E. coli DNA Replication: Our longest running program is directed toward understanding the enzymology of DNA replication with primary focus on the DNA polymerase III holoenzyme, the bacterial replicase. Because of the conserved nature of replication mechanisms, many of the central principles, applicable to all life forms, have been established in this system. Our current focus is on the signaling networks that lead to coordinated action of the leading and lagging strand replicase and the cycling of the lagging strand polymerase, during Okazaki fragment synthesis. We also want to understand the function of ATPase activity in replication complex assembly.
B. subtilis DNA Replication: The low GC Gram-positive bacteria diverged from E. coli approximately two billion years ago. This model organism provides an opportunity to learn how the central themes of DNA replication vary in highly divergent organisms. We have established a full in vitro system that requires 13 purified proteins. Our current focus is upon learning how two distinct replicases coordinate their activities during lagging strand replication.
Chemical Biology of DNA Replication: This program is directed toward the discovery and development of small molecules that block each of the steps of the complex replication process. These will provide tools for studying the mechanism of DNA replication and for blocking specific steps in vivo for cell physiology studies. A subset of the compounds discovered will provide leads for development of novel antibacterials.
Within this program, we have also developed tools for biochemically identifying the target of antibacterial agents that block DNA replication. We are willing to collaborate with industry to apply these tools.
Highly Processive Thermophilic Replication System: We have discovered a novel highly processive thermophilic replicase that, coupled with replication fork proteins, should be capable of replicating entire chromosomes and megabase segments of DNA. We are willing to collaborate with industry to apply this system to the development of new technologies.
Yuan, Q. and McHenry, C. (2014) Cycling of the E. coli lagging strand polymerase is triggered exclusively by the availability of a new primer at the replication fork. Nucleic Acids Res., 42, 1747-1756.
Antony, E., Weiland, E., Yuan, Q., Manhart, C.M., Nguyen, B., Kozlov, A.G., McHenry, C. and Lohmman, T.M. (2013) Multiple C-Terminal Tails within a Single E. coli SSB Homotetramer Ordinate DNA Replication and Repair. J. Mol. Bio. 425, 4802-4819.
Ungermannova, D., Lee, J., Zhang, G., Dallmann, H.G., McHenry, C. and Liu, X. (2013) High-Throughput Screening AlphaScreen Assay for identification of Small-Molecule Inhibitors of Ubiquitin E3 Ligase SCFkp2-Cks1. J. Biomol. Screen., 8, 910-920.
Seco, E., Zinder, J., Manhart, C.M., Piano, A.L., McHenry, C. and Ayora, S. (2013) Bacteriophage SPP1 in vitro DNA replication strategies promote viral and disable host replication. Nucleic Acids Res. 41,1711-1721.
Manhart, C.M., McHenry, C.S. (2013) "The PriA Replication Restart Protein Blocks Replicase Access Prior to Helicase Assembly and Directs Template Specificity through its ATPase Activity." J. Biol. Chem. 288, 3989-3999.
McHenry, C.S."Replicative DNA Polymerases." In E. Bell, J. Bond, J. Klinman, B. Siler Masters, and R. Wells (Eds.), Molecular Life Sciences: An Encyclopedic Reference. New York: Springer. In press.
Dohrmann, PR, Manhart, CM, Downey,CD, McHenry,CS (2011) "The Rate of Polymerase Release upon Filling the Gap between Okazaki Fragments is Inadequate to Support Cycling During Lagging Strand Synthesis." J. Mol. Biol, 414, 15-27.
McHenry, CS (2011) "Bacterial replicases and related polymerases." Curr.Opin.Chem Biol, 15, 587-594.
Downey, C.D., Crooke, E., McHenry, C.S. (2011) "Polymerase Chaperoning and Multiple ATPase Sites Enable the E. coli DNA Polymerase III Holoenzyme to Rapidly Form Initiation Complexes." J. Mol. Biol., 412, 340-353.
McHenry, CS (2011) "Breaking the rules: bacteria that use several DNA polymerase IIIs." EMBO Rep. 12, 408-414.
McHenry, CS (2011) DNA Replicases from a Bacterial Perspective. Annu Rev Biochem, 80, 403-436.
Wieczorek A, Downey CD, Dallmann HG, McHenry CS.(2010) "Only one ATP-binding DnaX subunit is required for initiation complex formation by the Escherichia coli DNA polymerase III holoenzyme." J Biol Chem. 285, 29049-29053.
Dallmann, H.G.,Fackelmayer, O.J.. Tomer, G., Chen, J.Y., Wiktor-Becker, A., Ferrara, T., Pope, C., Oliveira, M.T., Burgers, P.M.J., Kaguni, L.K., and McHenry, C.S. (2010) "Parallel Multiplicative Target Screening Against Divergent Bacterial Replicases: Identification of Specific Inhibitors with Broad Spectrum Potential." Biochemistry, 49, 2551-2562.
Downey, C.D. and McHenry, C.S. (2010) "Chaperoning of a Replicative Polymerase onto a Newly-Assembled DNA Bound Sliding Clamp by the Clamp Loader." Mol. Cell, 37,481-491.
Sanders, G.M., Dallmann, H.G. and McHenry, C.S. (2010) "Reconstitution of the B. subtilis Replisome with 13 Proteins including Two Distinct Replicases." Mol. Cell, 37, 273-281.