Both High-Fidelity Replicative and Low-Fidelity Y-Family Polymerases Are Involved in DNA Rereplication

DNA rereplication is a major form of aberrant replication that causes genomic instabilities, such as gene amplification. However, little is known about which DNA polymerases are involved in the process. Here, we report that low-fidelity Y-family polymerases (Y-Pols), Pol η, Pol ι, Pol κ, and REV1, significantly contribute to DNA synthesis during rereplication, while the replicative polymerases, Pol δ and Pol ε, play an important role in rereplication, as expected. When rereplication was induced by depletion of geminin, these polymerases were recruited to rereplication sites in human cell lines. This finding was supported by RNA interference (RNAi)-mediated knockdown of the polymerases, which suppressed rereplication induced by geminin depletion. Interestingly, epistatic analysis indicated that Y-Pols collaborate in a common pathway, independently of replicative polymerases. We also provide evidence for a catalytic role for Pol η and the involvement of Pol η and Pol κ in cyclin E-induced rereplication. Collectively, our findings indicate that, unlike normal S-phase replication, rereplication induced by geminin depletion and oncogene activation requires significant contributions of both Y-Pols and replicative polymerases. These findings offer important mechanistic insights into cancer genomic instability.

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Effect of Proliferating Cell Nuclear Antigen Ubiquitination and Chromatin Structure on the Dynamic Properties of the Y-family DNA Polymerases

Molecular Biology of the Cell ◽

10.1091/mbc.e08-07-0724 ◽

2008 ◽

Vol 19 (12) ◽

pp. 5193-5202 ◽

Cited By ~ 64

Author(s):

Simone Sabbioneda ◽

Audrey M. Gourdin ◽

Catherine M. Green ◽

Angelika Zotter ◽

Giuseppina Giglia-Mari ◽

...

Keyword(s):

Chromatin Structure ◽

Proliferating Cell Nuclear Antigen ◽

Dynamic Properties ◽

Human Fibroblasts ◽

Dna Polymerases ◽

S Phase ◽

Nuclear Antigen ◽

Replication Foci ◽

Y Family ◽

Proliferating Cell

Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) η and ι are usually uniformly distributed through the nucleus but accumulate in replication foci during S phase. DNA-damaging treatments result in an increase in S phase cells containing polymerase foci. Using photobleaching techniques, we show that polη is highly mobile in human fibroblasts. Even when localized in replication foci, it is only transiently immobilized. Although ubiquitination of proliferating cell nuclear antigen (PCNA) is not required for the localization of polη in foci, it results in an increased residence time in foci. polι is even more mobile than polη, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both polη and polι diffuse through the cell but that they are transiently immobilized for ∼150 ms, with a larger proportion of polη than polι immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of polη but not polι. Our data are consistent with a model in which the polymerases are transiently probing the DNA/chromatin. When DNA is exposed at replication forks, the polymerase residence times increase, and this is further facilitated by the ubiquitination of PCNA.

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Mechanistic Comparison of High-Fidelity and Error-Prone DNA Polymerases and Ligases Involved in DNA Repair

ChemInform ◽

10.1002/chin.200621259 ◽

2006 ◽

Vol 37 (21) ◽

Author(s):

Alexander K. Showalter ◽

Brandon J. Lamarche ◽

Marina Bakhtina ◽

Mei-I Su ◽

Kuo-Hsiang Tang ◽

...

Keyword(s):

Dna Repair ◽

Dna Polymerases ◽

High Fidelity

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Elevated somatic mutation burdens in normal human cells due to defective DNA polymerases

10.1101/2020.06.23.167668 ◽

2020 ◽

Cited By ~ 2

Author(s):

Philip S. Robinson ◽

Tim H.H. Coorens ◽

Claire Palles ◽

Emily Mitchell ◽

Federico Abascal ◽

...

Keyword(s):

Somatic Mutation ◽

Normal Tissue ◽

Familial Cancer ◽

Dna Polymerases ◽

Cell Types ◽

Early Embryogenesis ◽

High Fidelity ◽

Mutational Signatures ◽

Exonuclease Domain ◽

Normal Human

ABSTRACTMutation accumulation over time in normal somatic cells contributes to cancer development and is proposed as a cause of ageing. DNA polymerases Pol ε and Pol δ replicate DNA with high fidelity during normal cell divisions. However, in some cancers defective proofreading due to acquired mutations in the exonuclease domains of POLE or POLD1 causes markedly elevated somatic mutation burdens with distinctive mutational signatures. POLE and POLD1 exonuclease domain mutations also cause familial cancer predisposition when inherited through the germline. Here, we sequenced normal tissue DNA from individuals with germline POLE or POLD1 exonuclease domain mutations. Increased mutation burdens with characteristic mutational signatures were found to varying extents in all normal adult somatic cell types examined, during early embryogenesis and in sperm. Mutation burdens were further markedly elevated in neoplasms from these individuals. Thus human physiology is able to tolerate ubiquitously elevated mutation burdens. Indeed, with the exception of early onset cancer, individuals with germline POLE and POLD1 exonuclease domain mutations are not reported to show abnormal phenotypic features, including those of premature ageing. The results, therefore, do not support a simple model in which all features of ageing are attributable to widespread cell malfunction directly resulting from somatic mutation burdens accrued during life.

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Hairpin structure facilitates high-fidelity DNA amplification reactions in both qPCR and high-throughput sequencing

10.1101/2021.11.10.21266179 ◽

2021 ◽

Author(s):

Kerou Zhang ◽

Alessandro Pinto ◽

Peng Dai ◽

Michael Wang ◽

Lauren Yuxuan Cheng ◽

...

Keyword(s):

Dna Sequences ◽

High Throughput Sequencing ◽

Limit Of Detection ◽

Dna Polymerases ◽

Dna Amplification ◽

Cost Effective ◽

High Fidelity ◽

Resistance Mutations ◽

Chain Reactions ◽

Early Cancer Diagnosis

Effective polymerase chain reactions (PCR) are important in bio-laboratories. It is essential to detect rare DNA-sequence variants for early cancer diagnosis or for drug-resistance mutations identification. Some of the common detection quantitative PCR (qPCR) methods are restricted in the limit of detection (LoD) because of the high polymerase misincorporation rate in Taq DNA polymerases. High-fidelity (HiFi) DNA polymerases have a 50- to 250-fold higher fidelity. Yet, there are currently no proper designs for multiplexed HiFi qPCR reactions. Moreover, the popularity of targeting highly multiplex DNA sequences requires minimizing PCR side products, as the potential of dimerization grows quadratically as the plexes of primers increases. Efforts tried before were either an add-on step, or technology-specific, or requiring high-level computing skills. There lacks an easy-to-apply and cost-effective method for dimerization reduction. Here, we presented the Occlusion System, composed of a 5'-overhanged primer and a probe with a short-stem hairpin. We demonstrated that it allowed multiplexing high-fidelity qPCR reaction, it was also compatible with the current variant-enrichment method to improve the LoD by 10-fold. Further, we found that the Occlusion System reduced the dimerization up to 10-fold in highly multiplexed PCR. Thus, the Occlusion System satisfactorily improved both qPCR sensitivity and PCR efficiency.

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