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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Bibliographic Details
Published in:Nature structural & molecular biology Vol. 21; no. 10; pp. 884 - 892
Main Authors: Giannattasio, Michele, Zwicky, Katharina, Follonier, Cindy, Foiani, Marco, Lopes, Massimo, Branzei, Dana
Format: Journal Article
Language:English
Published: New York Nature Publishing Group US 01.10.2014
Nature Publishing Group
Subjects:
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
Online Access:Get full text
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Summary: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.
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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