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Author Budiman, Michael E
Title DNA specificity and biological impact of a platinum acridinylthiourea conjugate
book jacket
Descript 123 p
Note Source: Dissertation Abstracts International, Volume: 67-02, Section: B, page: 0865
Adviser: Rebecca W. Alexander
Thesis (Ph.D.)--Wake Forest University, 2006
This dissertation focuses on the biological and chemical aspects of a novel platinum-acridinylthiourea conjugate, PT-ACRAMTU. This unique platinum compound is found to break the long-lasting paradigm of DNA-binding platinum compounds in that it modifies adenine in the minor groove. The capacity for minor groove modification lies in the novel design of PT-ACRAMTU itself, which instead of having two leaving groups attached to the platinum has only one leaving group and an intercalator. Interestingly, it has also been shown to possess anti-cancer activity against the human ovarian carcinoma cancer cell line C13*, which is cisplatin resistant. These findings prompted us to investigate how PT-ACRAMTU differs from cisplatin with respect to sequence specificity of platination. Furthermore, we set out to characterize the interactions of several cellular proteins with PT-ACRAMTU-modified DNA
In vitro biological studies such as enzymatic cleavage protection and footprinting assays were employed to answer questions regarding sequence preference. Several restriction endonucleases were used to compare cisplatin and PT-ACRAMTU mediated cleavage protection. When platination took place at certain sequences in the DNA, inhibition of cleavage was observed. Following treatment of plasmid DNA with PT-ACRAMTU, linearization by either EcoRI (G↓AATTC) or DraI (TTT↓AAA) was inhibited. To further elucidate the DNA damage profile, RNA transcriptional footprinting was employed. RNA transcriptional footprinting was chosen instead of standard DNA footprinting as the former does not require a denaturing step and because PT-ACRAMTU has been shown to inhibit T7 RNA polymerase-catalyzed in vitro transcription. The footprinting profile of PT-ACRAMTU damage, with modification at 5'-TA, 5'-GA, and 5'-CG, is complementary to cisplatin damage, which occurs at 5'-GG, 5'-GC, and 5'-GA sites. Transcriptional footprinting also reveals that PT-ACRAMTU adduct formation is faster than cisplatin, suggesting that PT-ACRAMTU binding is dictated more by the acridine intercalator than the platinum moiety
Alternating TA sequences are critical for transcription initiation of both prokaryotic and eukaryotic genes. The conserved TATA sequence within the eukaryotic RNA polymerase II promoter serves as a binding site for RNA polymerase and transcription factors such as the TATA binding protein (TBP) subunit of transcription factor IIID. (Abstract shortened by UMI.)
School code: 0248
DDC
Host Item Dissertation Abstracts International 67-02B
Subject Chemistry, Biochemistry
Chemistry, Pharmaceutical
0487
0491
Alt Author Wake Forest University
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