Elena I Stepchenkova, Anna S Zhuk, Jian Cui, Elena R Tarakhovskaya, Stephanie R Barbari, Polina V Shcherbakova, Dmitrii E Polev, Roman Fedorov, Eugenia Poliakov, Igor B Rogozin, Artem G Lada, Youri I Pavlov
{"title":"CDC28正选择突变补偿酵母中DNA聚合酶ε催化活性的缺失。","authors":"Elena I Stepchenkova, Anna S Zhuk, Jian Cui, Elena R Tarakhovskaya, Stephanie R Barbari, Polina V Shcherbakova, Dmitrii E Polev, Roman Fedorov, Eugenia Poliakov, Igor B Rogozin, Artem G Lada, Youri I Pavlov","doi":"10.1093/genetics/iyab060","DOIUrl":null,"url":null,"abstract":"<p><p>Current eukaryotic replication models postulate that leading and lagging DNA strands are replicated predominantly by dedicated DNA polymerases. The catalytic subunit of the leading strand DNA polymerase ε, Pol2, consists of two halves made of two different ancestral B-family DNA polymerases. Counterintuitively, the catalytically active N-terminal half is dispensable, while the inactive C-terminal part is required for viability. Despite extensive studies of yeast Saccharomyces cerevisiae strains lacking the active N-terminal half, it is still unclear how these strains survive and recover. We designed a robust method for constructing mutants with only the C-terminal part of Pol2. Strains without the active polymerase part show severe growth defects, sensitivity to replication inhibitors, chromosomal instability, and elevated spontaneous mutagenesis. Intriguingly, the slow-growing mutant strains rapidly accumulate fast-growing clones. Analysis of genomic DNA sequences of these clones revealed that the adaptation to the loss of the catalytic N-terminal part of Pol2 occurs by a positive selection of mutants with improved growth. Elevated mutation rates help generate sufficient numbers of these variants. Single nucleotide changes in the cell cycle-dependent kinase gene, CDC28, improve the growth of strains lacking the N-terminal part of Pol2, and rescue their sensitivity to replication inhibitors and, in parallel, lower mutation rates. Our study predicts that changes in mammalian homologs of cyclin-dependent kinases may contribute to cellular responses to the leading strand polymerase defects.</p>","PeriodicalId":12706,"journal":{"name":"Genetics","volume":"218 2","pages":""},"PeriodicalIF":3.3000,"publicationDate":"2021-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/genetics/iyab060","citationCount":"0","resultStr":"{\"title\":\"Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations in CDC28.\",\"authors\":\"Elena I Stepchenkova, Anna S Zhuk, Jian Cui, Elena R Tarakhovskaya, Stephanie R Barbari, Polina V Shcherbakova, Dmitrii E Polev, Roman Fedorov, Eugenia Poliakov, Igor B Rogozin, Artem G Lada, Youri I Pavlov\",\"doi\":\"10.1093/genetics/iyab060\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Current eukaryotic replication models postulate that leading and lagging DNA strands are replicated predominantly by dedicated DNA polymerases. The catalytic subunit of the leading strand DNA polymerase ε, Pol2, consists of two halves made of two different ancestral B-family DNA polymerases. Counterintuitively, the catalytically active N-terminal half is dispensable, while the inactive C-terminal part is required for viability. Despite extensive studies of yeast Saccharomyces cerevisiae strains lacking the active N-terminal half, it is still unclear how these strains survive and recover. We designed a robust method for constructing mutants with only the C-terminal part of Pol2. Strains without the active polymerase part show severe growth defects, sensitivity to replication inhibitors, chromosomal instability, and elevated spontaneous mutagenesis. Intriguingly, the slow-growing mutant strains rapidly accumulate fast-growing clones. Analysis of genomic DNA sequences of these clones revealed that the adaptation to the loss of the catalytic N-terminal part of Pol2 occurs by a positive selection of mutants with improved growth. Elevated mutation rates help generate sufficient numbers of these variants. Single nucleotide changes in the cell cycle-dependent kinase gene, CDC28, improve the growth of strains lacking the N-terminal part of Pol2, and rescue their sensitivity to replication inhibitors and, in parallel, lower mutation rates. 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Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations in CDC28.
Current eukaryotic replication models postulate that leading and lagging DNA strands are replicated predominantly by dedicated DNA polymerases. The catalytic subunit of the leading strand DNA polymerase ε, Pol2, consists of two halves made of two different ancestral B-family DNA polymerases. Counterintuitively, the catalytically active N-terminal half is dispensable, while the inactive C-terminal part is required for viability. Despite extensive studies of yeast Saccharomyces cerevisiae strains lacking the active N-terminal half, it is still unclear how these strains survive and recover. We designed a robust method for constructing mutants with only the C-terminal part of Pol2. Strains without the active polymerase part show severe growth defects, sensitivity to replication inhibitors, chromosomal instability, and elevated spontaneous mutagenesis. Intriguingly, the slow-growing mutant strains rapidly accumulate fast-growing clones. Analysis of genomic DNA sequences of these clones revealed that the adaptation to the loss of the catalytic N-terminal part of Pol2 occurs by a positive selection of mutants with improved growth. Elevated mutation rates help generate sufficient numbers of these variants. Single nucleotide changes in the cell cycle-dependent kinase gene, CDC28, improve the growth of strains lacking the N-terminal part of Pol2, and rescue their sensitivity to replication inhibitors and, in parallel, lower mutation rates. Our study predicts that changes in mammalian homologs of cyclin-dependent kinases may contribute to cellular responses to the leading strand polymerase defects.
期刊介绍:
GENETICS is published by the Genetics Society of America, a scholarly society that seeks to deepen our understanding of the living world by advancing our understanding of genetics. Since 1916, GENETICS has published high-quality, original research presenting novel findings bearing on genetics and genomics. The journal publishes empirical studies of organisms ranging from microbes to humans, as well as theoretical work.
While it has an illustrious history, GENETICS has changed along with the communities it serves: it is not your mentor''s journal.
The editors make decisions quickly – in around 30 days – without sacrificing the excellence and scholarship for which the journal has long been known. GENETICS is a peer reviewed, peer-edited journal, with an international reach and increasing visibility and impact. All editorial decisions are made through collaboration of at least two editors who are practicing scientists.
GENETICS is constantly innovating: expanded types of content include Reviews, Commentary (current issues of interest to geneticists), Perspectives (historical), Primers (to introduce primary literature into the classroom), Toolbox Reviews, plus YeastBook, FlyBook, and WormBook (coming spring 2016). For particularly time-sensitive results, we publish Communications. As part of our mission to serve our communities, we''ve published thematic collections, including Genomic Selection, Multiparental Populations, Mouse Collaborative Cross, and the Genetics of Sex.