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.
Highlights
Encoded by bacteriophage P1, the recombination enhancement function (Ref) protein is a 21-kDa RecA-dependent HNH endonuclease that can be targeted to produce doublestrand breaks (DSBs) at any desired DNA sequence
We investigated P1 Ref homologs from bacteriophages P7 and W39 [17,18,19] to determine if targeting efficiency could be increased relative to reactions previously characterized with the Ref protein from bacteriophage P1
Overall we provide evidence that the N-terminal region of Ref is necessary for DNA binding, and the putative charge motifs may contribute collectively to that function
Summary
Encoded by bacteriophage P1, the recombination enhancement function (Ref) protein is a 21-kDa RecA-dependent HNH endonuclease that can be targeted to produce doublestrand breaks (DSBs) at any desired DNA sequence. Reports showed that expression of Ref enhanced recombination events in a RecA- and RecBCD-dependent manner in Escherichia coli [1,2,3]. The linear double-stranded (linear ds) genome is cyclized upon infection. This process occurs via the phageencoded Cre-lox site-specific recombination system, but it can occur by RecA-mediated homologous recombination [4]. Ref may play a role in this RecA-dependent cyclization of the genome [5]. Some phage HNH proteins partner with terminases in the genome packaging reaction [6]. Ref exhibits no homology to the terminaseassociated HNH protein nucleases
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
