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Mutants of phage bIL67 RuvC with enhanced Holliday junction binding selectivity and resolution symmetry

Green, V.; Curtis, F.A.; Sedelnikova, S.; Rafferty, J.B.; Sharples, G.

Mutants of phage bIL67 RuvC with enhanced Holliday junction binding selectivity and resolution symmetry Thumbnail


Authors

V. Green

F.A. Curtis

S. Sedelnikova

J.B. Rafferty



Abstract

Viral and bacterial Holliday junction resolvases differ in specificity with the former typically being more promiscuous, acting on a variety of branched DNA substrates, while the latter exclusively targets Holliday junctions. We have determined the crystal structure of a RuvC resolvase from bacteriophage bIL67 to help identify features responsible for DNA branch discrimination. Comparisons between phage and bacterial RuvC structures revealed significant differences in the number and position of positively-charged residues in the outer sides of the junction binding cleft. Substitutions were generated in phage RuvC residues implicated in branch recognition and six were found to confer defects in Holliday junction and replication fork cleavage in vivo. Two mutants, R121A and R124A that flank the DNA binding site were purified and exhibited reduced in vitro binding to fork and linear duplex substrates relative to the wild-type, while retaining the ability to bind X junctions. Crucially, these two variants cleaved Holliday junctions with enhanced specificity and symmetry, a feature more akin to cellular RuvC resolvases. Thus, additional positive charges in the phage RuvC binding site apparently stabilize productive interactions with branched structures other than the canonical Holliday junction, a feature advantageous for viral DNA processing but deleterious for their cellular counterparts.

Citation

Green, V., Curtis, F., Sedelnikova, S., Rafferty, J., & Sharples, G. (2013). Mutants of phage bIL67 RuvC with enhanced Holliday junction binding selectivity and resolution symmetry. Molecular Microbiology, 89(6), 1240-1258. https://doi.org/10.1111/mmi.12343

Journal Article Type Article
Acceptance Date Jul 23, 2013
Online Publication Date Aug 14, 2013
Publication Date Sep 1, 2013
Deposit Date Jul 25, 2013
Publicly Available Date Feb 4, 2014
Journal Molecular Microbiology
Print ISSN 0950-382X
Electronic ISSN 1365-2958
Publisher Wiley
Peer Reviewed Peer Reviewed
Volume 89
Issue 6
Pages 1240-1258
DOI https://doi.org/10.1111/mmi.12343

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Publisher Licence URL
http://creativecommons.org/licenses/by/4.0/

Copyright Statement
© 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly cited.






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