Molecular mimicry in the loading of a bacterial recombinase by a phage mediator

Lead Research Organisation: Durham University
Department Name: Biological and Biomedical Sciences

Abstract

The genetic material in our cells is subject to frequent threats to its integrity, especially while undergoing replication to produce new cell copies. Multiple repair and restoration pathways have evolved to ensure faithful transfer of information from generation to generation. Defects in these processes lead to cancer in higher organisms and significantly impair survival in simpler forms of life such as bacteria. Our group studies the recombinational repair pathway, which utilises the unique pattern of nucleotides stored in the DNA double helix as a template for repair of a damaged partner chromosome. In all organisms, the same enzyme (called RecA or Rad51) is utilised to exchange a single strand from one DNA helix to another as the first step in damage restoration. Both bacterial RecA and human Rad51 strand exchange recombinases polymerise on single-strands of DNA to form long filament-like structures. Unfortunately loading of these recombination enzymes is hindered by single-stranded DNA binding proteins that protect the template from further damage. To overcome this potentially serious predicament, cells contain specialized helper proteins that assist RecA and Rad51 in gaining access to the DNA strands. Despite considerable research effort, the detailed mechanism of RecA and Rad51 facilitated assembly remains unclear. We have discovered a new helper activity, from a virus infecting bacteria, with the capacity to hijack the bacterial RecA protein to promote repair of its own genetic material. This protein, Orf, has some interesting features in common with BRCA2, which helps load the Rad51 recombinase onto DNA. Mutations in the BRCA2 gene increase the likelihood of developing breast and ovarian cancers because of a reduced capacity to repair DNA damage. The research proposed in this study aims to identify the critical parts of Orf responsible for helping RecA overcome the obstruction posed by single-stranded DNA binding proteins. We will employ biochemical assays to investigate how RecA polymer formation is influenced by the presence of Orf. In addition, we will study how RecA filament assembly is enhanced or disrupted in different contexts by visualising the proteins by electron microscopy. Finally, we will investigate how the Orf protein, which exists as a dual subunit ring, can open up like a clamp to bind single-stranded DNA. The results will give fresh insight into the molecular mechanisms of genetic recombination and the contribution this repair process makes in evading the onset of cancer.

Technical Summary

Recombination enzymes need to gain access to single-stranded DNA to perform the transactions necessary for efficient genomic replication and repair. Template access, however, is hampered by the presence of single-stranded DNA binding (SSB) proteins essential for chromosome duplication. To solve this quandary, accessory proteins have evolved to promote assembly of the nucleoprotein filaments responsible for strand pairing and synapsis. Although these mediators have been extensively characterised, the mechanics of partner nucleation in the context of this significant protein barrier has yet to be explained. Our group studies a simple phage mediator system involving Orf facilitated assembly of the bacterial RecA recombinase onto SSB-coated single-stranded DNA. We have identified a heptapeptide motif in Orf that matches a region in RecA required for filament self-assembly. Strikingly, a similar signature (BRC) occurs in BRCA2 and serves to sequester and deliver RAD51, the eukaryotic counterpart of RecA, onto damaged DNA. This project aims to show how the BRC-like peptide in Orf constitutes a nucleation site for RecA and promotes its assembly at the junction between duplex and single-stranded DNA. The work will reveal insights into the mechanism of RecA family protein assembly likely to be applicable to all organisms.
 
Description DNA has been adopted as the favoured repository for transmitting the information necessary for life. Such nucleic acid, however, is not a stable molecule and is repeatedly damaged or broken. Numerous enzymes are employed to ensure accurate copying of the genetic material and restore integrity when problems occur. Deficiencies in these cellular repair processes lead to cancer in higher organisms and considerably reduced viability in microbes. Our research focuses on recombinational repair, which utilises the unique pattern of nucleotides stored in the double helix as a template for repair of damaged chromosomal partners. Work has concentrated on how the viruses that infect bacteria, known as phages, impose their own recombination systems to maximise production of viral progeny. Phage recombination enzymes are less accurate than their bacterial counterparts and this imprecision fuels genetic diversity. As a result, new gene combinations arise, dramatically enhancing the rate of evolution; consequently, pathogenic traits are readily transferred between bacteria harbouring these viruses. In this project we characterised a novel phage protein, Orf, that substitutes for three considerably more complex host proteins in the loading of the bacterial recombinase, RecA, onto DNA covered by a single-stranded DNA binding protein, SSB. In fact the way Orf is proposed to load RecA is analogous to BRCA2 loading the RecA-like Rad51 in humans, a key function in preventing malignancy. The protein-DNA and protein-protein interactions in the bacterial and viral equivalent of this reaction were the subject of this grant.

We discovered that Orf protein preferentially targets DNA bubbles, with a five or more nucleotide stretch being necessary for stable assembly. Bubble structures arise during replication or repair when the double helix is prised apart to allow the protein machinery to copy or repair genetic information. Specifically Orf targets the fork structures at either end of the bubble and hence Orf ensures that these regions serve as the focus of recombinational repair. It is still unclear whether it is RecA or the phage recombinase Beta that is loaded at this by Orf, although Orf has been shown to disrupt RecA polymer formation consistent with it recognising the interface between RecA subunits and facilitating polymer nucleation. Mutations in the predicted Orf-RecA interacting motif disrupted this protein-protein association, although only under certain conditions.

Orf is a ring shaped protein composed of two identical subunits; the aperture of the ring is too narrow to accommodate a DNA duplex and so we hypothesised that Orf opens via a hinge to clamp onto single-stranded DNA. Such a mechanism must occur since Orf binds to bubble structures with single-strands constrained by flanking duplexes. A large number of mutant Orf proteins have been tested to identify those portions of Orf important for DNA recognition. Significantly, alterations within the interior of the toroid were defective in DNA binding.

The analysis carried out offers significant insights into the mechanics of recombination in phages and applicable to higher organisms. Ongoing work from the project aims to develop the phage recombination pathway further as a tool for genetic engineering.

Work initiated on the phage proteins Rap and NinH has been extended and a paper on NinH has been submitted for publication and one on Rap is in preparation.
Exploitation Route Genetic recombination promotes diversity, determines genome organisation and maintains chromosome integrity. The results from this study, including analysis of phage Orf and Beta proteins, have helped to uncover the mechanistic basis of presynapsis, the universal first step in recombination reactions. A detailed understanding of the molecular processes involved is directly relevant to cross-disciplinary research on bacterial, archaeal and human DNA replication and repair and how defects in these pathways lead to cell death and cancer. The work also has important implications for understanding how phage enzymes collaborate with the systems of the host to drive the genome rearrangements that ultimately spawn new pathogenic bacteria. Biomedical researchers studying these complex processes in a wide range of model organisms will be the principal beneficiaries.

The work is of commercial interest in the molecular biology sector, offering potential enhancements to the widely used recombineering approach to modifying prokaryotic genomes.
A fuller understanding of the mechanisms involved in presynapisis may lead to harnessing mutant or modified derivatives of these genes to improve the accuracy and efficacy of gene knockouts.
Sectors Manufacturing, including Industrial Biotechology,Retail

 
Description John Rafferty Collaboration 
Organisation University of Sheffield
Country United Kingdom 
Sector Academic/University 
PI Contribution Supply of knowledge and plasmid constructs for X-ray crystallography.
Collaborator Contribution Purification and crystal structure determination of 67RvC protein (as part of a PhD studentship). Ongoing work with another PhD student on various other target proteins.
Impact Green, V., Curtis, F.A, Sedelnikova, S., Rafferty, J.B. and Sharples, G.J. (2013) Mutants of phage bIL67 RuvC with enhanced Holliday junction binding selectivity and resolution symmetry. Mol. Microbiol. 89: 1240-1258 Multi-disciplinary combining, bacterial genetics, molecular biology, biochemistry and structural biology.
 
Description Jonathan Heddle Collaboration 
Organisation RIKEN
Country Japan 
Sector Public 
PI Contribution Supply of knowledge, plasmid constructs, expertise in protein-DNA interaction assays.
Collaborator Contribution Expertise in structural biology and molecular modelling - postdoc from RIKEN spent 4 weeks in Durham performing experiments for the Orf paper.
Impact Matsubara, K., Malay, A.D., Curtis, F.A., Sharples, G.J. and Heddle, J.G. (2013) Structural and functional characterization of the Redß recombinase from bacteriophage ?. PLoS One 8: e78869 Curtis, F.A., Malay, A.D., Trotter, A.J., Wilson, L.A., Barradell-Black, M.M., Bowers, L.Y., Reed, P., Hillyar, C.R.T., Yeo, R.P., Sanderson, J.M., Heddle, J.G. and Sharples, G.J. (2014) Phage Orf family recombinases: conservation of activities and involvement of the central channel in DNA binding. PLoS One 9: e102454
Start Year 2009
 
Description K Muniyappa Collaboration 
Organisation Indian Institute of Science, Bangalore
Country India 
Sector Academic/University 
PI Contribution Knowledge, expertise on Holliday junction binding proteins, supply of plasmid constructs, experimental data on E. coli YqgF and UV sensitivity experimental analysis. Protein structural modelling.
Collaborator Contribution Supply of TB plasmid constructs for testing.
Impact Nautiyal, A., Rani, P.S., Sharples, G.J., and Muniyappa, K. (2016) Mycobacterium tuberculosis RuvX is a Holliday junction resolvase formed by dimerization of the monomeric YqgF nuclease domain. Mol. Microbiol. - in press Multidisciplinary across two bacterial species. Bacterial genetics, molecular biology and biochemistry.
Start Year 2014