Analysis of DNA replication in human cells: Revisiting origin firing and the replisome

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandlingForskning

Cells propagate by duplicating their genetic material and distributing each copy into two new daughter cells. DNA replication is a series of highly regulated steps by which cells duplicate their DNA. First, potential replication origin sites on the parental double-stranded DNA (dsDNA) are recognised and ‘licensed’. Then, replication begins when a subset of these origins are fired by the concerted assembly of origin firing factors mediated by CDK2 activity. This leads to the formation of bidirectional replication forks where DNA polymerases can synthesise nascent daughter DNA strands. Occasionally, forks may be stalled due to replication stress, leading to accumulation of single-stranded DNA (ssDNA) and a replication stress response mediated by the checkpoint kinases ATR and CHK1. Mechanistic details of all these complex processes have emerged mainly from seminal studies in yeast. While core principles of DNA replication are conserved, a full understanding of this process in humans is still missing. The broad aim of this PhD study was to investigate two key processes of DNA replication in human cells, specifically origin firing and ssDNA generation during replication stress. First, we developed a simple method to isolate proteins recruited to the replicating chromatin. By inducing different levels of origin firing using a time-course of a specific inhibitor against CHK1 (CHK1i), we constructed time-resolved chromatin loading dynamics for more than 3000 proteins during origin firing. These dynamics not only recapitulated the replication fork proteome but also provided functional insights. For instance, we found that surplus soluble levels of the essential replisome factor CDC45 determined dormant origin firing potential and this could be crucial for recovery from replication stress. We also found that transient origin firing factors TOPBP1 and RECQL4 are majorly dispensable for ongoing replication but become important during dormant origin firing. Finally, we also identified several previously uncharacterised factors with potentially novel roles at replication forks. Second, we hypothesised if specific helicases/nucleases regulate ssDNA generation during replication stress. To test this, we optimised and performed a phenotypic RNA interference (siRNA) screen on these factors in cells treated with hydroxyurea (HU), a replication blocker. Validation experiments for one of the hits, PPP1R8, implied potential roles for this factor as a ssDNA regulator during replication stress. Overall, our data provides a comprehensive resource to get functional insights into DNA replication and supports the idea that higher order regulation of DNA replication exists in human cells.
ForlagUniversity of Copenhagen
Antal sider201
StatusUdgivet - 2022

ID: 318039083