DNA helicases catalyze NTP hydrolysis-dependent unwinding of duplex DNA to provide single-stranded DNA (ssDNA) for use as a template or reaction intermediate in DNA transactions. Eleven helicases have been identified in E. coli; the cellular role of each is being elucidated and structure- function studies are providing information regarding the mechanism of the unwinding reaction. The long-range goal of this research program is to understand, in enzymatic and molecular terms, the mechanism of action and cellular role of several E. coli DNA helicases. The current focus is on DNA helicases I and II.
The first aim proposes structure-function studies of helicase II. Conserved amino acid residues in helicase motifs will be altered, the mutant protein purified and characterized, and the mutant allele evaluated in genetic assays. This approach provides detailed information on the mechanism and role of helicase II. The second and third aims focus on protein-protein interactions involving helicase II. A genetic screen for mutants that fail to dimerize has been devised. Characterization of these mutants will allow evaluation of the importance of dimerization in helicase reaction mechanisms and cellular roles. The PI will also identify and characterize proteins that interact directly with helicase II to shed additional light on the roles this protein plays in the cell.
The fourth aim addresses the role of helicase II in DNA replication. To date, this role is uncharacterized and genetic experiments are proposed to provide additional detail. The fifth aim proposes acquisition of high resolution structural information to complement the structure- function studies. The crystal structure of a helicase II-ssDNA complex will be determined. The sixth aim proposes reconstitution of the nicking/unwinding reaction catalyzed by DNA helicase I to initiate bacterial conjugation. The nicking and unwinding reactions catalyzed by this protein have been evaluated separately during the previous grant period. Surprisingly, it has not been possible to couple these reactions. Preliminary data indicate a requirement for a host protein to trigger unwinding. This protein will be purified, identified and its role in both the nicking/unwinding reaction and bacterial conjugation will be elucidated using biochemical and genetic approaches.
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