scholarly journals Both DNA Polymerases δ and ε Contact Active and Stalled Replication Forks differently

2017 ◽  
Author(s):  
Chuanhe Yu ◽  
Haiyun Gan ◽  
Zhiguo Zhang
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Genome Duplication ◽  
Dna Polymerases ◽  
Replication Forks ◽  
Okazaki Fragments ◽  
Dna Replication Stress ◽  
Leading Strand ◽  
Stalled Replication Forks

AbstractThree DNA polymerases (Pol α, Pol δ, and Pol ε) are responsible for eukaryotic genome duplication. When DNA replication stress is encountered, DNA synthesis stalls until the stress is ameliorated. However, it is not known whether there is a difference in the association of each polymerase with active and stalled replication forks. Here, we show that each DNA polymerase has distinct patterns of association with active and stalled replication forks. Pol α is enriched at extending Okazaki fragments of active and stalled forks. In contrast, although Pol δ contacts the nascent lagging strands of active and stalled forks, it binds to only the matured (and not elongating) Okazaki fragments of stalled forks. Pol ε has a greater contact with the nascent ssDNA of leading strand on active forks compared with stalled forks. We propose that the configuration of DNA polymerases at stalled forks facilitate resumption of DNA synthesis after stress removal.

scholarly journals Both DNA Polymerases δ and ε Contact Active and Stalled Replication Forks Differently

2017 ◽  
Vol 37 (21) ◽  
Author(s):  
Chuanhe Yu ◽  
Haiyun Gan ◽  
Zhiguo Zhang
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Genome Duplication ◽  
Dna Polymerases ◽  
Distinct Pattern ◽  
Replication Forks ◽  
Okazaki Fragments ◽  
Dna Replication Stress ◽  
Leading Strand ◽  
Stalled Replication Forks

ABSTRACT Three DNA polymerases, polymerases α, δ, and ε (Pol α, Pol δ, and Pol ε), are responsible for eukaryotic genome duplication. When DNA replication stress is encountered, DNA synthesis stalls until the stress is ameliorated. However, it is not known whether there is a difference in the association of each polymerase with active and stalled replication forks. Here, we show that each DNA polymerase has a distinct pattern of association with active and stalled replication forks. Pol α is enriched at extending Okazaki fragments of active and stalled forks. In contrast, although Pol δ contacts the nascent lagging strands of active and stalled forks, it binds to only the matured (and not elongating) Okazaki fragments of stalled forks. Pol ε has greater contact with the nascent single-stranded DNA (ssDNA) of the leading strand on active forks than on stalled forks. We propose that the configuration of DNA polymerases at stalled forks facilitates the resumption of DNA synthesis after stress removal.

scholarly journals DNA polymerase κ-dependent DNA synthesis at stalled replication forks is important for CHK1 activation

2013 ◽  
Vol 32 (15) ◽  
pp. 2172-2185 ◽  
Author(s):  
Rémy Bétous ◽  
Marie-Jeanne Pillaire ◽  
Laura Pierini ◽  
Siem van der Laan ◽  
Bénédicte Recolin ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Replication Forks ◽  
Stalled Replication Forks

scholarly journals Phosphorylation of PNKP mediated by CDKs promotes end-processing of Okazaki fragments during DNA replication

2021 ◽  
Author(s):  
Kaima Tsukada ◽  
Rikiya Imamura ◽  
Kotaro Saikawa ◽  
Mizuki Saito ◽  
Naoya Kase ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Dna Replication ◽  
Dna Synthesis ◽  
High Speed ◽  
Replication Forks ◽  
Cyclin Dependent Kinases ◽  
Okazaki Fragments ◽  
Dna Ligation ◽  
Polynucleotide Kinase ◽  
Dna Ends

Polynucleotide kinase phosphatase (PNKP) has enzymatic activities as 3′ phosphatase and 5′ kinase of DNA ends to promote DNA ligation. Here, we show that PNKP is involved in progression of DNA replication through end-processing of Okazaki fragments (OFs). Cyclin-dependent kinases (CDKs) regulate phosphorylation on threonine 118 (T118) of PNKP, and which phosphorylation allows it to be recruited to OFs. Loss of PNKP and T118 phosphorylation significantly increased unligated OFs and high-speed DNA synthesis in replication forks, suggesting that PNKP T118 phosphorylation is required for OFs ligation for its maturation. Furthermore, phosphatase-dead PNKP also exhibited an accumulation of unligated OFs and high-speed DNA synthesis. Overall, our data suggested that CDK-mediated PNKP phosphorylation at T118 is important for its recruitment to OFs and PNKP subsequently promotes end-processing for OFs maturation for stable cell proliferation.

scholarly journals DNA repair factor RAD18 and DNA polymerase Polκ confer tolerance of oncogenic DNA replication stress

2017 ◽  
Vol 216 (10) ◽  
pp. 3097-3115 ◽  
Author(s):  
Yang Yang ◽  
Yanzhe Gao ◽  
Liz Mutter-Rottmayer ◽  
Anastasia Zlatanou ◽  
Michael Durando ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Replication Stress ◽  
Neoplastic Cells ◽  
Oncogenic Ras ◽  
Signaling Axis ◽  
Dna Replication Stress ◽  
Dna Repair Protein

The mechanisms by which neoplastic cells tolerate oncogene-induced DNA replication stress are poorly understood. Cyclin-dependent kinase 2 (CDK2) is a major mediator of oncogenic DNA replication stress. In this study, we show that CDK2-inducing stimuli (including Cyclin E overexpression, oncogenic RAS, and WEE1 inhibition) activate the DNA repair protein RAD18. CDK2-induced RAD18 activation required initiation of DNA synthesis and was repressed by p53. RAD18 and its effector, DNA polymerase κ (Polκ), sustained ongoing DNA synthesis in cells harboring elevated CDK2 activity. RAD18-deficient cells aberrantly accumulated single-stranded DNA (ssDNA) after CDK2 activation. In RAD18-depleted cells, the G2/M checkpoint was necessary to prevent mitotic entry with persistent ssDNA. Rad18−/− and Polκ−/− cells were highly sensitive to the WEE1 inhibitor MK-1775 (which simultaneously activates CDK2 and abrogates the G2/M checkpoint). Collectively, our results show that the RAD18–Polκ signaling axis allows tolerance of CDK2-mediated oncogenic stress and may allow neoplastic cells to breach tumorigenic barriers.

scholarly journals DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (1) ◽  
pp. 496-505 ◽  
Author(s):  
M E Budd ◽  
J L Campbell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Nonpermissive Temperature ◽  
Dna Polymerase Delta ◽  
Dna Polymerase Epsilon ◽  
Dna Polymerase Alpha ◽  
Polymerase Epsilon

Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.

scholarly journals DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (1) ◽  
pp. 496-505
Author(s):  
M E Budd ◽  
J L Campbell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Nonpermissive Temperature ◽  
Dna Polymerase Delta ◽  
Dna Polymerase Epsilon ◽  
Dna Polymerase Alpha ◽  
Polymerase Epsilon

Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.

scholarly journals Alternative Mechanisms for Coordinating Polymerase α and MCM Helicase

2009 ◽  
Vol 30 (2) ◽  
pp. 423-435 ◽  
Author(s):  
Chanmi Lee ◽  
Ivan Liachko ◽  
Roxane Bouten ◽  
Zvi Kelman ◽  
Bik K. Tye
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Molecular Motors ◽  
Replication Fork ◽  
Dna Polymerases ◽  
Physical Stability ◽  
Dependent Manner ◽  
Replication Forks ◽  
Mcm Helicase ◽  
The Stability

ABSTRACT Functional coordination between DNA replication helicases and DNA polymerases at replication forks, achieved through physical linkages, has been demonstrated in prokaryotes but not in eukaryotes. In Saccharomyces cerevisiae, we showed that mutations that compromise the activity of the MCM helicase enhance the physical stability of DNA polymerase α in the absence of their presumed linker, Mcm10. Mcm10 is an essential DNA replication protein implicated in the stable assembly of the replisome by virtue of its interaction with the MCM2-7 helicase and Polα. Dominant mcm2 suppressors of mcm10 mutants restore viability by restoring the stability of Polα without restoring the stability of Mcm10, in a Mec1-dependent manner. In this process, the single-stranded DNA accumulation observed in the mcm10 mutant is suppressed. The activities of key checkpoint regulators known to be important for replication fork stabilization contribute to the efficiency of suppression. These results suggest that Mcm10 plays two important roles as a linker of the MCM helicase and Polα at the elongating replication fork—first, to coordinate the activities of these two molecular motors, and second, to ensure their physical stability and the integrity of the replication fork.

A mechanism for Rad53 to couple leading- and lagging-strand DNA synthesis under replication stress in budding yeast

2021 ◽  
Vol 118 (38) ◽  
pp. e2109334118
Author(s):  
Albert Serra-Cardona ◽  
Chuanhe Yu ◽  
Xinmin Zhang ◽  
Xu Hua ◽  
Yuan Yao ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Replication Stress ◽  
Replication Checkpoint ◽  
Checkpoint Pathway ◽  
Dna Replication Checkpoint ◽  
Dna Replication Stress ◽  
Leading Strand ◽  
Mutant Cells ◽  
Lagging Strand

In response to DNA replication stress, DNA replication checkpoint kinase Mec1 phosphorylates Mrc1, which in turn activates Rad53 to prevent the generation of deleterious single-stranded DNA, a process that remains poorly understood. We previously reported that lagging-strand DNA synthesis proceeds farther than leading strand in rad53-1 mutant cells defective in replication checkpoint under replication stress, resulting in the exposure of long stretches of the leading-strand templates. Here, we show that asymmetric DNA synthesis is also observed in mec1-100 and mrc1-AQ cells defective in replication checkpoint but, surprisingly, not in mrc1∆ cells in which both DNA replication and checkpoint functions of Mrc1 are missing. Furthermore, depletion of either Mrc1 or its partner, Tof1, suppresses the asymmetric DNA synthesis in rad53-1 mutant cells. Thus, the DNA replication checkpoint pathway couples leading- and lagging-strand DNA synthesis by attenuating the replication function of Mrc1-Tof1 under replication stress.

scholarly journals A mechanism for preventing asymmetric histone segregation onto replicating DNA strands

Science ◽  
2018 ◽  
Vol 361 (6409) ◽  
pp. 1386-1389 ◽  
Author(s):  
Chuanhe Yu ◽  
Haiyun Gan ◽  
Albert Serra-Cardona ◽  
Lin Zhang ◽  
Songlin Gan ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Histone H3 ◽  
Epigenetic Inheritance ◽  
Yeast Cells ◽  
Replication Forks ◽  
Dna Strands ◽  
Leading Strand ◽  
Lagging Strand

How parental histone (H3-H4)2 tetramers, the primary carriers of epigenetic modifications, are transferred onto leading and lagging strands of DNA replication forks for epigenetic inheritance remains elusive. Here we show that parental (H3-H4)2 tetramers are assembled into nucleosomes onto both leading and lagging strands, with a slight preference for lagging strands. The lagging-strand preference increases markedly in budding yeast cells lacking Dpb3 and Dpb4, two subunits of the leading strand DNA polymerase, Pol ε, owing to the impairment of parental (H3-H4)2 transfer to leading strands. Dpb3-Dpb4 binds H3-H4 in vitro and participates in the inheritance of heterochromatin. These results indicate that different proteins facilitate the transfer of parental (H3-H4)2 onto leading versus lagging strands and that Dbp3-Dpb4 plays an important role in this poorly understood process.

scholarly journals Genome Instability Induced by Low Levels of Replicative DNA Polymerases in Yeast

Genes ◽  
2018 ◽  
Vol 9 (11) ◽  
pp. 539 ◽  
Author(s):  
Dao-Qiong Zheng ◽  
Thomas Petes
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Genome Instability ◽  
Dna Polymerases ◽  
Replication Stress ◽  
Yeast Strains ◽  
Different Types ◽  
Dna Replication Stress ◽  
Low Levels ◽  
Very High

Most cells of solid tumors have very high levels of genome instability of several different types, including deletions, duplications, translocations, and aneuploidy. Much of this instability appears induced by DNA replication stress. As a model for understanding this type of instability, we have examined genome instability in yeast strains that have low levels of two of the replicative DNA polymerases: DNA polymerase α and DNA polymerase δ (Polα and Polδ). We show that low levels of either of these DNA polymerases results in greatly elevated levels of mitotic recombination, chromosome rearrangements, and deletions/duplications. The spectrum of events in the two types of strains, however, differs in a variety of ways. For example, a reduced level of Polδ elevates single-base alterations and small deletions considerably more than a reduced level of Polα. In this review, we will summarize the methods used to monitor genome instability in yeast, and how this analysis contributes to understanding the linkage between genome instability and DNA replication stress.

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