The structural and biochemical analysis of Escherichia coli RecQ and Deinococcus radiodurans SSB

DNA replication, recombination, and repair enzymes play intertwined roles in maintaining genomic integrity. Both the RecQ family of helicases and Single Strand DNA (ssDNA) Binding (SSB) proteins are vital components of cellular genomic maintenance machinery. Through structural and biochemical methods, I examined these protein families and gained insight to their roles in genome maintenance reactions.; E. coli RecQ is composed of two structural domains: an N-terminal catalytic core and a C-terminal HRDC domain. While the catalytic core can unwind DNA and hydrolyze ATP at near wild type levels, it's DNA binding is destabilized. Using X-ray crystallography I determined the high-resolution structure of both domains from E. coli RecQ. E. coli RecQ's catalytic core is a tri-lobed structure in which the N-terminal two lobes comprising the helicase domain abut to form a deep cleft lined with conserved helicase motifs. The third lobe of the structure is divided into winged-helix (WH) and Zn2+-binding subdomains. E. coli RecQ's HRDC domain forms a bundle of helices that is similar in structure to other known DNA binding domains. I have identified the ssDNA-binding site on the E. coli HRDC domain.; D. radiodurans SSB codes for two OB folds and dimerizes to form a functional unit containing four OB folds. The crystal structure of D. radiodurans SSB suggests that the two OB folds have distinct functions. The two OB folds of D. radiodurans SSB are connected by a beta-turn linker region, and crystal packing contacts suggests that the linker region could be important in forming higher-order structures of the protein bound to ssDNA.; A number of studies have suggested that E. coli RecQ and SSB functionally interact. E. coli RecQ binds to the C-terminal nine amino acids of SSB. In addition, the WH subdomain is the primary interaction site for the C-terminal tail of SSB.