Investigation of the pre‐sensor 1 hairpin in minichromosome maintenance proteins 2–7.

Abstract

The minichromosome maintenance proteins 2–7 (Mcm2–7) assemble into the replicative helicase in eukaryotes. To understand how this ring‐shaped helicase translocates along DNA to unwind the duplex nucleic acid, we have analyzed the structure/function relationships of the S. cerevisiae proteins. A pre‐sensor 1 (PS1) hairpin found in archaeal MCMs is thought to be involved in DNA translocation, with a conserved lysine residue predicted to contact the phosphate backbone. We mutated the analogous lysines to alanines in the putative PS1 hairpins of each of the yeast Mcm2–7 subunits. Mutation of the Mcm3 hairpin results in a loss of cellular viability. Its overexpression in the context of a wild‐type allele results in enlarged cells and a reduced growth rate. Purified Mcm2–7 complex containing the Mcm3 hairpin mutant does not unwind DNA in vitro, yet retains wild‐type levels of ATPase activity. Interestingly, individual mutation of the PS1 hairpins of the other subunits does not affect viability. However, these strains do have growth‐related sensitivity to genotoxic stress agents, and cold. Collectively, our data indicates the importance of the PS1 hairpin for Mcm2–7 function.Support from the CIHR