Biophysical analysis of Thermus aquaticus single-stranded DNA binding protein

¹ University of Munich
² Hannover Medical School

^* To whom correspondence should be addressed. E-mail: curth.ute{at}mh-hannover.de.

Submitted on September 7, 2007
Revised on October 17, 2007
Accepted on 7 November 2007

	Abstract

Due to their involvement in processes such as DNA replication, repair and recombination bacterial single-stranded DNA binding (SSB) proteins are essential for the survival of the bacterial cell. Whereas most bacterial SSB proteins form homotetramers in solution, dimeric SSB proteins were recently discovered in the Thermus/Deinococcus group. In this work we characterize the biophysical properties of the SSB protein from Thermus aquaticus (TaqSSB), which is structurally quite similar to the tetrameric SSB protein from E. coli (EcoSSB). Binding of TaqSSB and EcoSSB to single-stranded nucleic acids was found to be very similar in affinity and kinetics. Mediated by its highly conserved C-terminal region TaqSSB interacts with the subunit of E. coli DNA polymerase III with similar affinity as EcoSSB. Using analytical ultracentrifugation, we show that TaqSSB mutants are able to form tetramers in solution via arginine-mediated hydrogen bond interactions identified by us in the crystal packing of wild type TaqSSB. In EcoSSB, we identified a homologous arginine residue involved in the formation of higher aggregates and metastable highly cooperative ssDNA binding under low salt conditions.

Key Words: Analytical ultracentrifugation, Protein-DNA interaction, Protein-protein interaction, Structure-function relationship, X-ray analysis