120 repeats). A little fork stalling in the fragments was observed, but it occurred within a short 3′‐flanking region downstream of the repeats. This region contains another short TNR, (CCG/CGG)₇, and the sense strand containing CCG repeats appeared to impede the replicative DNA polymerase Pol III. Examining the behavior of the human leading and lagging replicative polymerases Pol epsilon (hPolε) and Pol delta (hPolδ) on this sequence, we found hPolδ replicating DNA across the CCG repeats but hPolε stalling at the CCG repeats even if the secondary structure is eliminated by a single‐stranded binding protein. These findings offer insights into the distinct behavior of leading and lagging polymerases at CCG/CGG repeats, which may be important for understanding the process of replication arrest and genome instability at the HTT gene.     

The DNA translocase activity of FANCM protects stalled replication forks

FANCM is the most highly conserved protein within the Fanconi anaemia (FA) tumour suppressor pathway. However, although FANCM contains a helicase domain with translocase activity, this is not required for its role in activating the FA pathway. Instead, we show here that FANCM translocaseactivity is essential for promoting replication fork stability. We demonstrate that cells expressing translocase-defective FANCM show altered global replication dynamics due to increased accumulation of stalled forks that subsequently degenerate into DNA double-strand breaks, leading to ATM activation, CTBP-interacting protein (CTIP)-dependent end resection and homologous recombination repair. Accordingly, abrogation of ATM or CTIP function in FANCM-deficient cells results in decreased cell survival. We also found that FANCM translocase activity protects cells from accumulating 53BP1-OPT domains, which mark lesions resulting from problems arising during replication. Taken together, these data show that FANCM plays an essential role in maintaining chromosomal integrity by promoting the recovery of stalled replication forks and hence preventing tumourigenesis.     

TRF1 negotiates TTAGGG repeat-associated replication problems by recruiting the BLM helicase and the TPP1/POT1 repressor of ATR signaling

The semiconservative replication of telomeres is facilitated by the shelterin component TRF1. Without TRF1, replication forks stall in the telomeric repeats, leading to ATR kinase signaling upon S-phase progression, fragile metaphase telomeres that resemble the common fragile sites (CFSs), and the association of sister telomeres. In contrast, TRF1 does not contribute significantly to the end protection functions of shelterin. We addressed the mechanism of TRF1 action using mouse conditional knockouts of BLM, TRF1, TPP1, and Rap1 in combination with expression of TRF1 and TIN2 mutants. The data establish that TRF1 binds BLM to facilitate lagging but not leading strand telomeric DNA synthesis. As the template for lagging strand telomeric DNA synthesis is the TTAGGG repeat strand, TRF1-bound BLM is likely required to remove secondary structures formed by these sequences. In addition, the data establish that TRF1 deploys TIN2 and the TPP1/POT1 heterodimers in shelterin to prevent ATR during telomere replication and repress the accompanying sister telomere associations. Thus, TRF1 uses two distinct mechanisms to promote replication of telomeric DNA and circumvent the consequences of replication stress. These data are relevant to the expression of CFSs and provide insights into TIN2, which is compromised in dyskeratosis congenita (DC) and related disorders.     

The S. cerevisiae Rrm3p DNA helicase moves with the replication fork and affects replication of all yeast chromosomes

The Saccharomyces cerevisiae DNA helicase Rrm3p is needed for normal fork progression through >1000 discrete sites scattered throughout the genome. Here we show that replication of all yeast chromosomes was markedly delayed in rrm3 cells. Delayed replication was seen even in a region that lacks any predicted Rrm3p-dependent sites. Based on the pattern of replication intermediates in two-dimensional gels, the rate of fork movement in rrm3 cells appeared similar to wild-type except at known Rrm3p-dependent sites. These data suggest that although Rrm3p has a global role in DNA replication, its activity is needed only or primarily at specific, difficult-to-replicate sites. By the criterion of chromatin immunoprecipitation, Rrm3p was associated with both Rrm3p-dependent and -independent sites, and moved with the replication fork through both. In addition, Rrm3p interacted with Pol2p, the catalytic subunit of DNA polymerase epsilon, in vivo. Thus, rather than being recruited to its sites of action when replication forks stall at these sites, Rrm3p is likely a component of the replication fork apparatus.     

Orientation specificity of the Lactococcus lactis Chi site

BACKGROUND: In Escherichia coli, the Chi sequence modulates the activity of RecBCD, a powerful double-stranded (ds) DNA exonuclease/helicase. Chi attenuates RecBCD exonuclease activity and stimulates homologous recombination in an orientation-dependent manner. ChiEc is frequent and over-represented on its genome, which is thought to be related to its role in dsDNA break repair. We previously identified a Chi-like sequence (referred to as ChiLl) and an exonuclease/helicase in the Gram-positive bacterium Lactococcus lactis. ChiLl and RexAB are functional analogues of ChiEc and RecBCD. RESULTS: We report that ChiLl attenuates RexAB exonuclease activity and stimulates homologous recombination in an orientation-dependent manner. Analysis of ChiLl distribution on the L. lactis chromosome reveals that ChiLl is frequent, highly over-represented, and oriented with respect to the direction of replication. CONCLUSION: Our results show that a single orientation of ChiLl interacts with RexAB. The active orientation is preferentially found on the replication leading strand of the L. lactis genome, consistent with a primary role of ChiLl in repair of dsDNA breaks at the replication fork. We propose that orientation-dependence of Chi activity and over-representation of Chi sequences on bacterial genomes may be conserved properties of exonuclease/helicase-Chi couples. Other properties of the Chi sequence distribution on the genomes might reflect more specific characteristics of each couple and of the host.