The mechanism of modulation of the Escherichia coli DNA helicase II (UvrD) unwinding activity, a study of the 2B subdomain

Helicases are ubiquitous motor proteins which catalyze the separation of double stranded DNA into single stranded DNA for the purposes of replication, recombination and repair. Escherichia coli DNA helicase II, also known as UvrD, is a Superfamily 1 helicase involved in post-replicative mismatch repair, nucleotide excision repair and conjugative recombination. These roles that UvrD fills in the cell require dramatically different unwinding activities to be performed accurately. Nucleotide excision repair only requires a 12-13 bp section of DNA to be unwound for efficient repair, while methyl-directed mismatch repair and recombination can require upwards of 1 kilobase of DNA to be traversed for the processes to be faithfully completed. These contrasting activity requirements are even more confusing when one examines the in vitro processivity of UvrD, which has been measured to be on the order of 40 of bp unwound in a single event. This begs the question how is the activity of UvrD regulated to perform these roles which require converse activity levels in the cell?;This dissertation offers evidence that the 2B subdomain of UvrD is involved in modulating the activity of the helicase. Mutations within this subdomain have been linked to dramatically stimulated helicase activity which I have shown is due to an increase in the processivity of the unwinding reaction. In addition, the data presented here suggest that this subdomain is the interaction site between UvrD and other proteins which have been shown to have a stimulatory effect on the unwinding activity of UvrD. However, deletion of this subdomain in its entirety does not seem to stimulate the helicase activity of UvrD as it has been shown to do in other Superfamily 1 helicases.;It can be concluded that the 2B subdomain plays a poorly understood role in regulating the unwinding activity of UvrD and this work offers an exploratory look in to the roles this subdomain performs in the regulation of the helicase.