Sexta fase: Estudio financiero del proyecto

The major groove of DNA most often provides the best site for recognition and binding of proteins. This is achieved through its larger size which allows interaction with a greater number of base pairs and therefore access to greater functionality. In comparison with the minor

groove, there is an increased number of hydrogen bonding sites and greater scope for van der Waal interactions in the major groove. Upon complex formation, molecules binding in the major groove can often induce significant structural changes in the DNA. In comparison, the minor groove is more rigid and molecules binding in it rarely induce structural changes.

The helix turn helix and zinc finger are two of the motifs found in nature to bind selectively to DNA. Transcription of DNA is regulated by proteins that recognise specific DNA sequences through discrete DNA-binding domains in their polypeptide chains. The helix-turn-helix (HTH) motif is a common feature of most prokaryotic DNA-binding domains. An extended turn chain of amino acids connects the two α-helices which are held at a fixed angle. The HTH motif is typically about 20 amino acids long, the first 7 amino acids for the first helix, the turn is formed by amino acids 8 to 11 and the second (recognition) helix is formed by the remaining 9 amino acids. While these proteins are too big to fit into the minor groove, the recognition helix fits into the major groove of B-DNA. The sequence specificity of the DNA binding is

determined by direct interactions with the nucleotides of the groove. These interactions involve hydrogen bonding between amino acid side chains and the edges of the base pairs.96

Transcription of DNA in eukaryotic cells is controlled by site-specific DNA-binding proteins called transcription factors. The DNA-binding domains of transcription factors are built up of a very limited number of structural motifs. These include the leucine zipper and zinc finger.

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The zinc finger motif first described in 1985 by Klug and Rhodes contains nine repeated amino acid sequences of about 30 residues each, with a total of 344 residues in the whole transcription factor, the proteins also contain intrinsic zinc atoms which are essential for transcriptional activity97. They suggested that each zinc finger binds into the major groove of DNA. Klug and Rhodes proposed a model for DNA binding of zinc fingers whereby the protein lies on one face of the DNA helix with successive fingers pointing into the major groove alternately from opposite directions. Subsequent experimental studies have shown this model to be quite accurate. A zinc finger DNA complex from a crystal structure is represented schematically below.

Figure 1-15 Cartoon representation of a complex between DNA and the ZIF268 protein, containing 3 zinc

finger motifs. The coordinating residues of the middle zinc finger are highlighted. Based on the x-ray structure of PDB 1A1L98_.

The major groove, while providing binding sites for protein molecules, can also accommodate smaller molecules such as the well know anti-cancer drug, cisplatin. Cisplatin was established as a potent anti-leukamic agent in 1969 after a series of tests on neutral complexes by

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it to interact with the base pairs100. Competition binding studies with Ethidium bromide showed it to bind covalently to the bases rather than intercalating.

Cisplatins mechanism of action has been the focus of much research over the past few decades. It has been shown that cisplatin binds to purine bases with high selectivity. Furthermore, it binds preferentially at the N7 position in guanine inducing unstacking of the bases. Unstacking occurs at the Pt-binding site, causing the DNA to kink. In 1988, Sherman et al. obtained a

crystal structure for the major cisplatin-DNA adduct, cis-[Pt(NH3)2{d(pGpG)}], showing the

platinum metal centre coordinated in a square planar mode to two cis-Ammine ligands and two guanine N7 atoms in a manner that would lead to unstacking of the bases in duplex DNA101.

While cisplatin has been used successfully to cure a large number of patients since its introduction in 1979, it does produce toxic side effects and certain tumours can develop resistance to the drug. Second generation platinum drugs such as carboplatin and oxaliplatin have been developed. Carboplatin is the most widely used second generation drug, and whilst it offers greatly reduced side effects it is not as potent an anti-tumour agent as cisplatin. Over the past few years third generation, polynuclear platinum complexes have been developed and currently the possibility of developing anti-cancer drugs using transition metals other than platinum is being investigated102.

Despite the success of cisplatin and subsequent transition metal complexes in the treatment of cancer there is still a need to develop drugs that will bind in a sequence specific way to DNA, are efficient at low doses, produce few side effects, bind strongly enough to prevent DNA replication and will resist displacement by proteins103.

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