Replication of cellular chromosomes is a vital process that is precisely coordinated with cell division. To learn more about the molecular events that underlie chromosomal DNA replication, we have developed an in vitro system, composed of 20 highly purified proteins, that specifically replicates supercoiled chromosomes that carry a bacteriophage lambda replication origin. Our studies of this prototype model system indicate that nucleoprotein structures with novel properties are assembled at the origin prior to replication initiation. We have discovered that partial disassembly of these multiprotein structures is required both for the unwinding of DNA and for initiation of DNA chain elongation and have demonstrated that this protein-remodeling event is mediated by a universally conserved molecular chaperone team.

Our current efforts are focused towards elucidating precisely how synergistic protein-protein interactions in complex nucleoprotein structures contribute to "activation" and opening of origin DNA and to the specific loading of a DNA helicase ring on each DNA strand at the origin. In other studies, we are examining how molecular chaperones (i) cooperate to select protein targets and (ii) harness the energy of ATP binding and hydrolysis to energize protein remodeling events. We combine biochemical, molecular biological, biophysical, kinetic, and genetic approaches in our analyses of the structure and function of key macromolecular assemblies.

Biochemistry and molecular biology, biochemical mechanisms, DNA replication, macromolecular assemblies, DNA supercoiling, DNA helicases, protein-DNA interactions, protein structure and function, molecular chaperones, heat shock proteins, remodeling of protein assemblies

Struble, E.B., Gittis, A.G., Bianchet, M.A., and McMacken, R. (2007) Crystallization and Preliminary Crystallographic Characterization of the Origin-binding Domain of the Bacteriophage Lambda O Replication Initiator. Acta Cryst. F63:542-545.

Leng, F. , Amado, L., and McMacken, R. (2003). Coupling DNA supercoiling to transcription in defined protein systems. J. Biol. Chem. 279:47564-47571.

Leng, F. and McMacken, R. (2002). Potent stimulation of transcription-coupled DNA supercoiling by sequence-specific DNA-binding proteins. Proc. Natl. Acad. Sci. USA 99:9139-9144.

Russell, R., Karzai, A.W., Mehl, A.F., and McMacken R. (1999). DnaJ dramatically stimulates ATP hydrolysis by DnaK: Insight into targeting of Hsp70 proteins to polypeptide substrates. Biochemistry 38:4165-4176.

Russell, R., Jordan, R., and McMacken R. (1998). Kinetic characterization of the ATPase cycle of the DnaK molecular chaperone. Biochemistry 37:596-607.

Stephens, K.M. and McMacken, R. (1997). Functional properties of replication fork assemblies established by the bacteriophage lambda O and P replication proteins. J. Biol. Chem. 272:28800-28813.

Learn, B.A., Um, S.J., Huang, L. and McMacken, R. (1997). Cryptic single-stranded-DNA binding activities of the phage lambda P and Escherichia coli DnaC replication initiation proteins facilitate the transfer of Escherichia coli DnaB helicase onto DNA. Proc. Natl. Acad. Sci. USA 94:1154-1159.

Karzai, A.W., and McMacken, R. (1996) A bipartite signaling mechanism involved in DnaJ-mediated activation of the Escherichia coli DnaK protein. J. Biol. Chem. 271:11236-11246.

Jordan, R., and McMacken, R. (1995) Modulation of the ATPase activity of the molecular chaperone DnaK by peptides and the DnaJ and GrpE heat shock proteins. J. Biol. Chem. 270:4563-4569.

Montgomery, D., Jordan, R., McMacken, R., and Freire, E. (1993) Thermodynamic and structural analysis of the folding/unfolding transitions of the Escherichia coli molecular chaperone DnaK. J. Mol. Biol. 232:680-692.

Learn, B., Karzai, A.W., and McMacken, R. (1993) Transcription stimulates the establishment of bidirectional lambda DNA replication in vitro. Cold Spring Harbor Symp. Quant. Biol. 58:389-402.

Landry, S.J., Jordan, R., McMacken, R., and Gierasch, L.M. (1992) Different conformations for the same polypeptide bound to chaperones DnaK and GroEL. Nature 355:455-457.

Mallory, J.B., Alfano, C. and McMacken, R. (1990) Host-virus interactions during the initiation of bacteriophage lambda DNA replication: Recruitment of Escherichia coli DnaB helicase by the bacteriophage lambda P replication protein. J. Biol. Chem. 265:13297-13307.

Mensa-Wilmot, K., Carroll, K., and McMacken, R. (1989) Transcriptional activation of bacteriophage lambda DNA replication in vitro. Regulatory role of histonelike protein HU of Escherichia coli. EMBO J. 8: 2393-2402.

Dodson, M., McMacken, R., and Echols, H. (1989) Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: Protein association and dissociation reactions responsible for localized initiation of replication. J. Biol. Chem. 264: 10719-10725.

Alfano, C. and McMacken, R. (1989) Heat shock protein-mediated disassembly of nucleoprotein structures is required for the initiation of bacteriophage lambda DNA replication. J. Biol. Chem. 264:10709-10718.

Alfano, C. and McMacken, R. (1989) Ordered assembly of nucleoprotein structures at the bacteriophage lambda replication origin during the initiation of DNA replication. J. Biol. Chem. 264: 10699-10708.

Mensa-Wilmot, K., Seaby, R., Alfano, C., Wold, M. S., Gomes, B. and McMacken, R. (1989) Reconstitution of a nine-protein system that initiates bacteriophage lambda DNA replication. J. Biol. Chem. 264: 2853-2861.

Dodson, M., Echols, H., Wickner, S., Alfano, C., Mensa Wilmot, K., Gomes, B., LeBowitz, J., Roberts, J. D. and McMacken, R. (1986) Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: Localized unwinding of duplex DNA by a six-protein reaction. Proc. Natl. Acad. Sci. USA 83:7638-7642.

LeBowitz, J. H. and McMacken, R. (1986) The Escherichia coli DnaB replication protein is a DNA helicase. J. Biol. Chem. 261:4738 4748.