STRUCTURE DETERMINATION OF PROTEINS INVOLVED IN DNA REPLICATION

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. An ongoing project aimed at confirming, with MALDI-MS, the identification of various proteins involved in DNA replication. These proteins are being overexpressed and reconstituted for the purpose of X-Ray crystallographic determination of their three-dimensional structures. MCM proteins from S. cerevisiae and B. subtilus, and ORC proteins from S. cerevisiae are among the protein complexes under study. The mini-chromosome maintenance (MCM) proteins serve as the replicative helicases in archaea and eukaryotes. Interestingly, an MCM homolog was identified, by BLAST analysis, within a phage integrated in the bacterium Bacillus cereus (Bc). BcMCM is only related to the AAA region of MCM-helicases; the typical amino-terminus is missing and is replaced by a segment with weak homology to primases. We show that BcMCM displays 3'-->5' helicase and ssDNA-stimulated ATPase activity, properties that arise from its conserved AAA domain. Isolated BcMCM is a monomer in solution but likely forms the functional oligomer in vivo. We found that the BcMCM amino-terminus can bind ssDNA and harbors a zinc atom, both hallmarks of the typical MCM amino-terminus. No BcMCM-catalyzed primase activity could be detected. We propose that the divergent amino-terminus of BcMCM is a paralog of the corresponding region of MCM-helicases. A divergent amino terminus makes BcMCM a useful model for typical MCM-helicases since it accomplishes the same function using an apparently unrelated structure. This work has been published: A biochemically active MCM-like helicase in Bacillus cereus. Samuels M, Gulati G, Shin JH, Opara R, McSweeney E, Sekedat M, Long S, Kelman Z, Jeruzalmi D. Nucleic Acids Res. 2009 Jul;37(13):4441-52