Understanding the function of the Rtt101 E3 ubiquitin ligase in response to replication stress
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Abstract
The duplication of the cellular genetic material has to be precisely regulated to maintain genome integrity. Damage caused by a wide range of exogenous factors, commonly summarized as replication stress, interferes with DNA replication. Genome integrity is further threatened by endogenously arising structures such as ribonucleotide monophosphates (rNMP) that are frequently misincorporated into genomic DNA. They are removed by the RNase H2 enzyme in a process termed ribonucleotide excision repair (RER). When RER is defective, rNMPs accumulate in the genome and induce replication stress. Cullin 4 (CUL4)-based E3 ubiquitin ligases are required for DNA replication and repair in the presence of replicative DNA damage. In Saccharomyces cerevisiae, the CUL4 ortholog Rtt101 promotes DNA replication through damaged templates. However, the underlying mechanism and relevant ubiquitylation targets are poorly understood.
In this thesis we characterized the mechanism by which Rtt101 promotes DNA replication in the presence of the alkylating drug methyl methanesulfonate (MMS). We found that Rtt101, in complex with the putative substrate adaptor Mms22 (Rtt101-Mms22), counteracts a replicative function of the replisome component Mrc1. However, this does not alter Mrc1 protein levels. Instead, our genetic data suggests that interactions of Mrc1 with other replisome proteins are modulated. We propose that Rtt101 allows recombination-mediated fork restart at MMS-induced DNA lesions.
We further uncovered a novel role of Rtt101-Mms22 in the tolerance of misincorporated rNMPs that accumulate in the absence of RER. Cells lacking both Rtt101 and RER display reduced viability, which is not caused by increased levels of genomic rNMPs and can be partially offset by deletion of MRC1. Ubiquitin remnant profiling, a mass spectrometry-based approach, identified the leading strand polymerase ε subunit Dpb2 as a potential target of Rtt101. We suggest that Rtt101 ubiquitylates Dpb2 at replication forks that stall or break due to unrepaired rNMPs. This Rtt101-dependent ubiquitylation might facilitate replication fork restart or DNA synthesis repriming downstream of the site of damage.
We present evidence that underlines an important function of the Rtt101 E3 ubiquitin ligase to tolerate several aspects of faulty RER. Our data indicates a similar mechanism as under genotoxin-induced replication stress.