Abstract
Photochemical cross-linking has been used to identify residues in the Escherichia coli RecA protein that are proximal to and may directly mediate binding of DNA. Ultraviolet irradiation promotes specific and efficient cross-linking of the RecA protein to poly(deoxythymidylic) acid. Cross-linked peptides remaining covalently attached to the polynucleotide following proteolytic digestion with trypsin correspond to amino acids 61-72, 178-183, and 233-243 of the RecA protein primary sequence. Their location and surface accessibility in the crystal structure, along with the behavior of various recA mutants, support the assignment of the cross-linked regions to the DNA binding site(s) of the RecA protein. Functional overlap of amino acids 61-72 with an element of the ATP binding site suggests a structural mechanism by which nucleotide cofactors allosterically affect the RecA nucleoprotein filament.
Highlights
Genetic recombination is a universal occurrence that serves to generate genetic diversity, to preserve genomic integrity, and to ensure proper partitioning of chromosomes
The cross-linking of a peptide that coincides with a portion of the nucleotide binding site may provide further insight as to how conformational changes in the RecA nucleoprotein filament elicited by the binding and hydrolysis of ATP are directly transduced to regions implicated in DNA binding
The active conformation of the RecA protein-ssDNA complex requires the binding of nucleotide cofactor, such as ATP, the RecA nucleoprotein filament is too dynamic for examination by cross-linking due to the potential for multiple ssDNA binding states that occur during the ssDNAdependent hydrolysis of ATP [10, 28]
Summary
Genetic recombination is a universal occurrence that serves to generate genetic diversity, to preserve genomic integrity, and to ensure proper partitioning of chromosomes. Nucleoprotein filament formation requires stoichiometric amounts of the RecA protein and the binding of ATP to induce an active conformation in which the DNA assumes a highly extended and unwound state [12,13,14] This unusual DNA conformation appears to be universal to the mechanism of genetic recombination because homologs such as the uvsX and Rad proteins from T4 bacteriophage and Saccharomyces cerevisiae, respectively, form complexes with DNA that are structurally [15, 16] and functionally analogous [17, 18]. The manner by which the RecA nucleoprotein filament recognizes sequence homology within a duplex DNA target remains unknown Understanding of this mechanism would be greatly facilitated by definition of those regions within the RecA protein polymer that interact with DNA and are central to the recognition process. The cross-linking of a peptide that coincides with a portion of the nucleotide binding site may provide further insight as to how conformational changes in the RecA nucleoprotein filament elicited by the binding and hydrolysis of ATP are directly transduced to regions implicated in DNA binding
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
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
