Molecular Recognition in Biological Systems and Bioinformatics

Protein biochemistry; bacterial genetics; DNA replication/repair/damage tolerance.

Regulation and Coordination of DNA Replication and DNA Repair

Chromosomal DNA replication is a highly regulated event that is tightly coupled to cell growth. This event requires the concerted actions of numerous proteins and can be divided into three distinct stages referred to as initiation, elongation, and termination of DNA replication. In addition to DNA replication, a variety of DNA repair pathways exist. The combination of accurate replication and efficient repair are crucial for maintaining the integrity of an organism's genetic material. Research in my lab is directed at understanding the mechanisms of, regulation of, and coordination of DNA replication and repair. We are particularly interested in the roles played in these processes by molecular machines.

My lab utilizes a combination of biochemical and genetic approaches to investigate the molecular mechanisms of DNA replication and DNA repair in Escherichia coli. We are currently concentrating our research efforts in two related areas. First, we are investigating the molecular mechanism of translesion DNA synthesis, the potentially mutagenic replication over lesions in the DNA. In E. coli, translesion DNA synthesis depends on the umuDC-encoded DNA polymerase V (Pol V), acting together with other cellular proteins as a molecular machine. In vitro, Pol V-dependent translesion DNA synthesis is stimulated by the b clamp and clamp loader complex of another molecular machine, the replicative DNA polymerase, DNA Pol III. The b clamp functions as a ring-shaped dimer that is loaded onto DNA by the clamp loader complex. Once loaded, the b clamp slides freely along the DNA. We are interested in understanding how specific interactions of both Pol III and Pol V with the b clamp help coordinate the actions of these two molecular machines to enable translesion DNA synthesis.

The second main area of our research involves investigation into how the b clamp coordinates initiation of DNA replication with elongation and certain DNA repair pathways. b interacts with proteins involved in each of these three events. We are interested in characterizing these various protein-protein interactions as part of our larger effort to understand how an organism coordinates DNA replication with repair. Two genetic selections we developed have resulted in identification of a collection of novel mutant b clamp proteins. Our ongoing structure-function studies of these mutant b clamps are providing insights into how b coordinates the actions of the molecular machines involved in DNA replication and repair.

Two FPLCs, BIAcore X

• Escherichia coli
• Pseudomonas aeruginosa

• Cancer
• Antimicrobial applications
• Cystic fibrosis

• SPR (BIAcore)
• FPLC (protein purification, protein-protein interactions)
• Bacterial genetics (strain construction, genetic screens)

• Various types of overproduction constructs
• Various reagents for bacterial genetics (bacterial viral vectors etc.), purified E. coli and P. aeruginosa DNA replication/repair factors (DNA polymerases sliding clamp proteins clamp loader accessory factors)

• Yeast strain HF7c
• Yeast two-hybrid vectors: pGAD424
• pGBT9
• pGAD EGFP (pGAD424 with EGFP fusion for reverse Y2H)

http://www.smbs.buffalo.edu/bch/faculty/sutton.html