TY - JOUR
T1 - Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase
AU - Xia, Jun
AU - Chen, Li Tzu
AU - Mei, Qian
AU - Ma, Chien Hui
AU - Halliday, Jennifer A.
AU - Lin, Hsin Yu
AU - Magnan, David
AU - Pribis, John P.
AU - Fitzgerald, Devon M.
AU - Hamilton, Holly M.
AU - Richters, Megan
AU - Nehring, Ralf B.
AU - Shen, Xi
AU - Li, Lei
AU - Bates, David
AU - Hastings, P. J.
AU - Herman, Christophe
AU - Jayaram, Makkuni
AU - Rosenberg, Susan M.
N1 - Publisher Copyright:
© 2016 The Authors, some rights reserved.
PY - 2016/11
Y1 - 2016/11
N2 - DNA repair by homologous recombination (HR) underpins cell survival and fuels genome instability, cancer, and evolution. However, themain 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 orthologsmay act similarly in human cancers overexpressing the RecA ortholog RAD51 and find that cancer genome expression data implicate the orthologs BLMand RECQL4 in conjunctionwith EME1 and GEN1 as probable HJ reducers in such cancers. Our results support RecA-overproducing E. coli as amodel of themany 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.
AB - DNA repair by homologous recombination (HR) underpins cell survival and fuels genome instability, cancer, and evolution. However, themain 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 orthologsmay act similarly in human cancers overexpressing the RecA ortholog RAD51 and find that cancer genome expression data implicate the orthologs BLMand RECQL4 in conjunctionwith EME1 and GEN1 as probable HJ reducers in such cancers. Our results support RecA-overproducing E. coli as amodel of themany 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.
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U2 - 10.1126/sciadv.1601605
DO - 10.1126/sciadv.1601605
M3 - Article
C2 - 28090586
AN - SCOPUS:85021755738
SN - 2375-2548
VL - 2
JO - Science Advances
JF - Science Advances
IS - 11
M1 - e1601605
ER -