Abstract
Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4), but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101Mms22 ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101Mms22 E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1’s replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS) complex at stalled forks.
Highlights
DNA replication is a process through which cells duplicate their entire genome prior to cell division
In order to understand how eukaryotes cope with DNA damage, we have investigated a conserved E3 ubiquitin ligase complex required for the resistance to carcinogenic chemicals
We found that the Rtt101Mms22 E3 ubiquitin ligase complex interacts with the replisome during S-phase, and orchestrates the repair/restart of DNA synthesis after
Summary
DNA replication is a process through which cells duplicate their entire genome prior to cell division. In Saccharomyces cerevisiae, this adaptation is regulated by proteins such as Mrc, Tof, Csm and Ctf, which assemble around the CMG (Cdc45-MCM-GINS) DNA helicase at replication forks. These components form the ‘Replisome Progression Complex’ (RPC), a replisome sub-assembly that exists exclusively at replication forks [2]. The RPC functions in coupling DNA polymerases to the CMG helicase [3,4], and in regulating fork progression [5,6,7,8,9] These replisome components limit mutagenic frequency and prevent unscheduled homologous recombination (HR) events at stalled forks [10,11,12]. Significant stretches of ssDNA generated by uncoupling can promote HR-mediated replication re-start via either a template switch or break-induced replication (BIR) [19]
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