Book of Abstracts: Albany 2003
June 17-21 2003
Distribution of p53 Sites in the Human Genome Reflects the Versatility of p53 Binding and Its Tumor Suppressor Functions
The tetrameric p53 binding to DNA plays a key role in tumor suppression. In response to DNA damage and other types of cellular stress, the p53 protein becomes activated and binds DNA sequence-specifically, functioning as a transcriptional factor or cell cycle regulator. p53 is unique in regulating a wide spectrum of genes: at least several hundred human genes are either activated or repressed by p53. Normally, the p53 tetramer binds to DNA response elements, consisting of two decamers RRRCWWGYYY (half-sites) separated by a spacer. (The length of the spacer, S, varies from 0 to 14 bp in the known functional binding sites, but in most cases S=0 or 1.) How many putative p53 binding sites, consistent with this scheme, are there in the human genome? What is the distribution of the spacer lengths?
With the human genome sequence we can directly answer these questions. The distribution of spacers proves to be extremely nonuniform, with strong peaks in the profile, exceeding the average background 3-4 fold. Note that the peaks at S=0 and 10 bp, and the gap at 4-5 bp are consistent with the lateral positioning of the p53 core domains on the outer side of the DNA loop (1, 2). Indeed, if the two half-sites are adjacent to each other (S=0) or separated by a single turn of DNA helix (~10 bp), it is easier for two p53 dimers to form a p53 tetramer (Figure 1), than if the two dimers were separated by a half-turn (~5 bp) and hence were located on the opposite sides of the helix. In general, these data agree with our prediction that the p53 tetramer can bind DNA specifically without unwrapping nucleosomes (1, 2) and the observation that p53 interacts directly with DNA without chromatin remodeling in the course of transcriptional activation of the chromatin-assembled p21 genes (3).
1National Cancer Institute
Bethesda, MD 20892
2Iowa State University
Ames, IA 50011
3The Institute for Genomic Research
Rockville, MD 20850
Figure 1. Occurrences of the putative p53 sites in the human genome for various spacer lengths, S. Each half-site is selected to be consistent with the consensus rRRCWWGYYy (W is A or T; R is purine; Y is pyrimidine). The low case ?r? and ?y? indicate that the ?R/Y rule? may be broken at one of the decamer ends (i.e., in addition to the ?perfect? decamers we consider also the nanomers RRRCWWGYY- and -RRCWWGYYY). On the right: Schematic representation of the p53 dimers binding to the outer surface of the nucleosomal DNA loop for S=0 and 75 bp. Notice that in both cases, the protein N-termini interact with the neighboring p53 core domains, thereby stabilizing the p53 tetramer (1).
In addition to transactivation, p53 is directly involved in the control of both the G1/S and G2/M transitions in the cell cycle. We suggest the two highest peaks in Figure 1 (S=27 and 75) are related to these p53 activities. There are two features distinguishing the sites having S=27 and 75 bp from those described above. First, they are localized far away from the starts of transcription, and thus are unlikely to be involved in its regulation. Second, many of them are organized in multiple repeats (rRRCWWGYYy-spacer)N, where N is usually 5-10, but in a few cases increases up to ~100. When the spacer S=75, the two half-sites are separated by a superhelical turn of DNA in the nucleosome, such that the two p53 dimers (bound to two DNA decamers) can form a tetramer (Figure 1). This novel p53 tetramer would bridge the two DNA superhelical turns on the nucleosomal surface, thus forming a stabilizing clamp, especially strong when several such nucleosomes are arranged in a multiple ?solenoidal? repeat. The latter may serve as an effective roadblock for the replication fork, preventing replication of damaged genes before DNA repair is completed (G1/S arrest).
In contrast, the response elements with S=27 bp may be operative in arresting the cell cycle after replication (G2/M arrest) ? this would prevent segregation of defective chromosomes. Specifically, the repeating sites with S=27 bp may form sandwich-type structures similar to those observed by electron microscopy (4). These assemblages contain two identical duplexes separated by ~100 Å, and are hold together by several dozen p53 tetramers, each of which links together two identical DNA decamers (half-sites) from the sister chromosomes. In other words, the ?sandwich? would play the role of the second (post-replication) line of defense, if the first line (G1/S) were broken.
A significant fraction of the sites with S=27 and 75 bp belongs to the same family of mammalian LTR-transposons, THE1-MaLR. Probably, these p53 binding sites (S=27, 75) co-migrated in genome(s) interdependently, and their organization in the human genome is apparently consistent with a highly effective cellular response to DNA damage.
Zhurkin V. B.1*