Faculty Opinions recommendation of Differential arrival of leading and lagging strand DNA polymerases at fission yeast telomeres.

AbstractTelomeres, the protective ends of eukaryotic chromosomes, are replicated through concerted actions by conventional DNA polymerases and telomerase, though the regulation of this process is not fully understood. Telomere replication requires (C)-Stn1-Ten1, a telomere ssDNA-binding complex that is homologous to RPA. Here, we show that the evolutionarily conserved phosphatase Ssu72 is responsible for terminating the cycle of telomere replication in fission yeast. Ssu72 controls the recruitment of Stn1 to telomeres by regulating Stn1 phosphorylation at S74, a residue that lies within the conserved OB fold domain. Consequently, ssu72Δ mutants are defective in telomere replication and exhibit long 3’ overhangs, which are indicative of defective lagging strand DNA synthesis. We also show that hSSU72 regulates telomerase activation in human cells by controlling the recruitment of hSTN1 to telomeres. Thus, in this study, we demonstrate a previously unknown yet conserved role for the phosphatase SSU72, whereby this enzyme controls telomere homeostasis by activating lagging strand DNA synthesis, thus terminating the cycle of telomere replication.

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3′ → 5′ Exonucleases of DNA Polymerases ϵ and δ Correct Base Analog Induced DNA Replication Errors on Opposite DNA Strands in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/142.3.717 ◽

1996 ◽

Vol 142 (3) ◽

pp. 717-726 ◽

Cited By ~ 11

Author(s):

Polina V Shcherbakova ◽

Youri I Pavlov

Keyword(s):

Dna Replication ◽

Dna Polymerases ◽

Replication Errors ◽

Dna Strands ◽

Base Analog ◽

Chromosome Iii ◽

Dna Strand ◽

Dna Replication Errors ◽

Yeast Mutants ◽

Lagging Strand

Abstract The base analog 6-N-hydroxylaminopurine (HAP) induces bidirectional GC → AT and AT → GC transitions that are enhanced in DNA polymerase ϵ and δ 3′ → 5′ exonuclease-deficient yeast mutants, pol2-4 and pol3-01, respectively. We have constructed a set of isogenic strains to determine whether the DNA polymerases δ and ϵ contribute equally to proofreading of replication errors provoked by HAP during leading and lagging strand DNA synthesis. Site-specific GC → AT and AT → GC transitions in a Pol→, pol2-4 or pol3-01 genetic background were scored as reversions of ura3 missense alleles. At each site, reversion was increased in only one proofreading-deficient mutant, either pol2-4 or pol3-01, depending on the DNA strand in which HAP incorporation presumably occurred. Measurement of the HAP-induced reversion frequency of the ura3 alleles placed into chromosome III near to the defined active replication origin ARS306 in two orientations indicated that DNA polymerases ϵ and δ correct HAP-induced DNA replication errors on opposite DNA strands.

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Mechanism of SSB Displacement by Replicative DNA Polymerases During Lagging Strand Synthesis

Biophysical Journal ◽

10.1016/j.bpj.2018.11.443 ◽

2019 ◽

Vol 116 (3) ◽

pp. 74a

Author(s):

Fernando Cerron ◽

Grzegorz L. Ciesielski ◽

Laurie S. Kaguni ◽

Francisco J. Cao ◽

Borja Ibarra

Keyword(s):

Dna Polymerases ◽

Lagging Strand

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Analysis of APOBEC-induced mutations in yeast strains with low levels of replicative DNA polymerases

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922472117 ◽

2020 ◽

Vol 117 (17) ◽

pp. 9440-9450 ◽

Cited By ~ 6

Author(s):

Yang Sui ◽

Lei Qi ◽

Ke Zhang ◽

Natalie Saini ◽

Leszek J. Klimczak ◽

...

Keyword(s):

Dna Polymerase ◽

Dna Polymerases ◽

Induced Mutations ◽

Single Stranded Dna ◽

Dna Polymerase Epsilon ◽

Dna Polymerase Alpha ◽

Yeast Strains ◽

Polymerase Epsilon ◽

Low Levels ◽

Lagging Strand

Yeast strains with low levels of the replicative DNA polymerases (alpha, delta, and epsilon) have high levels of chromosome deletions, duplications, and translocations. By examining the patterns of mutations induced in strains with low levels of DNA polymerase by the human protein APOBEC3B (a protein that deaminates cytosine in single-stranded DNA), we show dramatically elevated amounts of single-stranded DNA relative to a wild-type strain. During DNA replication, one strand (defined as the leading strand) is replicated processively by DNA polymerase epsilon and the other (the lagging strand) is replicated as short fragments initiated by DNA polymerase alpha and extended by DNA polymerase delta. In the low DNA polymerase alpha and delta strains, the APOBEC-induced mutations are concentrated on the lagging-strand template, whereas in the low DNA polymerase epsilon strain, mutations occur on the leading- and lagging-strand templates with similar frequencies. In addition, for most genes, the transcribed strand is mutagenized more frequently than the nontranscribed strand. Lastly, some of the APOBEC-induced clusters in strains with low levels of DNA polymerase alpha or delta are greater than 10 kb in length.

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Genetics of lagging strand DNA synthesis and maturation in fission yeast: suppression analysis links the Dna2-Cdc24 complex to DNA polymerase

Nucleic Acids Research ◽

10.1093/nar/gkh963 ◽

2004 ◽

Vol 32 (21) ◽

pp. 6367-6377 ◽

Cited By ~ 12

Author(s):

H. Tanaka

Keyword(s):

Dna Synthesis ◽

Fission Yeast ◽

Dna Polymerase ◽

Lagging Strand

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The intra-S phase checkpoint directly regulates replication elongation to preserve the integrity of stalled replisomes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2019183118 ◽

2021 ◽

Vol 118 (24) ◽

pp. e2019183118

Author(s):

Yang Liu ◽

Lu Wang ◽

Xin Xu ◽

Yue Yuan ◽

Bo Zhang ◽

...

Keyword(s):

Fission Yeast ◽

Dna Polymerases ◽

Replication Stress ◽

Underlying Mechanism ◽

S Phase ◽

Replication Forks ◽

Helicase Activity ◽

Replicative Helicase ◽

Checkpoint Kinases ◽

S Phase Checkpoint

DNA replication is dramatically slowed down under replication stress. The regulation of replication speed is a conserved response in eukaryotes and, in fission yeast, requires the checkpoint kinases Rad3ATR and Cds1Chk2. However, the underlying mechanism of this checkpoint regulation remains unresolved. Here, we report that the Rad3ATR-Cds1Chk2 checkpoint directly targets the Cdc45-MCM-GINS (CMG) replicative helicase under replication stress. When replication forks stall, the Cds1Chk2 kinase directly phosphorylates Cdc45 on the S275, S322, and S397 residues, which significantly reduces CMG helicase activity. Furthermore, in cds1Chk2-mutated cells, the CMG helicase and DNA polymerases are physically separated, potentially disrupting replisomes and collapsing replication forks. This study demonstrates that the intra-S phase checkpoint directly regulates replication elongation, reduces CMG helicase processivity, prevents CMG helicase delinking from DNA polymerases, and therefore helps preserve the integrity of stalled replisomes and replication forks.

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Ribonucleotide incorporation characteristics around yeast autonomously replicating sequences reveal the labor division of replicative DNA polymerases

10.1101/2020.08.27.270728 ◽

2020 ◽

Author(s):

Penghao Xu ◽

Francesca Storici

Keyword(s):

Dna Polymerase ◽

Genome Instability ◽

Dna Polymerases ◽

Yeast Cells ◽

Yeast Genome ◽

Wild Type ◽

Specific Sequence ◽

Leading Strand ◽

Mapping Techniques ◽

Lagging Strand

ABSTRACTRibonucleoside monophosphate (rNMP) incorporation in DNA is a natural and prominent phenomenon resulting in DNA structural change and genome instability. While DNA polymerases have different rNMP incorporation rates, little is known whether these enzymes incorporate rNMPs following specific sequence patterns. In this study, we analyzed a series of rNMP incorporation datasets, generated from three rNMP mapping techniques, and obtained from Saccharomyces cerevisiae cells expressing wild-type or mutant replicative DNA polymerase and ribonuclease H2 genes. We performed computational analyses of rNMP sites around early and late firing autonomously replicating sequences (ARS’s) of the yeast genome, from which bidirectional, leading and lagging DNA synthesis starts. We find the preference of rNMP incorporation on the leading strand in wild-type DNA polymerase yeast cells. The leading/lagging-strand ratio of rNMP incorporation changes dramatically within 500 nt from ARS’s, highlighting the Pol δ - Pol ε handoff during early leading-strand synthesis. Furthermore, the pattern of rNMP incorporation is markedly distinct between the leading the lagging strand. Overall, our results show the different counts and patterns of rNMP incorporation during DNA replication from ARS, which reflects the different labor of division and rNMP incorporation pattern of Pol δ and Pol ε.

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No support for the adaptive hypothesis of lagging-strand encoding in bacterial genomes

10.1101/2020.01.14.906818 ◽

2020 ◽

Cited By ~ 1

Author(s):

Haoxuan Liu ◽

Jianzhi Zhang

Keyword(s):

Dna Replication ◽

Comparative Genomics ◽

Dna Polymerases ◽

Purifying Selection ◽

Deleterious Mutations ◽

Bacterial Genomes ◽

Highly Expressed Genes ◽

Balance Hypothesis ◽

Leading Strand ◽

Lagging Strand

ABSTRACTGenes are preferentially encoded on the leading instead of the lagging strand of DNA replication in most bacterial genomes1. This bias likely results from selection against lagging-strand encoding, which can cause head-on collisions between DNA polymerases and RNA polymerases that induce transcriptional abortion, replication delay, and possibly mutagenesis1. But there are still genes encoded on the lagging strand, an observation that has been explained by a balance between deleterious mutations bringing genes from the leading to the lagging strand and purifying selection purging such mutations2. This mutation-selection balance hypothesis predicts that the probability that a gene is encoded on the lagging strand decreases with the detriment of its lagging-strand encoding relative to leading-strand encoding, explaining why highly expressed genes and essential genes are underrepresented on the lagging strand3,4. In a recent study, Merrikh and Merrikh proposed that the observed lagging-strand encoding is adaptive instead of detrimental, due to beneficial mutations brought by the potentially increased mutagenesis resulting from head-on collisions5. They reported empirical observations from comparative genomics that were purported to support their hypothesis5. Here we point out methodological flaws and errors in their analyses and logical problems of their interpretation. Our reanalysis of their data finds no evidence for the adaptive hypothesis.

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DNA Replication Through Strand Displacement During Lagging Strand DNA Synthesis in Saccharomyces cerevisiae

Genes ◽

10.3390/genes10020167 ◽

2019 ◽

Vol 10 (2) ◽

pp. 167 ◽

Cited By ~ 5

Author(s):

Michele Giannattasio ◽

Dana Branzei

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Dna Synthesis ◽

Dna Polymerases ◽

Experimental Results ◽

Genome Integrity ◽

Strand Displacement ◽

Viral Dna ◽

Okazaki Fragment Processing ◽

Lagging Strand

This review discusses a set of experimental results that support the existence of extended strand displacement events during budding yeast lagging strand DNA synthesis. Starting from introducing the mechanisms and factors involved in leading and lagging strand DNA synthesis and some aspects of the architecture of the eukaryotic replisome, we discuss studies on bacterial, bacteriophage and viral DNA polymerases with potent strand displacement activities. We describe proposed pathways of Okazaki fragment processing via short and long flaps, with a focus on experimental results obtained in Saccharomyces cerevisiae that suggest the existence of frequent and extended strand displacement events during eukaryotic lagging strand DNA synthesis, and comment on their implications for genome integrity.

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