Studies on DNA replication: From regulation of initiation to helicase function

Multiple origins of replication are employed by eukaryotes in order to efficiently replicate the entire genome and a unique initiation event must occur at each origin, resulting in melting and unwinding of the template followed by polymerase action. The timing and regulation of initiation of DNA replication is dependent on S-phase promoting kinase (SPK) activity at discrete origins. DNA damage prevents origin firing through ATM- and ATR-dependent inhibition of the SPKs Cdk2 and Cdc7.; We demonstrated, in Xenopus laevis egg extracts, that modulation of ATM- and ATR-signaling pathways controls origin firing in the absence of DNA damage. Inhibition of ATM/ATR by caffeine or specific neutralizing antibodies, or upregulation of Cdk2 or Cdc7, promotes rapid and synchronous origin firing. Conversely, inhibition of Cdc25A phosphatase slows down DNA replication. The concentration of single-stranded DNA correlates with origin firing inhibition. Therefore, SPK activity is limiting for replication and is regulated via a feedback mechanism originating from activated replicons through ATM/ATR. These observations suggest that ATM- and ATR-signaling pathways operate during an unperturbed cell cycle to regulate initiation and progression of DNA synthesis.; The Mcms form a family of conserved molecules that are essential for initiation of DNA replication and are activated following SPK action. All eukaryotes contain six orthologous Mcm proteins, while the sequencing of the genomes of several Archaea has shown that presence of Mcm genes. We expressed and purified the single Methanobacterium thermoautotrophicum Mcm protein (MtMcm) and demonstrated that MtMcm protein assembles in large macromolecular complexes consistent in size with double hexamers. MtMcm contains helicase activity and DNA-dependent ATPase activity, unwinds DNA duplexes in a 3' → 5' direction and up to 500 base pairs in vitro, consistent with Mcm functioning as the replicative helicase.; In Xenopus egg extracts, the Mcm proteins are required for template unwinding and replication. Specific neutralizing antibodies directed against all six Mcm proteins blocked Mcm function and demonstrated that the Mcm complex is explicitly required during template unwinding. These data strongly support the hypothesis that the Mcm complex is the replicative helicase and is therefore responsible for melting the DNA at the replication fork.