DNA helicases are motor proteins that couple the energy generated through nucleotide binding and hydrolysis to DNA unwinding and translocation. E. Coli Rep helicase is involved in chromosomal replication and is required for replication of a number of phages, including phiX174. The focus of this proposal is to examine the role of the 2B subdomain in Rep helicase function. Towards this goal, the candidate has created a deletion mutant of Rep helicase, Repdelta2B, where the 2B domain has been fully deleted. Surprisingly, Repdelta2B can support bacteriophage phiX174 replication and has higher unwinding activity than wild type Rep. Studies of other closely related helicases show that mutations located within the 2B domain can significantly alter helicase activity. These results indicate that although the 2B domain is not necessary for function, mutation or removal of this domain significantly alters helicase activity.
The specific aims of the proposed research are: (1) to determine if the 2B domain effects DNA induced Rep oligomerization; (2) to examine the effect of deleting the 2B domain on single-strand or double-strand DNA binding; (3) to examine the effects of deleting the 2B domain on the mechanism of DNA unwinding; (4) to determine the effect of removal of the 2B domain on nucleotide binding and hydrolysis; and (5) to characterize the effects of a variety of site-directed mutations made within the 2B domain.
| Brendza, Katherine M; Cheng, Wei; Fischer, Christopher J et al. (2005) Autoinhibition of Escherichia coli Rep monomer helicase activity by its 2B subdomain. Proc Natl Acad Sci U S A 102:10076-81 |
