TopBPI is a highly conserved protein with a sequence that contains eight reiterations of the BRCT domain, a sequence motif that is found almost exclusively in proteins involved in DNA replication and damage repair. Since its discovery, human TopBPI has been implicated in many different DNA metabolic processes in mammalian cells, including DNA replication, DNA damage checkpoint activation, and damage-induced transcriptional control. Preliminary experiments with the SV40 cell-free DNA replication system show that TopBPI plays a previously unknown role in the elongation phase of replication. In order to determine which BRCT domain or domains are required for the replication activity of TopBPI, a series of deletion mutants will be purified that each lack one BRCT domain. These mutants will be used in conjunction with the SV40 in vitro replication system and TopBPI immunodepleted HeLa cell extracts to show which domains are required to rescue TopBPI activity. I hypothesize that by delineating the biochemical requirement for TopBPI in SV40 replication I can acquire important new information on the mechanism of mammalian replication fork assembly and movement. ? ?
