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. 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., & 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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Works referenced in this record:
Conformational Adaptability of Redβ during DNA Annealing and Implications for Its Structural Relationship with Rad52
journal, August 2009
- Erler, Axel; Wegmann, Susanne; Elie-Caille, Celine
- Journal of Molecular Biology, Vol. 391, Issue 3
Crystal Structure of the Homologous-Pairing Domain from the Human Rad52 Recombinase in the Undecameric Form
journal, August 2002
- Kagawa, Wataru; Kurumizaka, Hitoshi; Ishitani, Ryuichiro
- Molecular Cell, Vol. 10, Issue 2
Identification of the SSB Binding Site on E. coli RecQ Reveals a Conserved Surface for Binding SSB's C Terminus
journal, February 2009
- Shereda, Robert D.; Reiter, Nicholas J.; Butcher, Samuel E.
- Journal of Molecular Biology, Vol. 386, Issue 3
Crystal structures of exonuclease in complex with DNA suggest an electrostatic ratchet mechanism for processivity
journal, July 2011
- Zhang, J.; McCabe, K. A.; Bell, C. E.
- Proceedings of the National Academy of Sciences, Vol. 108, Issue 29
Rings and filaments of protein from bacteriophage suggest a superfamily of recombination proteins
journal, April 1999
- Passy, S. I.; Yu, X.; Li, Z.
- Proceedings of the National Academy of Sciences, Vol. 96, Issue 8
Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity
journal, April 2015
- Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.
- Journal of Biological Chemistry, Vol. 290, Issue 23
Refinement of Macromolecular Structures by the Maximum-Likelihood Method
journal, May 1997
- Murshudov, G. N.; Vagin, A. A.; Dodson, E. J.
- Acta Crystallographica Section D Biological Crystallography, Vol. 53, Issue 3
Overview of the CCP 4 suite and current developments
journal, March 2011
- Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D.
- Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
Temperate Phages Acquire DNA from Defective Prophages by Relaxed Homologous Recombination: The Role of Rad52-Like Recombinases
journal, March 2014
- De Paepe, Marianne; Hutinet, Geoffrey; Son, Olivier
- PLoS Genetics, Vol. 10, Issue 3
Dali server update
journal, April 2016
- Holm, Liisa; Laakso, Laura M.
- Nucleic Acids Research, Vol. 44, Issue W1
Structure of the single-strand annealing domain of human RAD52 protein
journal, October 2002
- Singleton, M. R.; Wentzell, L. M.; Liu, Y.
- Proceedings of the National Academy of Sciences, Vol. 99, Issue 21
Structural and Functional Characterization of the Redβ Recombinase from Bacteriophage λ
journal, November 2013
- Matsubara, Kazuko; Malay, Ali D.; Curtis, Fiona A.
- PLoS ONE, Vol. 8, Issue 11
Crystal structures of Escherichia coli exonuclease I in complex with single-stranded DNA provide insights into the mechanism of processive digestion
journal, April 2013
- Korada, Sai Krishna C.; Johns, Trevor D.; Smith, Christopher E.
- Nucleic Acids Research, Vol. 41, Issue 11
The C-terminus of the phage λ Orf recombinase is involved in DNA binding
journal, December 2010
- Curtis, Fiona A.; Reed, Patricia; Wilson, Lindsay A.
- Journal of Molecular Recognition, Vol. 24, Issue 2
Phage Orf Family Recombinases: Conservation of Activities and Involvement of the Central Channel in DNA Binding
journal, August 2014
- Curtis, Fiona A.; Malay, Ali D.; Trotter, Alexander J.
- PLoS ONE, Vol. 9, Issue 8
Recombineering: a powerful new tool for mouse functional genomics
journal, October 2001
- Copeland, Neal G.; Jenkins, Nancy A.; Court, Donald L.
- Nature Reviews Genetics, Vol. 2, Issue 10
Programming cells by multiplex genome engineering and accelerated evolution
journal, July 2009
- Wang, Harris H.; Isaacs, Farren J.; Carr, Peter A.
- Nature, Vol. 460, Issue 7257, p. 894-898
The χψ Subunits of DNA Polymerase III Holoenzyme Bind to Single-stranded DNA-binding Protein (SSB) and Facilitate Replication of an SSB-coated Template
journal, September 1998
- Glover, Bradley P.; McHenry, Charles S.
- Journal of Biological Chemistry, Vol. 273, Issue 36
Recombineering with overlapping single-stranded DNA oligonucleotides: Testing a recombination intermediate
journal, May 2003
- Yu, D.; Sawitzke, J. A.; Ellis, H.
- Proceedings of the National Academy of Sciences, Vol. 100, Issue 12
A Single-Strand Annealing Protein Clamps DNA to Detect and Secure Homology
journal, August 2015
- Ander, Marcel; Subramaniam, Sivaraman; Fahmy, Karim
- PLOS Biology, Vol. 13, Issue 8
An Improved Recombineering Approach by Adding RecA to λ Red Recombination
journal, January 2006
- Wang, Junping; Sarov, Mihail; Rientjes, Jeanette
- Molecular Biotechnology, Vol. 32, Issue 1
Domain Structure of the Redβ Single-Strand Annealing Protein: the C-terminal Domain is Required for Fine-Tuning DNA-binding Properties, Interaction with the Exonuclease Partner, and Recombination in vivo
journal, February 2016
- Smith, Christopher E.; Bell, Charles E.
- Journal of Molecular Biology, Vol. 428, Issue 3
Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs
journal, June 2010
- Lopes, Anne; Amarir-Bouhram, Jihane; Faure, Guilhem
- Nucleic Acids Research, Vol. 38, Issue 12
Domain Structure and DNA Binding Regions of β Protein from Bacteriophage λ
journal, July 2006
- Wu, Zengru; Xing, Xu; Bohl, Casey E.
- Journal of Biological Chemistry, Vol. 281, Issue 35
Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones
journal, December 2016
- Wilkinson, Martin; Troman, Lucy; Wan Nur Ismah, Wan AK
- eLife, Vol. 5
A Structure–Activity Analysis for Probing the Mechanism of Processive Double-Stranded DNA Digestion by λ Exonuclease Trimers
journal, September 2015
- Pan, Xinlei; Smith, Christopher E.; Zhang, Jinjin
- Biochemistry, Vol. 54, Issue 39
Structure of the SSB-DNA polymerase III interface and its role in DNA replication: SSB-replisome interaction
journal, August 2011
- Marceau, Aimee H.; Bahng, Soon; Massoni, Shawn C.
- The EMBO Journal, Vol. 30, Issue 20
Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting
journal, April 2012
- Fu, Jun; Bian, Xiaoying; Hu, Shengbaio
- Nature Biotechnology, Vol. 30, Issue 5
Techniques: Recombinogenic engineering–new options for cloning and manipulating DNA
journal, May 2001
- Muyrers, Joep P. P.; Zhang, Youming; Stewart, A. Francis
- Trends in Biochemical Sciences, Vol. 26, Issue 5
The RecBC enzyme loads RecA protein onto ssDNA asymmetrically and independently of chi , resulting in constitutive recombination activation
journal, April 1999
- Churchill, J. J.; Anderson, D. G.; Kowalczykowski, S. C.
- Genes & Development, Vol. 13, Issue 7
HKL -3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes
journal, July 2006
- Minor, Wladek; Cymborowski, Marcin; Otwinowski, Zbyszek
- Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 8
Structural, functional, and evolutionary relationships between -exonuclease and the type II restriction endonucleases
journal, July 1998
- Kovall, R. A.; Matthews, B. W.
- Proceedings of the National Academy of Sciences, Vol. 95, Issue 14
Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages
journal, January 2008
- Datta, S.; Costantino, N.; Zhou, X.
- Proceedings of the National Academy of Sciences, Vol. 105, Issue 5
Features and development of Coot
journal, March 2010
- Emsley, P.; Lohkamp, B.; Scott, W. G.
- Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
Recombinant Protein Complex Expression in E. coli
journal, May 2008
- Selleck, William; Tan, Song
- Current Protocols in Protein Science, Vol. 52, Issue 1
DNA annealing by Redβ is insufficient for homologous recombination and the additional requirements involve intra- and inter-molecular interactions
journal, October 2016
- Subramaniam, Sivaraman; Erler, Axel; Fu, Jun
- Scientific Reports, Vol. 6, Issue 1
Dna2 nuclease-helicase structure, mechanism and regulation by Rpa
journal, November 2015
- Zhou, Chun; Pourmal, Sergei; Pavletich, Nikola P.
- eLife, Vol. 4
The Translocating RecBCD Enzyme Stimulates Recombination by Directing RecA Protein onto ssDNA in a χ-Regulated Manner
journal, July 1997
- Anderson, Daniel G.; Kowalczykowski, Stephen C.
- Cell, Vol. 90, Issue 1
Functional similarities between phage Orf and Escherichia coli RecFOR in initiation of genetic exchange
journal, August 2005
- Maxwell, K. L.; Reed, P.; Zhang, R. -g.
- Proceedings of the National Academy of Sciences, Vol. 102, Issue 32
A Central Role for SSB in Escherichia coli RecQ DNA Helicase Function
journal, May 2007
- Shereda, Robert D.; Bernstein, Douglas A.; Keck, James L.
- Journal of Biological Chemistry, Vol. 282, Issue 26
Improving Lambda Red Genome Engineering in Escherichia coli via Rational Removal of Endogenous Nucleases
journal, September 2012
- Mosberg, Joshua A.; Gregg, Christopher J.; Lajoie, Marc J.
- PLoS ONE, Vol. 7, Issue 9
Structural basis of Escherichia coli single-stranded DNA-binding protein stimulation of exonuclease I
journal, June 2008
- Lu, D.; Keck, J. L.
- Proceedings of the National Academy of Sciences, Vol. 105, Issue 27
An efficient recombination system for chromosome engineering in Escherichia coli
journal, May 2000
- Yu, D.; Ellis, H. M.; Lee, E-C.
- Proceedings of the National Academy of Sciences, Vol. 97, Issue 11, p. 5978-5983
PriA helicase and SSB interact physically and functionally
journal, November 2004
- Cadman, C. J.
- Nucleic Acids Research, Vol. 32, Issue 21
MOLREP an Automated Program for Molecular Replacement
journal, December 1997
- Vagin, A.; Teplyakov, A.
- Journal of Applied Crystallography, Vol. 30, Issue 6, p. 1022-1025
High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides
journal, May 2001
- Ellis, H. M.; Yu, D.; DiTizio, T.
- Proceedings of the National Academy of Sciences, Vol. 98, Issue 12, p. 6742-6746
The phage lambda orf gene encodes a trans-acting factor that suppresses Escherichia coli recO, recR, and recF mutations for recombination of lambda but not of E. coli.
journal, November 1994
- Sawitzke, J. A.; Stahl, F. W.
- Journal of Bacteriology, Vol. 176, Issue 21
Characterization of the DNA-binding domain of β protein, a component of phage λ Red-pathway, by UV catalyzed cross-linking
journal, December 1996
- Mythili, E.; Kumar, K. Anand; Muniyappa, K.
- Gene, Vol. 182, Issue 1-2
Binding and Recognition of GATATC Target Sequences by the Eco RV Restriction Endonuclease: A Study Using Fluorescent Oligonucleotides and Fluorescence Polarization †
journal, February 2001
- Reid, Sarah L.; Parry, Damian; Liu, Hsiao-Hui
- Biochemistry, Vol. 40, Issue 8