Research Projects

Canonical primase is responsible for synthesizing RNA primers complementary to the replicating DNA sequence, which are extended into short DNA chains called Okazaki fragments by DNA polymerase during lagging strand synthesis. However, a unique noncanonical primase function associated with Cdc23/Mcm10 has been associated with insertion of 1-2 ribonucleotide moieties into the newly synthesized DNA chain at the mat1 locus in fission yeast, Schizosaccharomyces pombe. Cdc23/Mcm10 is also associated with the catalytic subunit of DNA Pol, which extends the RNA primers into the DNA chain. Mcm10/Cdc23 also plays an essential role in DNA replication in all eukaryotes, associating with the MCM-helicase and DNA Pol in the elongation stage of DNA replication. The primase function of Cdc23/Mcm10 is ascribed to the presence of putative primase domains found in the dnaG, with a conserved Aspartate residue being critical for the primase function and ribonucleotide insertion at the mat1 locus in fission yeast. This project proposes to carry out structure-function analysis of at least one vertebrate ortholog of Mcm10, X. laevis(Xl), from X. laevis(Xl). Domains IV-VI of the T7 gene 4 primase-helicase also possess the primase activity. The corresponding homology regions are found within the residues 416 to 593 in SpMcm10 and residues 560-750 of the XlMcm10. The project aims to subclone regions of the XlMcm10 into an E. coli expression vector with a hexahistidine or GST tag and investigate the structure and primase function of different subdomains of the XlMcm10.