NDLI: Mechanistic analyses of site-specific degradation in DNA-RNA hybrids by prototypic DNA cleavers.

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Mechanistic analyses of site-specific degradation in DNA-RNA hybrids by prototypic DNA cleavers.

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Mechanistic analyses of site-specific degradation in DNA-RNA hybrids by prototypic DNA cleavers.

Content Provider	PubMed Central
Author	Bansal, M. Lee, J. S. Stubbe, J. Kozarich, J. W.
Abstract	Bleomycin (BLM) binding and chemistry are apparently sensitive to differences in nucleic acid conformation and could conceivably be developed as a probe for sequence-dependent elements of conformation. We report on the development of a new methodology to synthesize heterogeneous DNA-RNA hybrids of defined sequence and present the results of our comparative studies on the cleavage of DNA and DNA-RNA hybrids by four drugs: BLM, neocarzinostatin and esperamicins A1 and C. In the case of BLM with duplex DNA, purine-pyrimidine steps such as GT and GC, are consistently hit, as previously observed. However, in heterogeneous sequence hybrids, not all GC sites are recognized by the drug, although all GT sites are. Suppressed GC sites are consistently flanked by pyrimidines on both the 3' and 5' sides, suggesting that the BLM binding site in hybrids spans at least four bases. Kinetic isotope studies with specifically deuterated substrates (kH/kD = 1.2-4.0) and the effect of oxygen on the product profile are presented in support of a mechanism consistent with 4'-hydrogen abstraction in hybrids. The powerful double-labeled probe technique was extended to study the mechanism of action of other DNA degrading drugs on DNA-RNA hybrids. For neocarzinostatin, the sequence specificity lies in the AT-rich region for hybrids and is similar to that of DNA, however, the overall cleavage pattern for the hybrid is significantly different from that for the same sequence of DNA. In the hybrid, a stretch of AT residues is essential and the A sites are damaged to a greater extent than they are in DNA. However, no kinetic isotope effects are observed and, based on the product profile, the mechanism of degradation of the DNA strand of hybrids seems to be limited to abstraction of the 5'-hydrogen. For esperamicin A1, damage on the DNA strand of hybrids occurs exclusively via 5'-hydrogen abstraction in a non-rate determining step and primarily at A and T sites. Esperamicin C behaves similarly, exhibiting no isotope effects at 1', 4' and 5' positions. Overall, the differences observed in site-specific cleavage between the two substrates is proposed to be a result of conformational differences between the DNA strand of duplex DNA and DNA-RNA hybrids.
Starting Page	1836
File Format	PDF
ISSN	13624962
e-ISSN	13624962
Journal	Nucleic Acids Research
Issue Number	9
Volume Number	25
Language	English
Publisher Date	1997-05-01
Access Restriction	Open
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Genetics

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