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DNA polymerase ? contributes to genome-wide lagging strand synthesis.

ABSTRACT: DNA polymerase ? (pol ?) is best known for its ability to bypass UV-induced thymine-thymine (T-T) dimers and other bulky DNA lesions, but pol ? also has other cellular roles. Here, we present evidence that pol ? competes with DNA polymerases ? and ? for the synthesis of the lagging strand genome-wide, where it also shows a preference for T-T in the DNA template. Moreover, we found that the C-terminus of pol ?, which contains a PCNA-Interacting Protein motif is required for pol ? to function in lagging strand synthesis. Finally, we provide evidence that a pol ? dependent signature is also found to be lagging strand specific in patients with skin cancer. Taken together, these findings provide insight into the physiological role of DNA synthesis by pol ? and have implications for our understanding of how our genome is replicated to avoid mutagenesis, genome instability and cancer.

SUBMITTER: Kreisel K

PROVIDER: S-EPMC6411934 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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DNA polymerase η contributes to genome-wide lagging strand synthesis.

Kreisel Katrin K Engqvist Martin K M MKM Kalm Josephine J Thompson Liam J LJ Boström Martin M Navarrete Clara C McDonald John P JP Larsson Erik E Woodgate Roger R Clausen Anders R AR

Nucleic acids research 20190301 5

DNA polymerase η (pol η) is best known for its ability to bypass UV-induced thymine-thymine (T-T) dimers and other bulky DNA lesions, but pol η also has other cellular roles. Here, we present evidence that pol η competes with DNA polymerases α and δ for the synthesis of the lagging strand genome-wide, where it also shows a preference for T-T in the DNA template. Moreover, we found that the C-terminus of pol η, which contains a PCNA-Interacting Protein motif is required for pol η to function in l ...[more]

PMID: 30597049

Similar Datasets

DNA polymerase η contributes to lagging strand synthesis

Project description:DNA polymerase eta (pol eta) is best known for its ability to bypass UV-induced thymine-thymine (T-T) dimers and other bulky DNA lesions, but pol eta also has other cellular roles. Here, we present evidence that pol eta competes with DNA polymerases alpha and delta for the synthesis of the lagging strand genome-wide, where it also shows a preference for T-T in the DNA template. Moreover, we found that the C-terminus of pol eta which contains a PCNA-Interacting Protein motif is required for pol eta to function in lagging strand synthesis. Finally, we provide evidence that a pol η dependent signature is also found to be lagging strand specific in patients with skin cancer. Taken together, these findings provide insight into the physiological role of DNA synthesis by pol eta and have implications for our understanding of how our genome is replicated to avoid mutagenesis, genome instability and cancer.

2018-12-14 | GSE110241 | GEO

DNA polymerase η contributes to lagging strand synthesis

Project description:DNA polymerase η contributes to lagging strand synthesis

| PRJNA433239 | ENA

High-accuracy lagging-strand DNA replication mediated by DNA polymerase dissociation.

Project description:The fidelity of DNA replication is a critical factor in the rate at which cells incur mutations. Due to the antiparallel orientation of the two chromosomal DNA strands, one strand (leading strand) is replicated in a mostly processive manner, while the other (lagging strand) is synthesized in short sections called Okazaki fragments. A fundamental question that remains to be answered is whether the two strands are copied with the same intrinsic fidelity. In most experimental systems, this question is difficult to answer, as the replication complex contains a different DNA polymerase for each strand, such as, for example, DNA polymerases ? and ? in eukaryotes. Here we have investigated this question in the bacterium Escherichia coli, in which the replicase (DNA polymerase III holoenzyme) contains two copies of the same polymerase (Pol III, the dnaE gene product), and hence the two strands are copied by the same polymerase. Our in vivo mutagenesis data indicate that the two DNA strands are not copied with the same accuracy, and that, remarkably, the lagging strand has the highest fidelity. We postulate that this effect results from the greater dissociative character of the lagging-strand polymerase, which provides additional options for error removal. Our conclusion is strongly supported by results with dnaE antimutator polymerases characterized by increased dissociation rates.

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Separable, Ctf4-mediated recruitment of DNA Polymerase ? for initiation of DNA synthesis at replication origins and lagging-strand priming during replication elongation.

Project description:During eukaryotic DNA replication, DNA polymerase alpha/primase (Pol ?) initiates synthesis on both the leading and lagging strands. It is unknown whether leading- and lagging-strand priming are mechanistically identical, and whether Pol ? associates processively or distributively with the replisome. Here, we titrate cellular levels of Pol ? in S. cerevisiae and analyze Okazaki fragments to study both replication initiation and ongoing lagging-strand synthesis in vivo. We observe that both Okazaki fragment initiation and the productive firing of replication origins are sensitive to Pol ? abundance, and that both processes are disrupted at similar Pol ? concentrations. When the replisome adaptor protein Ctf4 is absent or cannot interact with Pol ?, lagging-strand initiation is impaired at Pol ? concentrations that still support normal origin firing. Additionally, we observe that activation of the checkpoint becomes essential for viability upon severe depletion of Pol ?. Using strains in which the Pol ?-Ctf4 interaction is disrupted, we demonstrate that this checkpoint requirement is not solely caused by reduced lagging-strand priming. Our results suggest that Pol ? recruitment for replication initiation and ongoing lagging-strand priming are distinctly sensitive to the presence of Ctf4. We propose that the global changes we observe in Okazaki fragment length and origin firing efficiency are consistent with distributive association of Pol ? at the replication fork, at least when Pol ? is limiting.

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Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis.

Project description:In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesize in the opposite direction. By extending RNA primers, the lagging-strand polymerase restarts at short intervals and produces Okazaki fragments. At least in prokaryotic systems, this directionality problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. Here we use single-molecule techniques to visualize, in real time, the formation and release of replication loops by individual replisomes of bacteriophage T7 supporting coordinated DNA replication. Analysis of the distributions of loop sizes and lag times between loops reveals that initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release. The presence of two triggers may represent a fail-safe mechanism ensuring the timely reset of the replisome after the synthesis of every Okazaki fragment.

| S-EPMC2651468 | biostudies-literature

Rad53 checkpoint kinase couples leading and lagging strand DNA synthesis

Project description:In response to DNA replication stress, DNA replication checkpoint is activated to maintain fork stability, a process critical for maintenance of genome stability. However, how DNA replication checkpoint regulates replication forks remain elusive. Here we show that Rad53, a highly conserved replication checkpoint kinase, functions to couple leading and lagging strand DNA synthesis. In wild type cells under HU induced replication stress, synthesis of lagging strand, which contains ssDNA gaps, is comparable to leading strand DNA. In contrast, synthesis of lagging strand is much more than leading strand, and consequently, leading template ssDNA coated with ssDNA binding protein RPA was detected in rad53-1 mutant cells, suggesting that synthesis of leading strand and lagging strand DNA is uncoupled. Mechanistically, we show that replicative helicase MCM and leading strand DNA polymerase Pole move beyond actual DNA synthesis and that an increase in dNTP pools largely suppresses the uncoupled leading and lagging strand DNA synthesis. Our studies reveal an unexpected mechanism whereby Rad53 regulates replication fork stability.

2018-02-11 | GSE89721 | GEO

Genome-wide tracking of Escherichia coli DNA polymerase V to the lagging strand during the SOS response

Project description:Ribonucleotides are frequently incorporated into DNA and can be used as a marker of DNA replication enzymology. To investigate on a genome-wide scale, how E. coli pol V accesses undamaged chromosomal DNA during the SOS response, we mapped the location of ribonucleotides incorporated by steric gate variants of pol V across the entire E. coli genome. To do so, we used strains that are deficient in ribonucleotide excision repair (DrnhB), constitutively express all SOS-regulated genes [lexA(Def)] and constitutively “activated” RecA* (recA730). The strains also harbor two steric gate variants of E. coli pol V (Y11A or F10L), or a homolog of pol V, (pol VR391-Y13A). Ribonucleotides are frequently incorporated by the pol V-Y11A and pol VR391-Y13A variants, with a preference to the lagging strand. In contrast, the pol V-F10L variant incorporates less ribonucleotides and no strand preference was observed. Sharp transitions in strand specificity are observed at replication origin (oriC), while a gradient is observed at the termination region. To activate RecA* in a recA+ strain, we treated the strains with ciprofloxacin and genome-wide mapped the location of the incorporated ribonucleotides. Again, the polV-Y11A steric gate variant, exhibited a lagging strand preference. Our data is consistent with a specific role for pol V in lagging strand DNA synthesis across the entire E. coli genome during the SOS response.

2021-03-22 | GSE158570 | GEO

DNA polymerase ? stalls on telomeric lagging strand templates independently from G-quadruplex formation.

Project description:Previous evidence indicates that telomeres resemble common fragile sites and present a challenge for DNA replication. The precise impediments to replication fork progression at telomeric TTAGGG repeats are unknown, but are proposed to include G-quadruplexes (G4) on the G-rich strand. Here we examined DNA synthesis and progression by the replicative DNA polymerase ?/proliferating cell nuclear antigen/replication factor C complex on telomeric templates that mimic the leading C-rich and lagging G-rich strands. Increased polymerase stalling occurred on the G-rich template, compared with the C-rich and nontelomeric templates. Suppression of G4 formation by substituting Li(+) for K(+) as the cation, or by using templates with 7-deaza-G residues, did not alleviate Pol ? pause sites within the G residues. Furthermore, we provide evidence that G4 folding is less stable on single-stranded circular TTAGGG templates where ends are constrained, compared with linear oligonucleotides. Artificially stabilizing G4 structures on the circular templates with the G4 ligand BRACO-19 inhibited Pol ? progression into the G-rich repeats. Similar results were obtained for yeast and human Pol ? complexes. Our data indicate that G4 formation is not required for polymerase stalling on telomeric lagging strands and suggest that an alternative mechanism, in addition to stable G4s, contributes to replication stalling at telomeres.

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Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.

Project description:The integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase ? function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo.

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Replicative DNA polymerases promote active displacement of SSB proteins during lagging strand synthesis.

Project description:Genome replication induces the generation of large stretches of single-stranded DNA (ssDNA) intermediates that are rapidly protected by single-stranded DNA-binding (SSB) proteins. To date, the mechanism by which tightly bound SSBs are removed from ssDNA by the lagging strand DNA polymerase without compromising the advance of the replication fork remains unresolved. Here, we aimed to address this question by measuring, with optical tweezers, the real-time replication kinetics of the human mitochondrial and bacteriophage T7 DNA polymerases on free-ssDNA, in comparison with ssDNA covered with homologous and non-homologous SSBs under mechanical tension. We find important differences between the force dependencies of the instantaneous replication rates of each polymerase on different substrates. Modeling of the data supports a mechanism in which strong, specific polymerase-SSB interactions, up to ∼12 kBT, are required for the polymerase to dislodge SSB from the template without compromising its instantaneous replication rate, even under stress conditions that may affect SSB-DNA organization and/or polymerase-SSB communication. Upon interaction, the elimination of template secondary structure by SSB binding facilitates the maximum replication rate of the lagging strand polymerase. In contrast, in the absence of polymerase-SSB interactions, SSB poses an effective barrier for the advance of the polymerase, slowing down DNA synthesis.

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