Nucleic Acids Research Advance Access published online on February 10, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp071
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Genome Integrity, Repair, and Replication |
Single-molecule analysis reveals two separate DNA-binding domains in the Escherichia coli UvrA dimer
1Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden and 2Physics of Life Processes, Leiden Institute of Physics, Leiden University, Niels Bohrweg 2, 2333 CA, Leiden, the Netherlands
*To whom correspondence should be addressed. Tel: +31715274773; Fax: +31715274340; Email: n.goosen{at}chem.leidenuniv.nl
Received December 17, 2008. Revised January 23, 2009. Accepted January 26, 2009.
The UvrA protein is the initial damage-recognizing factor in bacterial nucleotide excision repair. Each monomer of the UvrA dimer contains two ATPase sites. Using single-molecule analysis we show that dimerization of UvrA in the presence of ATP is significantly higher than with ADP or nonhydrolyzable ATP
S, suggesting that the active UvrA dimer contains a mixture of ADP and ATP. We also show that the UvrA dimer has a high preference of binding the end of a linear DNA fragment, independent on the presence or type of cofactor. Apparently ATP binding or hydrolysis is not needed to discriminate between DNA ends and internal sites. A significant number of complexes could be detected where one UvrA dimer bridges two DNA ends implying the presence of two separate DNA-binding domains, most likely present in each monomer. On DNA containing a site-specific lesion the damage-specific binding is much higher than DNA-end binding, but only in the absence of cofactor or with ATP. With ATPS no discrimination between a DNA end and a DNA damage could be observed. We present a model where damage recognition of UvrA depends on the ability of both UvrA monomers to interact with the DNA flanking the lesion.