T4 endonuclease V protects the DNA strand opposite a thymine dimer from cleavage by the footprinting reagents DNase I and 1,10-phenanthroline-copper.
The glycosylase/abasic lyase T4 endonuclease V initiates the repair of ultraviolet light-induced pyrimidine dimers. This enzyme forms an imino intermediate between its N-terminal alpha-NH2 group and C-1' of the 5'-residue within the dimer. Sodium borohydride was used to covalently trap endonuclease V to a 49-base pair oligodeoxynucleotide containing a site-specific cyclobutane thymine dimer. The bound and free oligonucleotides were then subjected to nuclease protection assays using DNase I and a complex of 1,10-phenanthroline-copper. There was a large region of protection from both nucleases produced by endonuclease V evident on the strand opposite and asymmetrically opposed to the dimer. Little protection was seen on the dimer-containing strand. The existence of a footprint with the 1,10-phenanthroline-copper cleavage agent indicated that endonuclease V was interacting with the DNA predominantly via the minor groove. Methylation by dimethyl sulfate yielded no areas of protection when endonuclease V was covalently attached to the DNA, indicating that the protein may closely approach the DNA without direct contact with the bases near the thymine dimer. The Escherichia coli proteins Fpg and photolyase display a very different pattern of nuclease protection on their respective substrates, implying that endonuclease V recognizes pyrimidine dimers by a novel mechanism.
Latham
K A
KA
Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232.
Taylor
J S
JS
Lloyd
R S
RS
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D043224:Deoxyribonuclease (Pyrimidine Dimer); D003850:Deoxyribonuclease I / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D006868:Hydrolysis; D008745:Methylation; D008969:Molecular Sequence Data; D010618:Phenanthrolines / Q000378:metabolism*; D013941:Thymine / Q000378:metabolism*; D014764:Viral Proteins
D010618:Phenanthrolines; D014764:Viral Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer); D013941:Thymine; C025205:1,10-phenanthroline