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 α-NH 2 group and C-1â² of the 5â²-residue within the dimer. Sodium borohydride was used to covalently trap endonucleas...
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Published in | The Journal of biological chemistry Vol. 270; no. 8; pp. 3765 - 3771 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Biochemistry and Molecular Biology
24.02.1995
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Subjects | |
Online Access | Get full text |
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Summary: | 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 α-NH 2 group and C-1â² of the 5â²-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. |
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Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.270.8.3765 |