Regulation and new partners of the mammalian replisome

Abstract

Stable genomic integrity requires the faithful and complete duplication of chromosomes once per cell cycle only.  This study aimed to define the collective proteins involved in DNA replication in mammalian cells, along with associated regulatory mechanisms.

To achieve this, I targeted the essential SLD5 subunit of GINS in human hTERT RPE1 cells for N-terminal tagging with GFP.  Along with CDC45, GINS joins loaded MCM2-7 on DNA to form the active Cdc45-MCM-GINS (CMG) helicase.  CMG unwinds DNA to make it accessible to replicative DNA polymerases, thus putting it at the core of DNA replication.  Therefore, GINS, as part of CMG, was a suitable candidate to target for isolation and subsequent mass spectrometry analysis to establish what associates with CMG to carry out mammalian DNA replication.

The approach taken here led to successful generation of endogenously expressed GFP-SLD5 in human RPE1 cells.  With complimentary work carried out in mouse ES cells, both focussing on the isolation of SLD5, this gave two mammalian systems that confirmed that tagged-SLD5 is viable.  Mass spectrometry analysis of immunoprecipitations of tagged-SLD5 from asynchronously grown cultures identified most of the previously known components of the core replisome from lower eukaryotes.  That is, across S. cerevisiae, Xenopus, C. elegans, and the mammalian models utilised here, CMG is joined by CTF4 / WDHD1, CLASPIN, TIMELESS-TIPIN, DNA polymerases α and ε, FACT, and CTF18-RFC, to form the core replisome. Beyond this, I observed that the cullin-RING E3 ligase, CUL2LRR1, associates with the mouse replisome, when disassembly of the complex is abrogated by inhibition of the AAA+ ATPase, p97 segregase.

Considering the regulation of human CMG disassembly, CMG-MCM7 was ubiquitylated when the p97 segregase was inhibited in RPE1 cells; a modification that was reduced in the presence of cullin inhibitor.  When this cullin inhibition was combined with LRR1 depletion in mouse ES cells, this led to abolishment of MCM7 ubiquitylation.  Together, it can be concluded that in mammalian systems there is conservation of the regulatory pathway of CMG disassembly from Xenopus and C. elegans, in which CUL2LRR1 ubiquitylates MCM7 of CMG, thus making it a target for disassembly by p97.

I then investigated novel factors observed in association with SLD5 that were not otherwise observed in equivalent mass spectrometry analyses carried out in S. cerevisiae.  DONSON was a candidate protein of interest as, though human patients survive with mutations in DONSON, mice offspring cannot be generated with homozygous deletion of Donson.  I therefore proceeded with two strategies and determined that even at the level of mouse ES cells, Donson is essential.  What critical role DONSON protein plays remains an active area of study.

Further to DONSON, PARP1 was a predominant associate of SLD5, even in unperturbed human RPE1 and mouse ES cells.  Association of PARP1 was confirmed by deletion in mouse ES cells.  Further, I found that combined depletion and degradation of the replisome component, TIMELESS, reduced PARP1 association with the mouse replisome, indicating that TIMELESS is necessary for the recruitment of PARP1 to the mouse replisome.

Date of Award	2023
Original language	English
Supervisor	Karim Labib (Supervisor) & John Rouse (Supervisor)