title: Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins.
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.
authors.affiliation: 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 78712;
authors.forename: Logan R
authors.identifier: http://orcid.org/0000-0003-0673-7049
authors.initials: LR
authors.lastname: Myler
authors.affiliation: 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;
authors.forename: Ignacio F
authors.initials: IF
authors.lastname: Gallardo
authors.affiliation: 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;
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712;
authors.forename: Fade
authors.initials: F
authors.lastname: Gong
authors.affiliation: 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;
authors.forename: Soo-Hyun
authors.initials: SH
authors.lastname: Yang
authors.affiliation: Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242.
authors.forename: Marc S
authors.initials: MS
authors.lastname: Wold
authors.affiliation: Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712;
authors.forename: Kyle M
authors.initials: KM
authors.lastname: Miller
authors.affiliation: 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.edu.
authors.forename: Tanya T
authors.initials: TT
authors.lastname: Paull
authors.affiliation: 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.
authors.forename: Ilya J
authors.identifier: http://orcid.org/0000-0002-9371-2431
authors.initials: IJ
authors.lastname: Finkelstein
mesh_terms: D055162:Biocatalysis; D004249:DNA Damage; D045643:DNA Repair Enzymes / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000502:physiology*; D005090:Exodeoxyribonucleases / Q000502:physiology*; D006801:Humans; D012441:Saccharomyces cerevisiae / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; C114331:EXO1 protein, human; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; D045643:DNA Repair Enzymes
keywords: DNA curtains; DNA repair; nuclease; resection; single-molecule