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.     

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.     

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.     

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.     

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.     

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.     

Cdc13 both positively and negatively regulates telomere replication

Cdc13 is a single-strand telomeric DNA-binding protein that positively regulates yeast telomere replication by recruiting telomerase to chromosome termini through a site on Cdc13 that is eliminated by the cdc13-2 mutation. Here we show that Cdc13 has a separate role in negative regulation of telomere replication, based on analysis of a new mutation, cdc13-5. Loss of this second regulatory activity results in extensive elongation of the G strand of the telomere by telomerase, accompanied by a reduced ability to coordinate synthesis of the C strand. Both the cdc13-5 mutation and DNA polymerase alpha mutations (which also exhibit elongated telomeres) are suppressed by increased expression of the Cdc13-interacting protein Stn1, indicating that Stn1 coordinates action of the lagging strand replication complex with the regulatory activity of CDC13. However, the association between Cdc13 and Stn1 is abolished by cdc13-2, the same mutation that eliminates the interaction between Cdc13 and telomerase. We propose that Cdc13 participates in two regulatory steps-first positive, then negative-as a result of successive binding of telomerase and the negative regulator Stn1 to overlapping sites on Cdc13. Thus, Cdc13 coordinates synthesis of both strands of the telomere by first recruiting telomerase and subsequently limiting G-strand synthesis by telomerase in response to C-strand replication.     

Roles of yeast DNA polymerases delta and zeta and of Rev1 in the bypass of abasic sites

Abasic (AP) sites are one of the most frequently formed lesions in DNA, and they present a strong block to continued synthesis by the replicative DNA machinery. Here we show efficient bypass of an AP site by the combined action of yeast DNA polymerases delta and zeta. In this reaction, Poldelta inserts an A nucleotide opposite the AP site, and Polzeta subsequently extends from the inserted nucleotide. Consistent with these observations, sequence analyses of mutations in the yeast CAN1s gene indicate that A is the nucleotide inserted most often opposite AP sites. The nucleotides C, G, and T are also incorporated, but much less frequently. Enzymes such as Rev1 and Poleta may contribute to the insertion of these other nucleotides; the predominant role of Rev1 in AP bypass, however, is likely to be structural. Steady-state kinetic analyses show that Polzeta is highly inefficient in incorporating nucleotides opposite the AP site, but it efficiently extends from nucleotides, particularly an A, inserted opposite this lesion. Thus, in eukaryotes, bypass of an AP site requires the sequential action of two DNA polymerases, wherein the extension step depends solely upon Polzeta, but the insertion step can be quite varied, involving not only the predominant action of the replicative DNA polymerase, Poldelta, but also the less prominent role of various translesion synthesis polymerases.     

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.     

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.     

A novel mechanism generating short deletion/insertions following slippage is suggested by a mutation in the human alpha2-globin gene

A novel mechanism generating short deletion/insertions is described based on a mutation in the human alpha2-globin gene. A deletion of 9 bp (codons 39-41) is replaced by an eight nucleotide insertion, duplicating the adjacent downstream sequence. We propose that the mutation arose by slipped strand mispairing (SSM), creating a single- stranded loop, followed by DNA elongation, strand breathing and the formation of a mismatch bubble. An extensive literature search has revealed six additional deletion/insertion mutations in humans in which the inserted nucleotides come from the same DNA strand. Our model explains all six mutations, suggesting that rearrangement of a mismatch loop or bubble during DNA replication may be not uncommon.      

Differences in DNA synthesis in vitro using isolated nuclei from regenerating livers of young and aged rats

To detect changes in DNA synthesis during ageing, we compare DNA synthesis in the livers of young and aged rats. As an intermediate between an in vivo system using intact cells and an in vitro system using purified DNA polymerases, isolated nuclei were prepared and used as the machinery for DNA synthesis. The DNA synthesizing capacity of nuclei from regenerating liver was higher than that of nuclei from normal liver and these capacities from liver and regenerating liver were lower in nuclear preparations from aged rats. DNA synthesis using isolated nuclei was stimulated by ATP and the cytoplasmic preparation. The cytoplasmic preparation from regenerating rat liver was found to stimulate DNA synthesis more than the preparation from normal liver. The activity in regenerating liver from young rats was also greater than in that from aged rats. It is well known that DNA replication is inhibited by aphidicolin and DNA repair by ddTTP. We examined the effects of aphidicolin and ddTTP on DNA synthesis using the nuclear system. Surprisingly, the inhibition by aphidicolin was 30% of total DNA synthesis using the nuclear system from young rats. On the other hand, the inhibition by ddTTP was approximately 80%. We measured the sizes of the DNA synthesized in the presence of both inhibitors. DNA synthesis was allowed to proceed for 10 min using isolated nuclei from regenerating liver of young rats and the size of the DNA was determined by sucrose density gradient centrifugation analysis. DNA products appeared in two fractions. Following a chase of 50 min in the presence or absence of aphidicolin, the short DNA product grew larger in both cases, although the amount of DNA in the presence of aphidicolin was approximately 90% that in its absence. In the same experiment using nuclei from aged rats, the amount in the presence of aphidicolin was approximately 60% that in its absence. These results suggest that DNA polymerase beta is closely related to abnormal replication when DNA polymerases alpha and delta are inhibited and that the effect of cytosol on DNA synthesis, as well as the DNA synthetic capacity of isolated nuclei, becomes lower in regenerating rat liver during ageing.     

Complex Multiple‐Nucleotide Substitution Mutations Causing Human Inherited Disease Reveal Novel Insights into the Action of Translesion Synthesis DNA Polymerases

Translesion synthesis (TLS) DNA polymerases allow the bypass of unrepaired lesions during DNA replication. Based upon mutational signatures of a subtype of multiple‐nucleotide substitution (MNS) mutations causing human inherited disease, we have recently postulated two properties of TLS DNA polymerases in DNA repair, namely, the generation of neo‐microhomologies potentiating strand‐misalignment, and additional microlesions within the templated inserts when recruited to stalled replication forks. To provide further support for this postulate, we analyzed the mutational signatures of a new and complex subtype of pathogenic MNS mutation. Several mutations containing long templated inserts (8–19 bp) that are highly informative with regard to their underlying mutational mechanisms, harbor imprints of TLS DNA polymerase action. Dissecting the mechanism underlying the generation of the 19‐bp insert implicated repeated participation of TLS DNA polymerases in the conversion of a damaged base into a complex MNS lesion through a process of successive template switching and bypass repair.     

Interactions of human Cdc45 with the Mcm2-7 complex, the GINS complex, and DNA polymerases delta and epsilon during S phase

Cdc45 is an essential cellular protein that functions in both the initiation and elongation of DNA replication. Here, we analyzed the localization of human Cdc45 and its interactions with other proteins during the cell cycle. Human Cdc45 showed a diffuse distribution in G1 phase, a spot-like pattern in S and G2, and again a diffuse distribution in M phase of the cell cycle. The co-localization of Cdc45 with active replication sites during S phase suggested that the human Cdc45 protein was part of the elongation complex. This view was corroborated by findings that Cdc45 interacted with the elongating DNA polymerases delta and epsilon, with Psf2, which is a component of the GINS complex as well as with Mcm5 and 7, subunits of the putative replicative DNA helicase complex. Hence, Cdc45 may play an important role in elongation of DNA replication by bridging the processive DNA polymerases delta and epsilon with the replicative helicase in the elongating machinery.     

Roles of histone chaperone CIA/Asf1 in nascent DNA elongation during nucleosome replication

The nucleosome, which is composed of DNA wrapped around a histone octamer, is a fundamental unit of chromatin and is duplicated during the eukaryotic DNA replication process. The evolutionarily conserved histone chaperone cell cycle gene 1 (CCG1) interacting factor A/anti-silencing function 1 (CIA/Asf1) is involved in histone transfer and nucleosome reassembly during DNA replication. CIA/Asf1 has been reported to split the histone (H3-H4)(2) tetramer into histone H3-H4 dimer(s) in vitro, raising a possibility that, in DNA replication, CIA/Asf1 is involved in nucleosome disassembly and the promotion of semi-conservative histone H3-H4 dimer deposition onto each daughter strand in vivo. Despite numerous studies on the functional roles of CIA/Asf1, its mechanistic role(s) remains elusive because of lack of biochemical analyses. The biochemical studies described here show that a V94R CIA/Asf1 mutant, which lacks histone (H3-H4)(2) tetramer splitting activity, does not form efficiently a quaternary complex with histones H3-H4 and the minichromosome maintenance 2 (Mcm2) subunit of the Mcm2-7 replicative DNA helicase. Interestingly, the mutant enhances nascent DNA strand synthesis in a cell-free chromosomal DNA replication system using Xenopus egg extracts. These results suggest that CIA/Asf1 in the CIA/Asf1-H3-H4-Mcm2 complex, which is considered to be an intermediate in histone transfer during DNA replication, negatively regulates the progression of the replication fork.     

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.