Journal article
Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins
Proceedings of the National Academy of Sciences - PNAS, Vol.113(9), pp.E1170-E1179
03/01/2016
DOI: 10.1073/pnas.1516674113
PMCID: PMC4780606
PMID: 26884156
Abstract
Exonuclease 1 (Exo1) is a 5'→3' exonuclease and 5'-flap endonuclease that plays a critical role in multiple eukaryotic DNA repair pathways. Exo1 processing at DNA nicks and double-strand breaks creates long stretches of single-stranded DNA, which are rapidly bound by replication protein A (RPA) and other single-stranded DNA binding proteins (SSBs). Here, we use single-molecule fluorescence imaging and quantitative cell biology approaches to reveal the interplay between Exo1 and SSBs. Both human and yeast Exo1 are processive nucleases on their own. RPA rapidly strips Exo1 from DNA, and this activity is dependent on at least three RPA-encoded single-stranded DNA binding domains. Furthermore, we show that ablation of RPA in human cells increases Exo1 recruitment to damage sites. In contrast, the sensor of single-stranded DNA complex 1-a recently identified human SSB that promotes DNA resection during homologous recombination-supports processive resection by Exo1. Although RPA rapidly turns over Exo1, multiple cycles of nuclease rebinding at the same DNA site can still support limited DNA processing. These results reveal the role of single-stranded DNA binding proteins in controlling Exo1-catalyzed resection with implications for how Exo1 is regulated during DNA repair in eukaryotic cells.
Details
- Title: Subtitle
- Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins
- Creators
- Logan R Myler - Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712; Howard Hughes Medical Institute, The University of Texas at Austin, Austin, TX 78712; Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX 78712Ignacio F Gallardo - Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712; Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX 78712Yi Zhou - Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712; Howard Hughes Medical Institute, The University of Texas at Austin, Austin, TX 78712Fade Gong - Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712Soo-Hyun Yang - Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712; Howard Hughes Medical Institute, The University of Texas at Austin, Austin, TX 78712Marc S Wold - Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242Kyle M Miller - Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712Tanya T Paull - Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712; Howard Hughes Medical Institute, The University of Texas at Austin, Austin, TX 78712; tpaull@utexas.edu ifinkelstein@cm.utexas.eduIlya J Finkelstein - Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712; Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX 78712; tpaull@utexas.edu ifinkelstein@cm.utexas.edu
- Resource Type
- Journal article
- Publication Details
- Proceedings of the National Academy of Sciences - PNAS, Vol.113(9), pp.E1170-E1179
- DOI
- 10.1073/pnas.1516674113
- PMID
- 26884156
- PMCID
- PMC4780606
- NLM abbreviation
- Proc Natl Acad Sci U S A
- ISSN
- 0027-8424
- eISSN
- 1091-6490
- Publisher
- National Academy of Sciences; United States
- Grant note
- R01 GM120554 / NIGMS NIH HHS R00 GM097177 / NIGMS NIH HHS GM097177 / NIGMS NIH HHS K99 GM097177 / NIGMS NIH HHS
- Language
- English
- Date published
- 03/01/2016
- Academic Unit
- Radiation Oncology; Biochemistry and Molecular Biology
- Record Identifier
- 9984024420802771
Metrics
12 Record Views