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J Biol Chem, Vol. 275, Issue 18, 13235-13242, May 5, 2000

Interaction of UvrA and UvrB Proteins with a Fluorescent Single-stranded DNA
IMPLICATION FOR SLOW CONFORMATIONAL CHANGE UPON INTERACTION OF UvrB WITH DNA^*

Atsushi Yamagata, Ryoji Masui, Ryuichi Kato, Noriko Nakagawa^§, Hiroaki Ozaki^¶, Hiroaki Sawai^¶, Seiki Kuramitsu^**, and Keiichi Fukuyama

From the  Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, ^¶ Department of Chemistry, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515,  Harima Institute/SPring-8, the Institute of Physical and Chemical Research (RIKEN), Sayo-gun, Hyogo 679-5148, and ^** Genomic Sciences Center, RIKEN, Tsukuba, Ibaraki 305-0074, Japan

UvrA and UvrB proteins play key roles in the damage recognition step in the nucleotide excision repair. However, the molecular mechanism of damage recognition by these proteins is still not well understood. In this work we analyzed the interaction between single-stranded DNA (ssDNA) labeled with a fluorophore tetramethylrhodamine (TMR) and Thermus thermophilus HB8 UvrA (ttUvrA) and UvrB (ttUvrB) proteins. TMR-labeled ssDNA (TMR-ssDNA) as well as UV-irradiated ssDNA stimulated ATPase activity of ttUvrB more strongly than did normal ssDNA, indicating that this fluorescent ssDNA was recognized as damaged ssDNA. The addition of ttUvrA or ttUvrB enhanced the fluorescence intensity of TMR-ssDNA, and the intensity was much greater in the presence of ATP. Fluorescence titration indicated that ttUvrA has higher specificity for TMR-ssDNA than for normal ssDNA in the absence of ATP. The ttUvrB showed no specificity for TMR-ssDNA, but it took over 200 min for the fluorescence intensity of the ttUvrB-TMR-ssDNA complex to reach saturation in the presence of ATP. This time-dependent change could be separated into two phases. The first phase was rapid, whereas the second phase was slow and dependent on ATP hydrolysis. Time dependence of ATPase activity and fluorescence polarization suggested that changes other than the binding reaction occurred during the second phase. These results strongly suggest that ttUvrB binds ssDNA quickly and that a conformational change in ttUrvB-ssDNA complex occurs slowly. We also found that DNA containing a fluorophore as a lesion is useful for directly investigating the damage recognition by UvrA and UvrB.

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