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Function of the N-terminal segment of the RecA-dependent nuclease Ref.

ABSTRACT: The bacteriophage P1 Ref (recombination enhancement function) protein is a RecA-dependent, HNH endonuclease. It can be directed to create targeted double-strand breaks within a displacement loop formed by RecA. The 76 amino acid N-terminal region of Ref is positively charged (25/76 amino acid residues) and inherently unstructured in solution. Our investigation of N-terminal truncation variants shows this region is required for DNA binding, contains a Cys involved in incidental dimerization and is necessary for efficient Ref-mediated DNA cleavage. Specifically, Ref N-terminal truncation variants lacking between 21 and 47 amino acids are more effective RecA-mediated targeting nucleases. We propose a more refined set of options for the Ref-mediated cleavage mechanism, featuring the N-terminal region as an anchor for at least one of the DNA strand cleavage events.

SUBMITTER: Gruber AJ

PROVIDER: S-EPMC4330346 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Function of the N-terminal segment of the RecA-dependent nuclease Ref.

Gruber Angela J AJ Olsen Tayla M TM Dvorak Rachel H RH Cox Michael M MM

Nucleic acids research 20150123 3

The bacteriophage P1 Ref (recombination enhancement function) protein is a RecA-dependent, HNH endonuclease. It can be directed to create targeted double-strand breaks within a displacement loop formed by RecA. The 76 amino acid N-terminal region of Ref is positively charged (25/76 amino acid residues) and inherently unstructured in solution. Our investigation of N-terminal truncation variants shows this region is required for DNA binding, contains a Cys involved in incidental dimerization and i ...[more]

PMID: 25618854

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