Characterization of the Role of RecQ helicases in human DNA replication

Cellular and biochemical studies support a role for all five human RecQ helicases in DNA replication, however their specific functions during this process are unclear. In my thesis, I investigated the in vivo association of the five human RecQ helicases with three well-characterized human replication origins. I showed that only RECQ1 and RECQ4 associate with replication origins in a cell cycle-regulated fashion in unperturbed cells, while other RecQ helicases interact with replication origins only under replication perturbed conditions. Under endogenous conditions, RECQ4 is recruited to origins at late G1 after ORC and MCM complex assembly, while RECQ1 and additional RECQ4 are loaded at origins at the onset of S phase when licensed origins begin firing. Both proteins are lost from origins after DNA replication initiation, indicating either disassembly or tracking with the newly formed replisome. Cell proliferation, DNA synthesis, nascent origin DNA synthesis and the frequency of origin firing are reduced after RECQ1 depletion, and to a greater extent after RECQ4 depletion. Depletion of RECQ1, though not RECQ4, also suppresses replication fork rates in otherwise unperturbed cells. Loading of PCNA during S phase is affected by RECQ1 depletion while the RECQ4 depleted cells show defect in RPA and PCNA loading during S phase of the cell cycle. These results indicate that RECQ1 and RECQ4 are integral components of the human replication complex, and play distinct roles in DNA replication initiation and replication fork progression in vivo.