Crystal structure of the Redβ C-terminal domain in complex with λ Exonuclease reveals an unexpected homology with λ Orf and an interaction with Escherichia coli single stranded DNA binding protein
Abstract
Bacteriophage λ encodes a DNA recombination system that includes a 5'-3' exonuclease (λ Exo) and a single strand annealing protein (Redβ). The two proteins form a complex that is thought to mediate loading of Redβ directly onto the single-stranded 3'-overhang generated by λ Exo. Here, we present a 2.3 Å crystal structure of the λ Exo trimer bound to three copies of the Redβ 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 Redβ CTD forms a three-helix bundle with unexpected structural homology to phage λ 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 Redβ 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 λ Exo to facilitate loading of Redβ directly 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.
- Authors:
-
- The Ohio State Univ., Columbus, OH (United States)
- Harvard Medical School, Cambridge, MA (United States)
- Harvard Medical School, Cambridge, MA (United States); Harvard Univ., Cambridge, MA (United States)
- Publication Date:
- Research Org.:
- Harvard Medical School, Cambridge, MA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division
- OSTI Identifier:
- 1528913
- Grant/Contract Number:
- FG02-02ER63445
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nucleic Acids Research
- Additional Journal Information:
- Journal Volume: 47; Journal Issue: 4; Journal ID: ISSN 0305-1048
- Publisher:
- Oxford University Press
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; Bacteriophage lambda; Red-beta; crystal structure
Citation Formats
Caldwell, Brian J., Zakharova, Ekaterina, Filsinger, Gabriel T., Wannier, Timothy M., Hempfling, Jordan P., Chun-Der, Lee, Pei, Dehua, Church, George M., and Bell, Charles E. Crystal structure of the Redβ C-terminal domain in complex with λ Exonuclease reveals an unexpected homology with λ Orf and an interaction with Escherichia coli single stranded DNA binding protein. United States: N. p., 2019.
Web. doi:10.1093/nar/gky1309.
Caldwell, Brian J., Zakharova, Ekaterina, Filsinger, Gabriel T., Wannier, Timothy M., Hempfling, Jordan P., Chun-Der, Lee, Pei, Dehua, Church, George M., & Bell, Charles E. Crystal structure of the Redβ C-terminal domain in complex with λ Exonuclease reveals an unexpected homology with λ Orf and an interaction with Escherichia coli single stranded DNA binding protein. United States. https://doi.org/10.1093/nar/gky1309
Caldwell, Brian J., Zakharova, Ekaterina, Filsinger, Gabriel T., Wannier, Timothy M., Hempfling, Jordan P., Chun-Der, Lee, Pei, Dehua, Church, George M., and Bell, Charles E. Wed .
"Crystal structure of the Redβ C-terminal domain in complex with λ Exonuclease reveals an unexpected homology with λ Orf and an interaction with Escherichia coli single stranded DNA binding protein". United States. https://doi.org/10.1093/nar/gky1309. https://www.osti.gov/servlets/purl/1528913.
@article{osti_1528913,
title = {Crystal structure of the Redβ C-terminal domain in complex with λ Exonuclease reveals an unexpected homology with λ Orf and an interaction with Escherichia coli single stranded DNA binding protein},
author = {Caldwell, Brian J. and Zakharova, Ekaterina and Filsinger, Gabriel T. and Wannier, Timothy M. and Hempfling, Jordan P. and Chun-Der, Lee and Pei, Dehua and Church, George M. and Bell, Charles E.},
abstractNote = {Bacteriophage λ encodes a DNA recombination system that includes a 5'-3' exonuclease (λ Exo) and a single strand annealing protein (Redβ). The two proteins form a complex that is thought to mediate loading of Redβ directly onto the single-stranded 3'-overhang generated by λ Exo. Here, we present a 2.3 Å crystal structure of the λ Exo trimer bound to three copies of the Redβ 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 Redβ CTD forms a three-helix bundle with unexpected structural homology to phage λ 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 Redβ 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 λ Exo to facilitate loading of Redβ directly 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.},
doi = {10.1093/nar/gky1309},
journal = {Nucleic Acids Research},
number = 4,
volume = 47,
place = {United States},
year = {2019},
month = {1}
}
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