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Global analysis of double-strand break processing reveals in vivo properties of the helicase-nuclease complex AddAB

Badrinarayanan, Anjana and Le, Tung B. K. and Spille, Jan-Hendrik and Cisse, Ibrahim I. and Laub, Michael T. (2017) Global analysis of double-strand break processing reveals in vivo properties of the helicase-nuclease complex AddAB. PLOS Genetics, 13 (5). Art. No. e1006783. ISSN 1553-7404. PMCID PMC5443536. doi:10.1371/journal.pgen.1006783. https://resolver.caltech.edu/CaltechAUTHORS:20201112-143948193

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Abstract

In bacteria, double-strand break (DSB) repair via homologous recombination is thought to be initiated through the bi-directional degradation and resection of DNA ends by a helicase-nuclease complex such as AddAB. The activity of AddAB has been well-studied in vitro, with translocation speeds between 400–2000 bp/s on linear DNA suggesting that a large section of DNA around a break site is processed for repair. However, the translocation rate and activity of AddAB in vivo is not known, and how AddAB is regulated to prevent excessive DNA degradation around a break site is unclear. To examine the functions and mechanistic regulation of AddAB inside bacterial cells, we developed a next-generation sequencing-based approach to assay DNA processing after a site-specific DSB was introduced on the chromosome of Caulobacter crescentus. Using this assay we determined the in vivo rates of DSB processing by AddAB and found that putative chi sites attenuate processing in a RecA-dependent manner. This RecA-mediated regulation of AddAB prevents the excessive loss of DNA around a break site, limiting the effects of DSB processing on transcription. In sum, our results, taken together with prior studies, support a mechanism for regulating AddAB that couples two key events of DSB repair–the attenuation of DNA-end processing and the initiation of homology search by RecA–thereby helping to ensure that genomic integrity is maintained during DSB repair.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1371/journal.pgen.1006783DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5443536/PubMed CentralArticle
ORCID:
AuthorORCID
Badrinarayanan, Anjana0000-0001-5520-2134
Le, Tung B. K.0000-0003-4764-8851
Spille, Jan-Hendrik0000-0001-8493-4721
Cisse, Ibrahim I.0000-0002-8764-1809
Laub, Michael T.0000-0002-8288-7607
Additional Information:© 2017 Badrinarayanan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: October 10, 2016; Accepted: April 26, 2017; Published: May 10, 2017. Editor: Michael Lichten, National Cancer Institute, UNITED STATES. This work was supported by a NIH grant (5R01GM082899) to MTL, who is also an Investigator of the Howard Hughes Medical Institute, a Human Frontier Science Program postdoctoral fellowship to AB, a Gordon and Betty Moore Foundation postdoctoral fellowship of the Life Sciences Research Foundation to TBKL and an MIT RSC grant (2654055) to IIC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability: All relevant data are within the paper and its Supporting Information files. Sequencing data are available in GEO, GSE86913. The authors have declared that no competing interests exist. Author Contributions: Conceptualization: AB MTL. Data curation: AB TBKL JHS. Formal analysis: AB TBKL JHS MTL. Funding acquisition: AB TBKL IIC MTL. Investigation: AB TBKL JHS. Methodology: AB TBKL JHS IIC MTL. Project administration: MTL. Resources: AB TBKL JHS. Software: JHS. Supervision: MTL. Validation: AB TBKL JHS IIC MTL. Visualization: AB TBKL MTL. Writing ± original draft: AB MTL. Writing ± review & editing: AB TBKL MTL.
Funders:
Funding AgencyGrant Number
NIH5R01GM082899
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Human Frontier Science ProgramUNSPECIFIED
Gordon and Betty Moore FoundationUNSPECIFIED
Life Sciences Research FoundationUNSPECIFIED
Massachusetts Institute of Technology (MIT)2654055
Issue or Number:5
PubMed Central ID:PMC5443536
DOI:10.1371/journal.pgen.1006783
Record Number:CaltechAUTHORS:20201112-143948193
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20201112-143948193
Official Citation:Badrinarayanan A, Le TBK, Spille J-H, Cisse II, Laub MT (2017) Global analysis of double-strand break processing reveals in vivo properties of the helicase-nuclease complex AddAB. PLoS Genet 13(5): e1006783. https://doi.org/10.1371/journal.pgen.1006783
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:106650
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:16 Nov 2020 16:23
Last Modified:16 Nov 2021 18:55

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