TY - JOUR
T1 - Interaction of T4 endonuclease V with DNA. Importance of the flexible loop regions in protein-DNA interaction
AU - Ahn, Hee Chul
AU - Ohkubo, Tadayasu
AU - Iwai, Shigenori
AU - Morikawa, Kosuke
AU - Lee, Bong Jin
PY - 2003/8/15
Y1 - 2003/8/15
N2 - T4 endonuclease V (T4 endo V), a thymine dimer-specific DNA repair enzyme, and its interaction with DNA were investigated by nuclear magnetic resonance (NMR) spectroscopy. Backbone resonance assignment, chemical shift mapping, and 15N relaxation measurements were employed to the free and DNA-bound enzymes. The secondary structure and the tertiary fold of T4 endo V in solution were consistent with those from the crystallographic study. The backbone 1H and 15N chemical shift perturbation upon the addition of DNA without a lesion revealed that the residues including Arg3, Arg22-Arg26, Lys45-Phe60, and Lys86-Thr88 participate in DNA binding. However, when DNA with a lesion was added to the enzyme and concomitantly the catalytic reaction was completed, the resonances of Arg22, Glu23, and Arg26, which constitute the catalytic active site, and the resonance of Thr88, were perturbed in a different manner. The region around Lys45-Ser47 was found to be involved in DNA binding, which have not been reported elsewhere. The backbone relaxation measurements of the free and DNA-bound enzymes indicated that two loop regions, Lys 45-Phe60 and Lyss86-Asp92, show the high degree of backbone flexibility. These results imply that two flexible loop regions may play an important role in DNA binding and in scanning along DNA duplex to search the thymine dimer sites in UV-damaged DNA.
AB - T4 endonuclease V (T4 endo V), a thymine dimer-specific DNA repair enzyme, and its interaction with DNA were investigated by nuclear magnetic resonance (NMR) spectroscopy. Backbone resonance assignment, chemical shift mapping, and 15N relaxation measurements were employed to the free and DNA-bound enzymes. The secondary structure and the tertiary fold of T4 endo V in solution were consistent with those from the crystallographic study. The backbone 1H and 15N chemical shift perturbation upon the addition of DNA without a lesion revealed that the residues including Arg3, Arg22-Arg26, Lys45-Phe60, and Lys86-Thr88 participate in DNA binding. However, when DNA with a lesion was added to the enzyme and concomitantly the catalytic reaction was completed, the resonances of Arg22, Glu23, and Arg26, which constitute the catalytic active site, and the resonance of Thr88, were perturbed in a different manner. The region around Lys45-Ser47 was found to be involved in DNA binding, which have not been reported elsewhere. The backbone relaxation measurements of the free and DNA-bound enzymes indicated that two loop regions, Lys 45-Phe60 and Lyss86-Asp92, show the high degree of backbone flexibility. These results imply that two flexible loop regions may play an important role in DNA binding and in scanning along DNA duplex to search the thymine dimer sites in UV-damaged DNA.
UR - http://www.scopus.com/inward/record.url?scp=0042232223&partnerID=8YFLogxK
U2 - 10.1074/jbc.M210939200
DO - 10.1074/jbc.M210939200
M3 - Article
C2 - 12783877
AN - SCOPUS:0042232223
SN - 0021-9258
VL - 278
SP - 30985
EP - 30992
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 33
ER -