RRC ID 53777
Author Xia J, Chen LT, Mei Q, Ma CH, Halliday JA, Lin HY, Magnan D, Pribis JP, Fitzgerald DM, Hamilton HM, Richters M, Nehring RB, Shen X, Li L, Bates D, Hastings PJ, Herman C, Jayaram M, Rosenberg SM.
Title Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase.
Journal Sci Adv
Abstract DNA repair by homologous recombination (HR) underpins cell survival and fuels genome instability, cancer, and evolution. However, the main kinds and sources of DNA damage repaired by HR in somatic cells and the roles of important HR proteins remain elusive. We present engineered proteins that trap, map, and quantify Holliday junctions (HJs), a central DNA intermediate in HR, based on catalytically deficient mutant RuvC protein of Escherichia coli. We use RuvCDefGFP (RDG) to map genomic footprints of HR at defined DNA breaks in E. coli and demonstrate genome-scale directionality of double-strand break (DSB) repair along the chromosome. Unexpectedly, most spontaneous HR-HJ foci are instigated, not by DSBs, but rather by single-stranded DNA damage generated by replication. We show that RecQ, the E. coli ortholog of five human cancer proteins, nonredundantly promotes HR-HJ formation in single cells and, in a novel junction-guardian role, also prevents apparent non-HR-HJs promoted by RecA overproduction. We propose that one or more human RecQ orthologs may act similarly in human cancers overexpressing the RecA ortholog RAD51 and find that cancer genome expression data implicate the orthologs BLM and RECQL4 in conjunction with EME1 and GEN1 as probable HJ reducers in such cancers. Our results support RecA-overproducing E. coli as a model of the many human tumors with up-regulated RAD51 and provide the first glimpses of important, previously elusive reaction intermediates in DNA replication and repair in single living cells.
Volume 2(11)
Pages e1601605
Published 2016-11-1
DOI 10.1126/sciadv.1601605
PII 1601605
PMID 28090586
PMC PMC5222578
MeSH DNA Breaks, Single-Stranded* DNA, Bacterial* / genetics DNA, Bacterial* / metabolism DNA, Cruciform* / genetics DNA, Cruciform* / metabolism DNA, Neoplasm / genetics DNA, Neoplasm / metabolism Escherichia coli* / genetics Escherichia coli* / metabolism Humans Neoplasm Proteins / genetics Neoplasm Proteins / metabolism Neoplasms / genetics Neoplasms / metabolism Rad51 Recombinase / genetics Rad51 Recombinase / metabolism RecQ Helicases* / genetics RecQ Helicases* / metabolism Recombination, Genetic*
IF 12.804
Times Cited 10
Resource
Prokaryotes E. coli