The Study On The MCM Complex Remodeling And Modifications During DNA Replication Initiation

DNA replication is one of the most fundamental and fateful biological processes in all species. The accurate genome duplication is the prerequisite to the genetic information transmission from generation to next generation. To ensure this accurate copying process, DNA replication need to be tightly controlled. In eukaryotes, the primary safeguard mechanism lies in coordinating the loading and activation of the replicative helicase during the initiation stage, which ensures the whole genome is replicated once and only once in each cell cycle. Mcm2-7, the core of eukaryotic replicative helicase, is loaded onto double-stranded origin DNA as an inactive double hexamer in G1 phase. However, the activation mechanisms of MCM helicase in S phase remain unknown.To investigate how MCM is activated in Saccharomyces cerevisiae, we first develop a new approach to directly detect and purify the endogenous MCM double hexamer (DH). Through chromatin fraction and subsequent affinity purification, the native MCM DH is purified to a nearly homogenous level. In G1 phase, DH Mcm2-7 is only detected in the high-salt resistant chromatin-bound fraction, but not in the nucleoplasmic fraction. Furthermore, we establish a novel DH Mcm2-7 splitting assay, through which we are able to show that the MCM DH becomes separated during S phase in vivo. Interestingly, Mcm 10 is co-purified exclusively with inactive DH Mcm2-7 on chromatin, which implies Mcm 10 might be involved in helicase activation. The C terminus of Mcm10 mediates the interaction between Mcm10 and Mcm2-7. Disruption of this interaction causes DNA replication and growth defects, and these defects can be suppressed by restoration of the interaction through the GFP-GBP mediated Mcm10-MCM fusion. These results provide direct evidence to the notion that the replication defects in mcmlOAC are indeed caused by the disruption of Mcm10-MCM interaction. mcmlOAC shows significant delay in DH Mcm2-7 dissolution during S phase. Based on these, we propose an essential role for Mcm10 in Mcm2-7 remodeling through Mcm10-MCM interaction during helicase activation.Meanwhile, we also find the novel posttranslational modification of MCM in hyperthermophilic Sulfolobus islandicus. In vitro results show that recombinant sisMCM, an archaeal homolog of Mcm2-7 eukaryotic replicative helicase, is methylated by methyltransferase aKMT4. Mono-methylation of 3 clusters of lysine residues occurs coincidently in the endogenous sisMCM protein as indicated by mass spectra. Strinkingly. the helicase activity of sisMCM is stimulated by methylation at temperatures close to the optimal growth conditionsof Sulfolobus. In addition, the half-life of sisMCM is dramatically extended at 80℃ after methylation. Moreover, the methylation-mimic mutants of sisMCM show heat resistant helicase activity comparable to the methylated one. These lysine residues are located on the accessible protein surface, the methylation may change the hydrophobicity and surface charge to modulate the intra- and inter- molecular interactions of sisMCM. Therefore, these results illustrate that methylation may enhance kinetic stability of MCM proteins to ensure the survival of hyperthermophiles under extreme high temperature.This study sheds new light on the structure and function of the crucial replicative helicase MCM in archaea and eukaryotes, which has been remaining as the most enigmatic essential enzyme involved in DNA replication. Our data will contribute to understanding DNA replication initiation and cell proliferation and growth in these organisms including human beings.