Endonuclease V from bacteriophage T4 interacts with its substrate in the minor groove.
The binding of bacteriophage T4 endonuclease V to its substrate has been studied using synthetic oligodeoxyribonucleotide duplexes containing a cis-syn thymine dimer. Substrate analogues containing a methylphosphonate linkage with a defined configuration at the thymine dimer site were prepared, and the binding of the enzyme to each diastereomer was analyzed by the filter-binding method. The duplex containing a methylphosphonate with the SP configuration formed a complex with the enzyme, although the dissociation constant for this substrate analogue was about 8 times larger than that for the 12-mer substrate containing a phosphodiester linkage at this site. In contrast, no binding was observed when a duplex containing the RP-methylphosphonate linkage was used. The glycosyl bond of the thymine dimer in the SP isomer was cleaved by the enzyme, while no incision was detected in the case of the RP isomer, even after alkali treatment. Another substrate analogue containing a sulfur atom in place of the 3'-oxygen of the 5'-component at the thymine dimer site showed a reduced affinity for the enzyme. These results suggest that T4 endonuclease V interacts with its substrate in the minor groove. This mode of binding was confirmed by methylation protection experiments.
Iwai
S
S
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Maeda
M
M
Shimada
Y
Y
Hori
N
N
Murata
T
T
Morioka
H
H
Ohtsuka
E
E
D017122:Bacteriophage T4 / Q000201:enzymology*; D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis / Q000378:metabolism*; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000378:metabolism*; D008745:Methylation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009943:Organophosphorus Compounds / Q000737:chemistry; D011485:Protein Binding; D013379:Substrate Specificity; D013455:Sulfur / Q000737:chemistry; D013941:Thymine / Q000737:chemistry; D014764:Viral Proteins
D009943:Organophosphorus Compounds; D014764:Viral Proteins; C032627:methylphosphonic acid; D013455:Sulfur; D004247:DNA; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer); D013941:Thymine