Is APIM functional in RAD5 homolog HLTF?
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To ensure secure transmission of genome to daughter cells, all cells carry out highly processive error-free replication of the genome. Proliferating cell nuclear antigen (PCNA) plays a central part as processivity factor for DNA replication and repair, by providing a structural platform that enables binding of essential proteins to the replication machinery. Protein interactions with PCNA are mediated through the two known protein-interacting sequences; the PCNA-interacting peptide (PIP)-box and AlkB homologue 2 PCNA-interacting motif (APIM). Over 200 proteins involved in DNA damage response pathways contain APIM, indicating that PCNA-APIM interactions are more involved in cellular stress responses. The yeast Rad5 homolog HLTF contains APIM. HLTF exhibits both E3 ubiquitin ligase and DNA helicase activities and is shown to be important for DNA damage by inhibiting stalling of replication forks. The aim of this study was to investigate whether the APIM sequence in HLTF is functional. In order to investigate whether APIM is functional in HLTF, the SupF mutagenesis assay was performed. Mutation frequencies obtained by cells overexpressing HLTF wildtype or HLTF containing a mutation in the APIM sequence were investigated by introducing UVB damage to a reporter plasmid. In addition, co-transfection of HLTF wildtype, mutated HLTF and PCNA were performed to investigate if HLTF localization is dependent on APIM. The mutation frequency of UVB damaged reporter plasmids were significantly increased in cells overexpressing HLTF wildtype compared to cells overexpressing APIM mutated HLTF. This suggests an error-prone TLS promotion by HLTF that is dependent on HLTF PCNA interaction via APIM. Our data confirms functionality of APIM in HLTF, however localization of HLTF in replication foci was not dependent on APIM.