Visualization of recombination-mediated damage bypass by template switching

A combination of two-dimensional gel electrophoresis and EM is used to isolate and characterize multiple template-switching intermediates generated in budding yeast during replication of damaged DNA. Template switching (TS) mediates damage bypass via a recombination-related mechanism involving PCNA...

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Vydané v:	Nature structural & molecular biology Ročník 21; číslo 10; s. 884 - 892
Hlavní autori:	Giannattasio, Michele, Zwicky, Katharina, Follonier, Cindy, Foiani, Marco, Lopes, Massimo, Branzei, Dana
Médium:	Journal Article
Jazyk:	English
Vydavateľské údaje:	New York Nature Publishing Group US 01.10.2014 Nature Publishing Group
Predmet:	631/114 631/1647/2210 631/337/100 631/337/572 Biochemistry Biological Microscopy Brewer's yeast Cell division Chromatids Deoxyribonucleic acid DNA DNA Damage DNA Polymerase III - genetics DNA Repair DNA Replication - genetics DNA, Cruciform DNA, Fungal - biosynthesis DNA, Fungal - genetics Electrophoresis Genetic aspects Genetic recombination Genetic research Genetics Life Sciences Membrane Biology Molecular biology Proliferating Cell Nuclear Antigen - metabolism Properties Protein Structure Quantitative trait loci Recombination, Genetic Saccharomyces cerevisiae Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae Proteins - genetics Templates, Genetic Toxicity Ubiquitination Yeast
ISSN:	1545-9993, 1545-9985, 1545-9985
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Shrnutí:	A combination of two-dimensional gel electrophoresis and EM is used to isolate and characterize multiple template-switching intermediates generated in budding yeast during replication of damaged DNA. Template switching (TS) mediates damage bypass via a recombination-related mechanism involving PCNA polyubiquitination and polymerase δ–dependent DNA synthesis. Using two-dimensional gel electrophoresis and EM, here we characterize TS intermediates arising in Saccharomyces cerevisiae at a defined chromosome locus, identifying five major families of intermediates. Single-stranded DNA gaps of 150–200 nt, and not DNA ends, initiate TS by strand invasion. This causes reannealing of the parental strands and exposure of the nondamaged newly synthesized chromatid, which serves as a replication template for the other blocked nascent strand. Structures resembling double Holliday junctions, postulated to be central double-strand break–repair intermediates but so far visualized only in meiosis, mediate late stages of TS before being processed to hemicatenanes. Our results reveal the DNA transitions accounting for recombination-mediated DNA-damage tolerance in mitotic cells and replication under conditions of genotoxic stress.
Bibliografia:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 AUTHOR CONTRIBUTIONS M.G. designed and executed the experiments, acquired the EM images, analyzed the data, and made the figures. K.Z. and C.F. acquired a subset of EM images and helped with EM data analysis. M.F. conceived the project and discussed the results. M.L. conceived the project, supervised the EM part, analyzed the EM data, and commented on the manuscript. D.B. conceived and supervised the project, designed the experiments, analyzed the data, and wrote the paper. Present address: Department of Molecular Biology, Princeton University, NJ, USA
ISSN:	1545-9993 1545-9985 1545-9985
DOI:	10.1038/nsmb.2888