RNase-sensitive DNA modification(s) initiates S. pombe mating-type switching

Mating-type switching in fission yeast depends on an imprint at the mat1 locus. Previous data showed that the imprint is made in the DNA strand replicated as lagging. We now identify this imprint as an RNase-sensitive modification and suggest that it consists of one or two RNA residues incorporated into the mat1 DNA. Formation of the imprint requires swi1- and swi3-dependent pausing of the replication fork. Interestingly, swi1 and swi3 mutations that abolish pausing do not affect the use of lagging-strand priming site during replication. We show that the pausing of replication and subsequent formation of the imprint occur after the leading-strand replication complex has passed the site of the imprint and after lagging-strand synthesis has initiated at this proximal priming site. We propose a model in which a swi1- and swi3-dependent signal during lagging-strand synthesis leads to pausing of leading-strand replication and the introduction of the imprint.     

UV lesions located on the leading strand inhibit DNA replication but do not inhibit SV40 T-antigen helicase activity

DNA replication in eucaryotic cells involves a variety of proteins which synthesize the leading and lagging strands in an asymmetric coordinated manner. To analyse the effect of this asymmetry on the translesion synthesis of UV-induced lesions, we have incubated SV40 origin-containing plasmids with a unique site-specific cis,syn-cyclobutane dimer or a pyrimidine-pyrimidone (6-4) photoproduct on either the leading or lagging strand template with DNA replication-competent extracts made from human HeLa cells. Two dimensional agarose gel electrophoresis analyses revealed a strong blockage of fork progression only when the UV lesion is located on the leading strand template. Because DNA helicases are responsible for unwinding duplex DNA ahead of the fork and are then the first component to encounter any potential lesion, we tested the effect of these single photoproducts on the unwinding activity of the SV40 T antigen, the major helicase in our in vitro replication assay. We showed that the activity of the SV40 T-antigen helicase is not inhibited by UV-induced DNA lesions in double-stranded DNA substrate.     

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.     

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.     

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.     

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.     

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.     

DNA polymerase delta in dna replication and genome maintenance

The eukaryotic genome is in a constant state of modification and repair. Faithful transmission of the genomic information from parent to daughter cells depends upon an extensive system of surveillance, signaling, and DNA repair, as well as accurate synthesis of DNA during replication. Often, replicative synthesis occurs over regions of DNA that have not yet been repaired, presenting further challenges to genomic stability. DNA polymerase δ (pol δ) occupies a central role in all of these processes: catalyzing the accurate replication of a majority of the genome, participating in several DNA repair synthetic pathways, and contributing structurally to the accurate bypass of problematic lesions during translesion synthesis. The concerted actions of pol δ on the lagging strand, pol ? on the leading strand, associated replicative factors, and the mismatch repair (MMR) proteins results in a mutation rate of less than one misincorporation per genome per replication cycle. This low mutation rate provides a high level of protection against genetic defects during development and may prevent the initiation of malignancies in somatic cells. This review explores the role of pol δ in replication fidelity and genome maintenance. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

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.     

Recombination-dependent concatemeric viral DNA replication

The initiation of viral double stranded (ds) DNA replication involves proteins that recruit and load the replisome at the replication origin (ori). Any block in replication fork progression or a programmed barrier may act as a factor for ori-independent remodelling and assembly of a new replisome at the stalled fork. Then replication initiation becomes dependent on recombination proteins, a process called recombination-dependent replication (RDR). RDR, which is recognized as being important for replication restart and stability in all living organisms, plays an essential role in the replication cycle of many dsDNA viruses. The SPP1 virus, which infects Bacillus subtilis cells, serves as a paradigm to understand the links between replication and recombination in circular dsDNA viruses. SPP1-encoded initiator and replisome assembly proteins control the onset of viral replication and direct the recruitment of host-encoded replisomal components at viral oriL. SPP1 uses replication fork reactivation to switch from ori-dependent θ-type (circle-to-circle) replication to σ-type RDR. Replication fork arrest leads to a double strand break that is processed by viral-encoded factors to generate a D-loop into which a new replisome is assembled, leading to σ-type viral replication. SPP1 RDR proteins are compared with similar proteins encoded by other viruses and their possible in vivo roles are discussed.     

Distinct roles of DNA polymerases delta and epsilon at the replication fork in Xenopus egg extracts

DNA polymerases delta and epsilon (Poldelta and Polepsilon) are widely thought to be the major DNA polymerases that function in elongation during DNA replication in eukaryotic cells. However, the precise roles of these polymerases are still unclear. Here we comparatively analysed DNA replication in Xenopus egg extracts in which Poldelta or Polepsilon was immunodepleted. Depletion of either polymerase resulted in a significant decrease in DNA synthesis and accumulation of short nascent DNA products, indicating an elongation defect. Moreover, Poldelta depletion caused a more severe defect in elongation, as shown by sustained accumulation of both short nascent DNA products and single-stranded DNA gaps, and also by elevated chromatin binding of replication proteins that function more frequently during lagging strand synthesis. Therefore, our data strongly suggest the possibilities that Poldelta is essential for lagging strand synthesis and that this function of Poldelta cannot be substituted for by Polepsilon.     

Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae

Replication fork stalling at a DNA lesion generates a damage signal that activates the Rad53 kinase, which plays a vital role in survival by stabilizing stalled replication forks. However, evidence that Rad53 directly modulates the activity of replication forks has been lacking, and the nature of fork stabilization has remained unclear. Recently, cells lacking the Psy2-Pph3 phosphatase were shown to be defective in dephosphorylation of Rad53 as well as replication fork restart after DNA damage, suggesting a mechanistic link between Rad53 deactivation and fork restart. To test this possibility we examined the progression of replication forks in methyl-methanesulfonate (MMS)-damaged cells, under different conditions of Rad53 activity. Hyperactivity of Rad53 in pph3Delta cells slows fork progression in MMS, whereas deactivation of Rad53, through expression of dominant-negative Rad53-KD, is sufficient to allow fork restart during recovery. Furthermore, combined deletion of PPH3 and PTC2, a second, unrelated Rad53 phosphatase, results in complete replication fork arrest and lethality in MMS, demonstrating that Rad53 deactivation is a key mechanism controlling fork restart. We propose a model for regulation of replication fork progression through damaged DNA involving a cycle of Rad53 activation and deactivation that coordinates replication restart with DNA repair.     

Normal human chromosomes have long G-rich telomeric overhangs at one end

Telomeres protect the ends of linear chromosomes from degradation and abnormal recombination events, and in vertebrates may be important in cellular senescence and cancer. However, very little is known about the structure of human telomeres. In this report we purify telomeres and analyze their termini. We show that following replication the daughter telomeres have different terminal overhangs in normal diploid telomerase-negative human fibroblasts. Electron microscopy of those telomeres that have long overhangs yields 200±75 nucleotides of single-stranded DNA. This overhang is four times greater than the amount of telomere shortening per division found in these cells. These results are consistent with models of telomere replication in which leading-strand synthesis generates a blunt end while lagging-strand synthesis produces a long G-rich 3′ overhang, and suggest that variations in lagging-strand synthesis may regulate the rate of telomere shortening in normal diploid human cells. Our results do not exclude the possibility that nuclease processing events following leading strand synthesis result in short overhangs on one end.     

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 SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart

The integrity of genomic DNA is continuously challenged by the presence of DNA base lesions or DNA strand breaks. Here we report the identification of a new DNA damage response protein, SMARCAL1 (SWI/SNF-related, matrix associated, actin-dependent regulator of chromatin, subfamily a-like 1), which is a member of the SNF2 family and is mutated in Schimke immunoosseous dysplasia (SIOD). We demonstrate that SMARCAL1 directly interacts with Replication protein A (RPA) and is recruited to sites of DNA damage in an RPA-dependent manner. SMARCAL1-depleted cells display sensitivity to DNA-damaging agents that induce replication fork collapse, and exhibit slower fork recovery and delayed entry into mitosis following S-phase arrest. Furthermore, SIOD patient fibroblasts reconstituted with SMARCAL1 exhibit faster cell cycle progression after S-phase arrest. Thus, the symptoms of SIOD may be caused, at least in part, by defects in the cellular response to DNA replication stress.