Sequence-specific DNA Binding of

Central to the function of the p53 protein is its abUity, as a transcription activator, to specifically bind to corresponding cis elements in the promoter region of various genes and to activate their transcription. The importance of sequence-specific DNA binding for the tumor-suppressing function of p53 became clear when the crystal structure of the complex of p53 protein and a corresponding DNA element were resolved and this structure was compared with the spectrum of known mutations of p53 protein occurring in human tumors (Cho et al., 1994). 

The p53 protein is a tetrameric protein that binds to DNA elements with the consensus sequence 5 -RRRC(A/T)(T/A)GYYY-3 (R = pmine, Y = pyrimidine). The struc- 

The p53 protein contacts the recognition sequence by two means. A loop-helix-loop motif is placed in the large groove of the DNA and makes contact with the bases. In addition, another loop (L3) forms a contact via an Arg side chain (R248) to the minor groove of the DNA. 

The large number of known sequences of the p53 gene from tumor patients was particularly valuable for interpretation of the crystal structure since a spectrum could be assembled for p53 mutation in association with tumor formation. The mutation spectrum shown in Fig. 14.9 shows hotspots , positions at which p53 mutations are seen particularly frequently in tumor patients. 

Comparison of the mutation spectrum with the structure of the p53-DNA complex indicates that the positions of frequent mutations coincide with the conserved structu- 

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Acetylation ofp53 at distinct Lys-residues of the C-terminus is observed concomitant with transcriptional activation. This modification has been reported to enhance the sequence-specific DNA binding. It remains to be shown whether this acetylation is a side effect of recruitment of histone acetylases or whether it serves directly to modulate sequence-specific DNA binding of p53. 

One of the most important molecular functions of p53 is therefore to act as an activator of p21 transcription. The wild-type protein binds to specific DNA sequences, whereas tumor-derived p53 mutants are defective in sequence-specific DNA binding and consequently cannot activate the transcription of p5 3-con trolled genes. As we will see more than half of the over one thousand different mutations found in p53 involve amino acids which are directly or indirectly associated with DNA binding. 

The polypeptide chain of p53 is divided in three domains, each with its own function (Figure 9.16). Like many other transcription factors, p53 has an N-terminal activation domain followed by a DNA-binding domain, while the C-terminal 100 residues form an oligomerization domain involved in the formation of the p53 tetramers. Mutants lacking the C-terminal domain do not form tetramers, but the monomeric mutant molecules retain their sequence-specific DNA-binding properties in vitro. 

Larson, J.S., Schuetz, T.J., Kingston, R.E. (1988). Activation in vitro of sequence specific DNA binding by a human regulatory factor. Nature 335, 372-375. 

The amino acid 58 was used in the solid-phase synthesis of sequence-specific DNA binding polyamides containing N-methylimidazole and N-methylpyrrole amino acids <96JACS6141> and it was also reported that the imidazole-acridine conjugate 59 could effectively catalyze the cleavage of t-RNA <96TL4417>. 

The availability of sequence-specific DNA binding molecules led to the development of bifunctional polyamides that covalently react with the minor groove of DNA. Two classes of alkylating agents were conjugated to the hairpin turn unit and bound proximal to their alkylation sites by a DNA-binding polyamide [58, 59]. 

Song, Z., et al., A novel cysteine-ridi sequence-specific DNA-binding protein interacts with the conserved X-box motif of the human major histocompatibility complex dass II genes via a repeated Cys-His domain and functions as a transcriptional repressor. / Exp Med, 1994, 180(5), 1763-74. 

Gagne et al, 2003). Some non-sequence-specific DNA-binding proteins, such as the linker histone HI, may even have a greater affinity for PAR than DNA (Malanga et al., 1998). Thus, an understanding of PAR is an important aspect of understanding the biology of PARP-1 and other PARPs. 

Gu W, Roeder RG (1997) Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 90(4) 595-606... 

HMG-box proteins stimulate the binding of certain sequence-specific DNA-binding proteins to their DNA targets... 

The specific transcriptional activators (see 1.4.3.1), represented by the GAL4 protein of yeast, are sequence-specific DNA-binding proteins. They possess both a DNA-binding domain and a trans-activating domain to allow them to interact directly with the transcription apparatus. 

The principle means by which the activity of sequence-specific DNA-binding proteins is controlled have aheady been presented in section 1.2. The importance of these mechanisms for regulation in eucaryotes will be discussed below. Altogether, the demands on eucaryotic organisms with regard to the regulation of transcription activity are much more complex than for procaryotes. This tenet holds for the structure of the transcription apparatus as well as for the mechanism of transcription regulation. 

The steroid hormone receptors are sequence specific DNA binding proteins whose cognate DNA elements are termed hormone responsive elements" (HREs). The HREs known to date possess a common structure. They are composed primarily of two copies of a hexamer sequence. In table 4.1 are listed the hexamer sequences of the HREs of important nuclear receptors. 

Structure-function investigations have shown that the Smads have a sequence-specific DNA binding domain and two regulatory domains known as the MHl and MH2 domains. It is assiuned that the pathway-restricted Smads are kept in an inactive form by intramolecular interactions between the MHl and MH2 domains. The inactive conformation is lifted by phosphorylation at the C-terminus upon activation of the TGPp receptor, and an interaction with the conunon mediator Smad 4 becomes possible. According to these observations, the MHl domain functions as a negative regulator of the MH2 domain. 

Jayaraman, L. and Prives, C. Activation of p53 sequence-specific DNA binding by short single strands of DNA requires the p53 C-terminus (1995) Cell 81, 1021-1029... 

An important clue to c-myc function was the discovery in the conserved carboxy-terminal regions of three structural motifs, the leucine zipper (LZ), helix-loop-helix (HLH) and basic region (B). These motifs were originally defined in a number of other sequence-specific DNA-bind-ing proteins but had not previously been found within a single protein. 

Fu, Y.-H. Marzluf, G.A. (1990). nit-2, the major positively-acting nitrogen regulatory gene of Neurospora crassa, encodes a sequence-specific DNA-binding protein. Proceedings of the National Academy of Sciences (USA) 87, 5331-4. 

Hainaut, P. and Miller, J. 1993. Redox modulation of p53 conformation and sequence-specific DNA binding. Cancer Res. 53, 4469 1473. 

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