Photo-cross-linking and identification of nuclear proteins that bind to DNA containing a site-specific adduct of cis-[Pt(NH₃)(N-6-aminohexyl)-4-benzophenonamide)CI₂]

cis-Diamminedichloroplatinum(II), or cisplatin, is the most successful cancer drug ever discovered. It has been used for over 30 years in the treatment of several types of malignancies, with the highest success rates in testicular cancer patients. Despite the triumphs of this drug, the exact mechanisms of cell-killing by cisplatin are not fully understood. A more complete understanding of the ability of this drug to target and destroy cancer cells will lead to advances in overcoming the intrinsic and acquired resistance that many cancer patients encounter with cisplatin. Cisplatin can form several distinct DNA adducts, which are toxic to cells if not repaired. By identifying proteins that bind to platinum-modified DNA, the work in this thesis focuses on the early stages of DNA adduct processing. The first chapter of this thesis gives a review of the identification of proteins with an affinity for platinum-modified DNA in the literature. Different techniques are discussed, and the proteins identified by these methodologies are discussed. The methodologies previously used to identify proteins with an affinity for platinum-modified DNA have led to important advances in our knowledge of the cellular processing of these adducts. The development of an assay that can survey the entire nuclear milieu, allowing for multiprotein complexes to stay intact and proteins to compete for binding to the damaged DNA has been difficult. These challenges were addressed by using a photo-active cisplatin analogue, cis-[Pt(NH3)(N-(6-aminohexyl)-4-benzophenonamide)C12] (PtBP6). Site-specifically modified DNA probes were synthesized and used to identify proteins photo-cross-linked by the benzophenonemodified platinum complex. A 25-bp probe containing a 1,2-d(GpG) and 1,3-d(GpTpG) intrastrand adduct of PtBP6 were synthesized and used in photo-cross-linking experiments. The results demonstrated that the 1,2-d(GpG) adduct binds more strongly to HMG-domain proteins, and the 1,3-d(GpTpG) adduct exclusively binds a nucleotide excision repair protein RPA1.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2008

Author dc:creator

• Guggenheim, Evan R

Advisor dc:contributor.advisor

• Stephen J. Lippard.

• 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/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/46040

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/46040