A Study of Loading of Base Excision Repair Proteins at Replication Locus
Abstract
Our genome is constantly exposed to endogenous and exogenous DNA damaging factors. In order to prevent accumulation of mutations and to maintain genome integrity, cells must repair induced lesions. This occurs through a DNA damage response involving a wide variety of proteins. One of these responses has been classified as base excision repair (BER), mainly responsible for removal of damaged and mis-incorporated bases. XRCC1 and PCNA are proteins with no enzymatic activity that act as scaffolding proteins in separate BER pathways, but PCNA is also essential in normal replication. The DNA glycosylase UNG2 interacts with PCNA and carries out post-replicative removal of uracil. Additionally, XRCC1 interacts and co-localizes with PCNA in replication foci. As UNG2 creates a single-strand break (SSB) and XRCC1 is central in single-strand break repair (SSBR) a model where XRCC1 complexes were localized close to replication forks were suggested. In this thesis, isolation of proteins on nascent DNA (iPOND) has been conducted to identify BER proteins on nascent DNA. In particular, it has been of interest to investigate the presence of XRCC1 on nascent DNA, as it functions as a binding platform for many core BER proteins and a post-replicative function of these has not been elucidated. UNG2 interacts with PCNA through the PIP-box similar to many replication proteins. Recently, another PCNA interacting motif, APIM, was identified. APIM is found in many proteins involved in defence against cellular stress. One of the goals of this thesis has been to mutate the PIP-box to APIM in UNG2 and investigate how this mutation affects the affinity to PCNA and loading on nascent DNA. BER proteins, including XRCC1, were verified in close proximity of replication forks, indicating that XRCC1 protein complexes are involved in post-replicative repair. This was supported by coupling iPOND with both Western blotting and mass spectrometry. Furthermore, the mutation from PIP to APIM in UNG2 most likely decreased its affinity to PCNA in absence of DNA damage. However, after stalling of replication by MMS and MMC the mutant seemed to have increased affinity to a fraction of the PCNAs in replication foci, indicating that APIM-PCNA interactions have greater importance upon cellular stress. Both the wild type and mutant fusion proteins were poorly loaded on nascent DNA by iPOND, indicating that the smaller endogenous UNG2 more easily access PCNA