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]
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008.In title on title page, "cis" appears as italic. Vita.Includes bibliographical references.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.(cont.) A probe containing a mismatched 1,2-d(GpG) adduct was also synthesized, and this DNA photo-cross-linked more strongly to the mismatch repair protein, Msh2. These results demonstrate that PtBP6-modified DNA mimics the behavior of cisplatin-modified DNA published in the literature. These experiments also identify PARP-1 as a protein with an affinity for platinum-modified DNA. This protein will bind to other types of DNA damage, but had not been previously implicated in platinum-DNA damage binding. Photo-cross-linking experiments were carried out using nuclear extracts from cancer cells with varying sensitivity to cisplatin. The results indicate that expression levels of PARP-1 may effect the cell's ability to survive cisplatin exposure. The proteins photo-cross-linked by different nuclear extracts were consistent, however, indicating that DNA damage recognition is not the mechanism for cisplatin resistance in these cell lines. In order to ensure that the proteins identified using the 25-bp probe were not binding to the blunt ends of the DNA and being photo-cross-linked, a 90 base dumbbell probe was synthesized. In this probe, the DNA contains no blunt ends but two loops, which are 19 bases from the platinum adduct. Photo-cross-linking experiments using this probe confirmed the binding of each of the proteins identified using the 25-bp probe. The photo-cross-linking of DDBI, RFC1 and RFC2 demonstrates that a longer platinummodified DNA probe is a better substrate for recognition by nucleotide excision repair proteins. This work also indicates that both mismatch repair recognition complexes Msh2/Msh3 and Msh2/Msh6 bind to platinum-damaged DNA. The affinity of chromatin remodeling proteins for platinum-damaged DNA was also established in this work. The characterization of PARP-1 as a protein with an affinity for platinumdamaged DNA was first discovered using photo-cross-linking experiments with PtBP6. Prior to this discovery, the ability of PARP inhibitors to sensitize cells to cisplatin had already been investigated.(cont.) Conflicting results are reported in the literature about the efficacy of these inhibitors as potentiators of cisplatin toxicity. In order to probe the mechanism of the interaction, assays were carried out using PARP inhibitors synthesized by Alison Ondrus of the Movassaghi lab. These experiments indicated that the activity of PARP proteins following exposure to platinum-modified DNA may be cell-line dependent. We were unable to detect a significant increase in PARP activity following exposure to cisplatin, however, in any cell line tested. These results suggest that more mechanistic studies into the effect of the activity of PARP proteins on cancer cells exposed to cisplatin are warranted. Initial photo-cross-linking experiments using a duplex containing a benzophenone moiety on the DNA indicated that this type of probe could be used to identify proteins with an affinity for various types of DNA lesions. Also, photo-cross-linking using a 157bp probe, site-specifically modified with PtBP6, indicated that there are some obstacles to overcome when performing these types of experiments with longer DNA probes. Initial NMR spectroscopic studies of a PtBP6-modified d(GpG) were carried out to characterize the orientational isomers of this type of DNA lesion.by Evan Ross Guggenheim.Ph.D
