Unlike most tumor suppressor genes, the most common genetic alterations in tumor protein p53 (TP53) are missense mutations. Mutant p53 protein is often abundantly expressed in cancers and specific allelic variants exhibit dominant-negative or gain-of-function activities in experimental models. To gain a systematic view of p53 function, we interrogated loss-of-function screens conducted in hundreds of human cancer cell lines and performed TP53 saturation mutagenesis screens in an isogenic pair of TP53 wild-type and null cell lines. We found that loss or dominant-negative inhibition of wild-type p53 function reliably enhanced cellular fitness. By integrating these data with the Catalog of Somatic Mutations in Cancer (COSMIC) mutational signatures database, we developed a statistical model that describes the TP53 mutational spectrum as a function of the baseline probability of acquiring each mutation and the fitness advantage conferred by attenuation of p53 activity. Collectively, these observations show that widely-acting and tissue-specific mutational processes combine with phenotypic selection to dictate the frequencies of recurrent TP53 mutations.

SignificanceThe “centromere paradox” refers to rapidly evolving and highly diverse centromere DNA sequences even in closely related eukaryotes. However, factors contributing to this rapid divergence are largely unknown. Here, we identified large regional, LTR retrotransposon-rich centromeres in a group of human fungal pathogens belonging to the Cryptococcus species complex. We provide evidence that loss-of-functional RNAi machinery and possibly cytosine DNA methylation trigger instability of the genome by activation of centromeric retrotransposons presumably suppressed by RNAi. We propose that RNAi, together with cytosine DNA methylation, serves as a critical determinant that maintains repetitive transposon-rich centromere structures. This study explores the direct link between RNAi and centromere structure evolution.

The stochasticity of chromosome organization was investigated by fluorescently labeling genetic loci in live Escherichia coli cells. In spite of the common assumption that the chromosome is well modeled by an unstructured polymer, measurements of the locus distributions reveal that the E. coli chromosome is precisely organized into a nucleoid filament with a linear order. Loci in the body of the nucleoid show a precision of positioning within the cell of better than 10% of the cell length. The precision of interlocus distance of genomically-proximate loci was better than 4% of the cell length. The measured dependence of the precision of interlocus distance on genomic distance singles out intranucleoid interactions as the mechanism responsible for chromosome organization. From the magnitude of the variance, we infer the existence of an as-yet uncharacterized higher-order DNA organization in bacteria. We demonstrate that both the stochastic and average structure of the nucleoid is captured by a fluctuating elastic filament model. chromosome segregation | chromosome structure | nucleoid structure | polymer physics P rokaryotic chromosomes are organized into a compact DNA-protein complex called the nucleoid (1, 2). The physical structure of chromosomes has functional consequences, for example it affects gene regulation from the simplest prokaryotes (1) to multicellular organisms (3). Nucleoid organization and condensation also appear to play a central role in chromosome segregation: Mutants with defective chromosome segregation are typically accompanied by abnormal nucleoid organization or condensation (2). Although a significant number of such genes have been identified by genetic screens, the mechanism by which these molecular players effect the cellular-scale nucleoid structure is not yet understood (2). Similarly, the mechanism by which prokaryotic chromosomes are segregated is still hotly debated (2, 4, 5). The apparent dispensability of a mitotic-spindle-like mechanism in chromosome segregation in Escherichia coli (2, 4) has led to speculation that nucleoid organization and segregation may be the result of several redundant mechanisms, including polymer physics-embodied by the combined effects of entropy, confinement, and excluded volume-rather than resulting from the action of dedicated cellular machinery alone (2, 6, 7). This paper complements earlier work by focusing on the measurement and theoretical interpretation of two classes of statistical measures of chromosome organization: (i) the distributions of the positions of individual loci within the cell; and (ii) the distributions of displacements between pairs of genetic loci. We argue that the measurement and analysis of these distributions sheds light on the mechanisms of chromosomal positioning that have not been revealed in earlier measurements. The cellular-scale structure of the circular Caulobacter crescentus chromosome has already been shown to be linearly organized between replication cycles, with the origin of replication at one pole and the ter...

A widely distributed family of small regulators, called C proteins, controls a subset of restriction-modification systems. The C proteins studied to date activate transcription of their own genes and that of downstream endonuclease genes; this arrangement appears to delay endonuclease expression relative to that of the protective methyltransferase when the genes enter a new cell. C proteins bind to conserved sequences called C boxes. In the PvuII system, the C boxes have been reported to extend from ؊23 to ؉3 relative to the transcription start for the gene for the C protein, an unexpected starting position relative to a bound activator. This study suggests that transcript initiation within the C boxes represents initial, C-independent transcription of pvuIICR. The major C protein-dependent transcript appears to be a leaderless mRNA starting farther downstream, at the initiation codon for the pvuIIC gene. This conclusion is based on nuclease S1 transcript mapping and the effects of a series of nested deletions in the promoter region. Furthermore, replacing the region upstream of the pvuIIC initiation codon with a library of random oligonucleotides, followed by selection for C-dependent transcription, yielded clones having sequences that resemble ؊10 promoter hexamers. The ؊35 hexamer of this promoter would lie within the C boxes. However, the spacing between C boxes/؊35 and the apparent ؊10 hexamer can be varied by ؎4 bp with little effect. This suggests that, like some other activator-dependent promoters, PpvuIICR may not require a ؊35 hexamer. Features of this transcription activation system suggest explanations for its broad host range.The genera hosting restriction-modification (RM) systems appear to comprise the majority of the prokaryotic world. RM systems play a variety of roles, ranging from the simply selfish (44, 54) through defense against bacteriophages (42, 51) to facilitating recombination (34, 36). While much remains to be learned about the functions of RM systems, our understanding of how they are regulated is even more limited. Most, if not all, RM system genes can move from cell to cell, either by residing on a plasmid or because of transduction, transformation, or Hfr-type conjugation. As a result, they all face a critical regulatory problem-how to ensure that a new host's DNA is protectively methylated before endonuclease activity appears. This problem is particularly acute for the type II RM systems, in which the methyltransferase (MTase) and endonuclease (REase) function independently.A subset of type II RM systems includes, in addition to genes for the MTase and REase, a third gene for a conserved regulator called the C (controller) protein. C proteins were originally discovered in the PvuII (58, 59) and BamHI (22, 57) RM systems and were then noted in several other systems as well