Author: Tyler H. Stanage

Publisher:

Published: 2017

Total Pages: 0

ISBN-13:

In each cell cycle, the replisome encounters replication blocks that include DNA lesions, tightly bound DNA-protein complexes, and breaks in the DNA. In Escherichia coli, many pathways have evolved to recognize, respond to, and repair these replication blocks so that replication can be completed in a timely and accurate manner. The highly conserved RarA (Replication associated recombination protein A) protein family has been implicated in the faithful resolution of these replication conflicts. Despite high conservation and extensive work conducted on this protein family, the overall function of RarA remains enigmatic. Here, we carefully describe two novel DNA-dependent functions for the E. coli RarA protein. First, we demonstrated that RarA binds to and hydrolyzes ATP at double-stranded DNA ends in order to generate single-stranded DNA flaps. Second, using a single-molecule reconstituted DNA replication assay, we show that RarA generated single-stranded DNA gaps behind active replisomes in an ATP-dependent manner. This activity is achieved by RarA disengaging DNA polymerase III cores from their [beta]2 processivity clamps. In vivo, we demonstrate that RarA--presumably using its gap creation activity--generates suitable substrates for post-replication repair pathways including RecFOR-mediated homologous recombination and translesion DNA synthesis. Loss of this function results in impaired growth and varied DNA replication phenotypes. Overexpression of rarA is toxic due to its ATPase activity and the resulting overuse of mutagenic TLS polymerases. Together, these results further elucidate the role of the RarA protein family at the interface of DNA replication and DNA repair.