The role of ATP in the regulation of Escherichia coli DNA polymerase V activity.

摘要

In Escherichia coli, DNA damage elicits the well regulated SOS response, which occurs in two phases. The first phase of SOS is dominated by accurate repair processes such as excision repair and recombinational DNA repair, while the second phase is characterized by a large increase in mutations caused by error-prone replication of damaged DNA templates. This phase of the SOS response is mediated by DNA polymerases that replicate past template lesions in a process called translesion synthesis (TLS). It is DNA polymerase V (UmuD'2C), encoded by the LexA-regulated umuDC operon, that is responsible for most of the damage-induced chromosomal mutations. It is therefore not surprising that DNA polymerase V (pol V) expression and activity are tightly regulated so as to restrict the time and place the polymerase can perform DNA synthesis. In addition to transcriptional regulation by the LexA protein, pol V is post-transcriptionally regulated through proteolysis and by the RecA nucleoprotein filament (RecA*). UmuD2 undergoes RecA*-mediated autodigestion, resulting in the mutagenically active form UmuD2&feet; which forms a stable heterotrimeric complex with UmuC resulting in UmuD'2C. We have previously shown that the final conversion of the UmuD'2C complex to a highly active TLS enzyme requires the transfer of a RecA subunit from the 3' end of the RecA* filament to form UmuD' 2C-RecA-ATP, which we refer to as pol V Mut. ATP is part of the active complex, with approximately one molecule of ATP per active enzyme, and until now ATP has played an enigmatic role in the activation process. This thesis establishes three features of the roles of ATP in pol V Mut activity; 1) bound ATP is required for DNA synthesis; 2) pol V Mut is a DNA-dependent ATPase; 3) ATP is required to bind primer/template (p/t) DNA and ATP hydrolysis triggers dissociation of pol V Mut from the DNA. Pol V Mut formed with an ATPase-deficient RecA E38K/K72R mutant hydrolyzes ATP rapidly, establishing the DNA-dependent ATPase as an intrinsic property of pol V Mut distinct from the ATP hydrolytic activity of RecA*. No similar ATPase activity or autoregulatory mechanism has previously been found for a DNA polymerase. The autoregulatory ATPase function provides a novel mechanistic explanation for the confinement of mutagenic DNA synthesis to short DNA segments surrounding lesions. The cellular risk of excessive and untargeted mutation is correspondingly reduced.;The SOS response functions not only repair the DNA damage and restore the replication fork but also drive mutagenesis, thereby enhancing survival and contributing to bacterial evolution. Mobile genetic elements frequently found in virulent bacterial strains often carry SOS response genes homologous to pol V as well as antibiotic-resistance genes. DNA-damage induced by antibiotics sets off the SOS response including the expression of Umu-like polymerases in bacteria, which likely increases the genetic repertoire and disseminates pathogenic traits. This thesis also focuses on the characterization of a highly mutagenic homolog of pol V named Rum polymerase, which is encoded on a Resistance factor, and the role of ATP in Rum's activity.