Investigating the interaction between the alpha subunit of DNA polymerase III and UmuD

Abstract

The alpha subunit of DNA polymerase III (DNA pol III) is the main replicative polymerase in Escherichia coli. Because alpha cannot copy damaged DNA, replication is stalled in presence of DNA lesions. To compensate, the cell initiates the SOS response, inducing the expression of at least 57 genes. The products of these genes are involved in DNA damage tolerance mechanisms, one of which is translesion synthesis, a process by which lesions are bypassed by specialized DNA polymerases with the ability to copy damaged DNA. DNA pol V, one of these polymerases, is composed of two proteins: UmuD'2, the RecA/ssDNA‐facilitated cleavage product of UmuD2, and UmuC, the polymerase subunit. UmuD and UmuD’ both interact with the alpha, beta processivity clamp, and epsilon proofreading subunits of DNA polymerase III, likely regulating DNA pol III activity in response to DNA damage (Sutton et al PNAS 1999 v 96 p 12373–12378). We are currently investigating the interaction between alpha and UmuD2. Thermofluor data suggest that UmuD2 binds alpha in a specific conformation, while UmuD cleavage assays show that alpha competes with the RecA/ssDNA filament for UmuD2 binding. We are identifying the binding site for UmuD2 by using a number of alpha mutations and truncations. Research supported by NSF Career MCB‐0845033.