E.V. McCollum
Professor and Chair
Department of Biochemistry and Molecular Biology
Bloomberg School of Public Health

rmcmacken@jhsph.edu

W8041 Hygiene Building
615 N. Wolfe Street
Baltimore, MD 21205

Office: 410-955-3949
Lab: 410-955-3534

This laboratory is interested in the workings of two types of macromolecular assemblies. One assembly, composed of three universally conserved molecular chaperones, is required for protein folding, polypeptide transport across membranes, cellular tolerance of thermal stress, and disassembly of protein aggregates. These molecular chaperones also act in a diverse variety of protein remodeling events, including specific steps in cell cycle regulation, oncogenic transformation, DNA replication, vesicle fusion, insulin secretion, neurotransmission, and activation of steroid hormone receptors. The second assembly is composed of viral and cellular proteins that direct the initiation of chromosomal DNA replication.

Our primary goal for the molecular chaperone system is to define the mechanistic basis for the action of the DnaK (Hsp70) and DnaJ (Hsp40) heat shock proteins. It is known that DnaK contains a weak intrinsic ATPase activity and it is thought that cycles of ATP hydrolysis drive association and dissociation of polypeptide substrates to and from DnaK. We are examining how the DnaJ and GrpE cochaperones and polypeptide substrates influence passage of DnaK through this cycle. Additionally, stopped-flow fluorescence and biosensor technologies are being used to focus on important unsolved problems such as (i) how is polypeptide binding and release coupled to the ATPase cycle of DnaK; (ii) how do molecular chaperones identify specific target proteins and modify their structures; and (iii) how do interactions of DnaJ with both DnaK and with substrate polypeptides facilitate chaperone action?

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 l replication origin. Our goals are to understand the molecular mechanisms and energetics involved in a well-defined DNA replication machine. Currently, we are focusing our investigative efforts on a preinitiation nucleoprotein complex that has the capacity to trap the energy of DNA supercoiling. We wish to learn how protein-protein and protein-DNA interactions in this complex contribute to "activation" and opening of origin DNA. In collaboration with Ed Lattman and colleagues in the Department of Biophysics, we have crystallized the DNA-binding domain of the l O replication initiator and are presently engaged in the determination of its three-dimensional structure. An important unsolved problem in DNA replication is how replicative DNA helicases are recruited to act at chromosomal DNA replication origins. To this end, we are currently examining the molecular events involved in the delivery of the DnaB replicative helicase to the viral replication origin by the l O and P replication proteins. Finally, we are also investigating in vitro the molecular mechanisms responsible for "transcriptional activation of DNA replication", an intriguing long-range phenomenon in which RNA transcription activates replication at distant chromosomal replication origins.

Selected Publications
Leng, F., L. Amado, and R. McMacken. (2004) Coupling DNA supercoiling to transcription in defined protein systems. J. Biol. Chem. 279:47564-47571.

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

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

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

Stephens, K.M., and R. McMacken. (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., S.-J. Um, L. Huang, and R. McMacken. (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.