Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase
| Autor: | Lei Li, Chien-Hui Ma, Jennifer A. Halliday, John P. Pribis, Christophe Herman, Devon M. Fitzgerald, Holly M. Hamilton, Li-Tzu Chen, David Bates, Megan Richters, Xi Shen, Makkuni Jayaram, Hsin Yu Lin, Susan M. Rosenberg, Qian Mei, Jun Xia, P. J. Hastings, Ralf B. Nehring, David Magnan |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Genome instability DNA Bacterial RuvC DNA repair DNA damage RecQ helicase RAD51 homologous recombination Biology DNA replication spontaneous DNA damage holliday junctions 03 medical and health sciences Neoplasms Holliday junction Escherichia coli Genetics Humans cancer DNA Breaks Single-Stranded Research Articles Recombination Genetic DNA Cruciform Multidisciplinary RecQ Helicases RecQ family proteins SciAdv r-articles DNA Neoplasm Neoplasm Proteins 030104 developmental biology Rad51 Recombinase Homologous recombination BLM Research Article |
| Zdroj: | Science Advances |
| ISSN: | 2375-2548 |
| Popis: | Freeze-frame synthetic proteins trap DNA reaction intermediates in single live cells, revealing origins of genome instability. 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. |
| Databáze: | OpenAIRE |
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