Topic Overview:
The ability of DNA to replicate and repair itself is critical. Alterations or disruptions in these functions can lead to various disorders like Werner’s syndrome, which results in premature aging and cancer, and xeroderma pigmentosum, which causes extreme ultraviolet sensitivity and produces cancer. Chromosome replication involves not only duplication of the genetic information present on each of the component DNA strands but also the precise replication of the chromosome’s functional state. Any mistakes in this complex process may cause an alteration of the genetic code or the expression of the component genes, resulting in cell death or genomic vulnerability leading to cancer.

One essential enzyme needed for DNA replication is the replicative helicase, a molecular motor that unwinds the DNA double helix to generate a single-stranded DNA template required for DNA polymerase interaction. In eukaryotes, considerable in vivo data indicates that a ring-shaped complex called the minichromosome maintenance complex (Mcm2-7) fulfills this function. Although all other known hexameric helicases are formed from six identical subunits, Mcm2-7 is formed from six different, essential, and evolutionarily conserved subunits that are each AAA+ ATPases. In addition to Mcm2-7’s enzymatic function, both the chromosome loading and activation of this complex are central regulatory features of eukaryotic DNA replication. However, mechanistic studies of this complex have been hindered by an inability to reconstitute in vitro DNA unwinding activity.

To gain a mechanistic understanding of Mcm2-7 function, Dr. Schwacha uses the yeast Saccharomyces cerevisiae as a tool to study both the genetics and biochemistry of eukaryotic chromosome replication. Through careful biochemical studies comparing purified recombinant Mcm2-7 to an informative Mcm subcomplex, Dr. Schwacha’s laboratory reconstituted DNA unwinding activity from Mcm2-7. Various studies examining the effects of numerous ATPase mutations among the six Mcm subunits have shown—both in vivo and in vitro—that these subunits each contribute different functions: three are specialized for DNA unwinding and the other three are regulatory, perhaps forming an ATP-dependent discontinuity or “gate” within the circular complex. Dr. Schwacha’s laboratory is now investigating the potential in vivo role for this gate. Understanding the regulation and function of Mcm2-7 helicase is a critical foundation for future mechanistic studies of the replication fork.