STRUCTURE AND FUNCTION OF BACTERIAL RECQ PROTEIN

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. RecQ is a multi-component enzyme and is an ATP-dependent DNA helicase. Three human syndromes, Bloom's, Werner's, and Rothmun-Thompson's syndromes, arise from mutations within the closely related BLM, WRN, and RECQ4 recQ genes, respectively, highlighting the importance of the RecQ family of proteins. One of the sub-domains of RecQ contains a winged-helix (WH) structural motif, containing a recognition alpha-helix which makes extensive major groove contacts with double stranded DNA (dsDNA) and a pair of flanking beta-strands (forming the "wing"), which typically interact on the minor groove side of dsDNA. The WH domain of RecQ helicases and its variants are known to bind a variety of DNA substrates and has also been implicated as a protein-protein interaction domain. It has been proposed that this sub-domain of RecQ may transiently interact with multiple key proteins thus mediating DNA replication, recombination, and repair mechanisms. Evidence suggests that the WH domain binds tightly and specifically to the C-terminus of the single stranded DNA binding (SSB) protein. In addition, the C-terminal domain of the SSB protein is disordered when bound to ssDNA (Savvides et al., 2004) and it is therefore possible that this protein-protein interaction may be critical for modulating SSB function as well as perhaps regulating the enzymatic acitivity of RecQ. The goal in this collaboration (between the Keck and the Butcher laboratories) is to map the protein-protein interaction between the WH domain of RecQ helicase and a critical 10 amino acid stretch within the C-terminus of the SSB protein by NMR spectroscopy. In addition, we aim to identify the ssDNA, dsDNA, and SSB binding sites on the WH domain of the E. Coli RecQ protein.