Abstract 352: DNA replication licensing factor MCM5 is a target for SETD8 mediated methylation

SETD8 methyltransferase activity is implicated in several fundamental cellular processes such as transcriptional regulation and heterochromatin formation as well as processes that ensures genomic stability including DNA replication and the DNA damage response. It has been proposed that SETD8 affects DNA replication as a positive regulator of origin licensing through H4K20 methylation and by supporting Okazaki fragment processing through PCNA methylation. However, there is no evidence whether other key element in the replication machinery is directly modified by SETD8. To address this question, we used a combination of SILAC-based BPPM (Bioorthogonal Profiling of Protein Methylation) to profile new substrates for SETD8. We genetically engineered SETD8 and identified mutants amenable to accommodate non-native SAM analogues containing a terminal alkyne moiety for click chemistry. The engineered SETD8 can transfer this unique chemical moiety into target proteins for subsequent identification of the modified substrates. Stable cell lines expressing catalytically active or catalytically dead SETD8 mutants were systematically cultured with distinct stable isotope labeled amino acids allowing quantitative proteomic analysis of the identified substrates. With this method we could validate previously known substrates of SETD8 and identify novel targets, including MCM5, a subunit of the hexameric minichromosome maintenance (MCM) DNA helicase complex. MCM5 directly interacts with MCM2 and 3 to form the MCM2-7 hexamer which associates with the origins of DNA replication to form part of the pre-replicative complex (preRC), playing a major role during replication initiation and elongation. We found that SETD8 mediated methylation of MCM5 directly affects its binding affinity to MCM2 and 3. Amino acid substitutions at the methylated lysine further evidenced a stronger binding to its interacting partners contributing to the formation of the MCM hexamer. In addition, CRISPR based modifications on MCM5 methylated lysine affected the replication process resulting in defective cell cycle progression. Taken together our results indicate that MCM5 methylation contributes to the assembly of the MCM complex revealing a novel role for SETD8 in replication initiation. Our findings brings new perspectives on the biological importance of SETD8 during DNA replication. Citation Format: Fabio Pittella, Gil Blum, Yongxia Zhu, Chamara Senevirathne, Minkui Luo. DNA replication licensing factor MCM5 is a target for SETD8 mediated methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 352.