The role of McmD and the C-terminal of McmD in Methanococcus maripaludis

The minichromosome maintenance (MCM) proteins play an essential role in the replication of DNA, functioning as the genomic helicase in eukaryotes and archaea. In eukaryotes the MCMs comprise a family of six highly related proteins; MCM 2-7, which interact together to form heterohexameric ring complexes. The hexameric structure of MCM contributes to the highly complex and tightly regulated assembly and processivity of the replisome. Significant similarities can be observed between the structure of eukaryotic MCM 2-7 and homologous MCM complexes found in archaea, presenting an intriguing, simplified and biochemically tractable model. The majority of archaeal species have been shown to possess a single functional MCM that forms homohexameric complexes. In previous studies we have discovered that species in the order Methanococcales possess between two and eight MCM genes. Our model organism Methanococcus maripaludis S2 possesses four MCM genes (McmA, B, C & D), which may offer insight into additional motifs required for MCM function. We have generated deletions of individual and combinations of MCM genes, and determined that McmA appears to be essential. Previous studies have shown that McmD may be implicated in DNA damage response and the deletion of McmD causes a DNA damage repair defect. A series of experiments employing UV exposure and ionising radiation as agents of DNA damage were planned, designed to investigate possible roles the protein may play in DNA damage repair. This study was focused around McmD and the insert in the C-terminal of the protein; investigations found that, extrachromosomal overexpression of McmD caused colonies to become sick and possibly caused lethality to cells. Therefore ‘rescue’ of the McmD mutant under a strong constitutive promoter located on plasmid clones (pJB002/pJB003) could not be investigated. McmD gene (MMP1024) and MMP1025 is present in 16 Methanococcales species. MMP1024 and MMP1025 were included in operonic arrangements in 8 of the Methanococcales species analysed. It was possible to produce double and triple M. maripaludis MCM knockout strains (BDΔ, CDΔ, BCΔ, and BCDΔ). For purposes of the domain swap / expression of fusion protein investigation; a clone comprising the N-terminal of McmB and the C-terminal of McmD in commercial expression vector pPROEXHTa was produced. Future work would continue with cloning, co-expression, co-purification of MMP1024 and MMP1025 and biochemical characterisation of McmD.