4.8 Article

Crystal structure of the Red C-terminal domain in complex with Exonuclease reveals an unexpected homology with Orf and an interaction with Escherichia coli single stranded DNA binding protein

Journal

NUCLEIC ACIDS RESEARCH
Volume 47, Issue 4, Pages 1950-1963

Publisher

OXFORD UNIV PRESS
DOI: 10.1093/nar/gky1309

Keywords

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Funding

  1. National Science Foundation (NSF) [MCB-1616105, DGE1745303]
  2. National Institutes of Health [T32GM118291, GM122459]
  3. United States Department of Energy [DE-FG02-02ER63445]
  4. NSF [MCB-1616105]

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Bacteriophage encodes a DNA recombination system that includes a 5-3 exonuclease ( Exo) and a single strand annealing protein (Red). The two proteins form a complex that is thought to mediate loading of Red directly onto the single-stranded 3-overhang generated by Exo. Here, we present a 2.3 angstrom crystal structure of the Exo trimer bound to three copies of the Red C-terminal domain (CTD). Mutation of residues at the hydrophobic core of the interface disrupts complex formation in vitro and impairs recombination in vivo. The RedCTD forms a three-helix bundle with unexpected structural homology to phage Orf, a protein that binds to E. coli single-stranded DNA binding protein (SSB) to function as a recombination mediator. Based on this relationship, we found that Red binds to full-length SSB, and to a peptide corresponding to its nine C-terminal residues, in an interaction that requires the CTD. These results suggest a dual role of the CTD, first in binding to Exo to facilitate loading of Reddirectly onto the initial single-stranded DNA (ssDNA) at a 3-overhang, and second in binding to SSB to facilitate annealing of the overhang to SSB-coated ssDNA at the replication fork.

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