Mechanistic studies of bleomycin-mediated double-stranded DNA cleavage and structural studies of DNA containing normal and 4'-oxidized abasic sites
Name
70852081-MIT.pdf
Description
Full printable version
Size
29.26 MB
Format
Adobe PDF
Checksum (MD5)
18c72eb5151642bbccf52c03552e4e7e
Author(s)
Chen, Jingyang, Ph. D. Massachusetts Institute of Technology
Advisor(s)
JoAnne Stubbe.
Alternative Title
Structural studies of DNA containing normal and 4'-oxidized abasic sites
Date Issued
2006
Publisher
Massachusetts Institute of Technology
Abstract
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.
(cont.) In order to understand the mechanism(s) of recognition and repair of DNA lesions generated by BLM, a duplex DNA containing a 4'-oxidized abasic site (X) in d(CCAAAGXACCGGG)-d(CCCGGTACTTTGG) (1) was synthesized and characterized by 2D NMR spectroscopy and molecular modeling. The results indicate that the 4'- oxidized abasic site adopts an intrahelical conformation, in contrast to a normal abasic site in the same sequence context, which is partially extrahelical. A systematic structural characterization was performed using 2D NMR methods and molecular modeling on an oligonucleotide containing a normal abasic site (Y) with four different bases (A, G, T, or C) opposite the lesion in d(CCAAAGYACCGGG). The results suggest that the conformation in the abasic site region is more perturbed with a pyrimidine opposite the abasic site. This study provides the first structural insight into the dynamics of abasic sites that are intrinsically modulated by the opposite and neighboring bases.
Description
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2006.
Vita.
Includes bibliographical references.
Subjects
Chemistry.
MIT Department
Massachusetts Institute of Technology. Department of Chemistry
Terms of Use
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.
Persistent DSpace Link
