λ Rap protein is a structure-specific endonuclease involved in phage recombination

λ Rap protein is a structure-specific endonuclease involved in phage recombination

Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, United Kingdom

Edited by Charles M. Radding, Yale University School of Medicine, New Haven, CT, and approved September 29, 1998 (received for review May 20, 1998)

Abstract

Bacteriophage λ encodes a number of genes involved in the recombinational repair of DNA double-strand breaks. The product of one of these genes, rap, has been purified. Truncated Rap proteins that copurify with the full-length form are derived, at least in part, from a ρ-dependent transcription terminator located within its coding sequence. Full-length and certain truncated Rap polypeptides bind preferentially to branched DNA substrates, including synthetic Holliday junctions and D-loops. In the presence of manganese ions, Rap acts as an endonuclease that cleaves at the branch point of Holliday and D-loop substrates. It shows no obvious sequence preference or symmetry of cleavage on a Holliday junction. The biochemical analysis of Rap gives an insight into how recombinants could be generated by the nicking of a D-loop without the formation of a classical Holliday junction.

Footnotes

↵ * To whom reprint requests should be addressed. e-mail: gary.sharples{at}nottingham.ac.uk.
↵ † Present address: School of Biological Sciences, Division of Biochemistry, Royal Holloway, University of London, Egham, TW20 0EX, United Kingdom.
This paper was submitted directly (Track II) to the Proceedings Office.
ABBREVIATIONS:

wt,

wild-type;

IPTG,

isopropyl β-d-thiogalactoside