Mechanistic studies of bleomycin-mediated double-stranded DNA cleavage and structural studies of DNA containing normal and 4'-oxidized abasic sites

In order to examine the role of partial intercalation in double-stranded (ds) DNA cleavage mediated by a single bleomycin (BLM), a bulky group ([-cyclodextrin) was chemically attached to the polyamine tail of BLM A5 to prevent the intercalation of the bithiazole tail. The ability of this analog (CD-BLM) to effect ds-DNA cleavage was quantitatively analyzed using the internally [132P-labeled hairpin DNA technology and the supercoilded DNA relaxation assay. CD-BLM can mediate both ss and ds-DNA cleavage, although 5-fold less efficient than BLM A5. Analysis of DNA cleavage by CD-BLM with competitive BLM-Co(III)-OOH indicates that the ds-DNA cleavage is mediated by two CD-BLM molecules, suggesting that the partial intercalation is essential for one BLM molecule to mediate ds-DNA cleavage. A "hot spot" for blunt-ended ds-cleavage by BLM (5'-GTCA-3'/3'-CAGT-5') has been identified in an effort to obtain structural insights into the mechanism of the blunt-ended ds-DNA cleavage. A 3'-phosphoglycolate/5'-phosphate (3'PG/5'P) gapped lesion in this "hot spot" has been synthesized to probe the structural basis for re-organization of BLM to the second cleavage site. This lesion was titrated with BLM-Co(III)-OOH. The resulting mixture of complexes was in fast exchange on the NMR time scale, which precluded further structural characterization.

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Chemistry

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2006

Author dc:creator

• Chen, Jingyang, Ph. D. Massachusetts Institute of Technology

Advisor dc:contributor.advisor

• JoAnne Stubbe.

• Chemistry.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/34498
• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Identifier URI

http://dspace.mit.edu/handle/1721.1/34498

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/34498