Kip E. Guja

1st Year Medical Student

Department: School of Medicine

Graduate Program: TBD

Advisor: TBD

Abstract - Master's Research (undergraduate):

Advisor: Dr. Joel F. Schildbach, Department of Biology, Johns Hopkins University

Title:  Specificity determinants of F transfer initiation and of F TraI relaxase DNA recognition and cleavage

Kip Guja1, Matthew Harley, Katherine Hekman2, Chris Larkin3 and Joel F. Schildbach
Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA
Abstract

Conjugative plasmid transfer is an important mechanism for diversifying prokaryotic genomes and disseminating antibiotic resistance. Relaxases are conjugative plasmid-encoded proteins essential for plasmid transfer. Relaxases bind and cleave one plasmid strand site- and sequence-specifically before transfer of the cleaved strand. TraI36, a domain of F plasmid TraI that contains relaxase activity, binds a plasmid sequence in single-stranded form with subnanomolar KD and high sequence specificity. Despite 91% amino acid sequence identity, TraI36 domains from plasmids F and R100 discriminate between binding sites. The binding sites differ by 2 of 11 bases, but both proteins bind their cognate site with three orders of magnitude higher affinity than the other site. To identify specificity determinants, we generated several F and R100 TraI36 variants. Our results demonstrate that F-like relaxases can switch between highly sequence-specific recognition of different sequences with minimal amino acid substitution. The crystal structure of the F TraI36 with bound single-stranded DNA suggests binding specificity is also partly determined by an intrastrand three-way base-pairing interaction. We showed previously that single substitutions for the three interacting bases could significantly reduce binding. Here we demonstrate that many single and double base substitutions at these positions also reduce plasmid transfer, although the detrimental effects of some substitutions can be partially overcome by substitutions at a second site. We measured the affinity of the F TraI relaxase domain for several DNA sequence variants. While reduced transfer generally correlates with reduced binding affinity, some oriT variants transfer with an efficiency different than expected from their binding affinities, indicating ssDNA binding and cleavage do not correlate absolutely. Oligonucleotide cleavage assay results suggest the essential function of the three-base interaction may be to position the scissile phosphate for cleavage, rather than to directly contribute to binding affinity.

Present address: 1School of Medicine at Stony Brook University Medical Center, Stony Brook, NY 11794, USA. 2Pritzker School of Medicine, The University of Chicago, 924 East 57th Street, Chicago, IL 60637, USA. 3Laboratory of Molecular Biology, NIDDK, NIH, Bethesda, MD 20892, USA.

Publications:
(MSTP-supported publications indicated with an *)

Guja, K. and Schildbach, J.F. Fine Specificity Determinants of Single-stranded DNA Recognition by F and R100 Relaxases. BMC Microbiology. 2009, in press.

Anderson, B. J., Larkin, C., Guja, K., and Schildbach, J. F. Using fluorophore-labeled oligonucleotides to measure affinities of protein-DNA interactions. Methods in Enzymology. 2008, 450:253-272.

Hekman, K., Guja, K., Larkin, C., and Schildbach, J. F. An intrastrand three-DNA-base interaction is a key specificity determinant of F transfer initiation and of F TraI relaxase DNA recognition and cleavage. Nucleic Acids Research. 2008, 36(14):4565-4572.