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MutL assists the mismatch recognition protein MutS to initiate and coordinate mismatch repair in species ranging from bacteria to humans. The MutL N-terminal ATPase domain is highly conserved, but the C-terminal region shares little sequence similarity among MutL homologs. We report here the crystal structure of the Escherichia coli MutL C-terminal dimerization domain and the likelihood of its conservation among MutL homologs. A 100-residue proline-rich linker between the ATPase and dimerization domains, which generates a large central cavity in MutL dimers, tolerates sequence substitutions and deletions of one-third of its length with no functional consequences in vivo or in vitro. Along the surface of the central cavity, residues essential for DNA binding are located in both the N- and C-terminal domains. Each domain of MutL interacts with UvrD helicase and is required for activating the helicase activity. The DNA-binding capacity of MutL is correlated with the level of UvrD activation. A model of how MutL utilizes its ATPase and DNA-binding activities to mediate mismatch-dependent activation of MutH endonuclease and UvrD helicase is proposed

The Polycomb Repressive Complex 2 (PRC2) mediates epigenetic gene silencing by trimethylating histone H3 lysine 27 (H3K27me3) and is known to aberrantly silence tumor suppressor genes in cancer. EZH2, the catalytic subunit of PRC2, enhances tumorigenesis and is commonly overexpressed in several types of cancer. Our microRNA profiling of bladder transitional cell carcinoma (TCC) patient samples revealed that microRNA-101 (miR-101) is down-regulated in TCC, and we showed that miR-101 inhibits cell proliferation and colony formation in TCC cell lines. Furthermore, our results confirm that miR-101 directly represses EZH2 and stable EZH2 knockdowns in TCC cell lines create a similar growth suppressive phenotype. This suggests that abnormal down-regulation of miR-101 could lead to the overexpression of EZH2 frequently seen in cancer. We conclude that miR-101 may be a potent tumor suppressor by altering global chromatin structure through repression of EZH2. [Cancer Res 2009;69(6):2623-9]

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It was recently shown that a large portion of the human transcriptome can originate from within repetitive elements, leading to ectopic expression of protein-coding genes. However the mechanism of transcriptional activation of repetitive elements has not been definitively elucidated. For the first time, we directly demonstrate that hypomethylation of retrotransposons can cause altered gene expression in humans. We also reveal that active LINE-1s switch from a tetranucleosome to dinucleosome structure, acquiring H2A.Z- and nucleosome-free regions upstream of TSSs, previously shown only at active single-copy genes. Hypomethylation of a specific LINE-1 promoter was also found to induce an alternate transcript of the MET oncogene in bladder tumors and across the entire urothelium of tumor-bearing bladders. These data show that, in addition to contributing to chromosomal instability, hypomethylation of LINE-1s can alter the functional transcriptome and plays a role not only in human disease but also in disease predisposition.

Urothelial cancer (UC) develops along two different genetic pathways, resulting in noninvasive or invasive tumors. However, it is unknown whether there are also different epigenetic pathways in UC. UC is also characterized by a high rate of recurrence, and the presence of a field defect has been postulated. In this study, we compared the DNA methylation patterns between noninvasive and invasive UC and the DNA methylation patterns between normal-appearing urothelium from bladders with cancer and urothelium from cancer-free bladders. We used the Illumina GoldenGate methylation assay at 1,370 loci in 49 noninvasive urothelial tumors, 38 invasive tumors with matched normal-appearing urothelium, and urothelium from 12 age-matched UC-free patients. We found distinct patterns of hypomethylation in the noninvasive tumors and widespread hypermethylation in the invasive tumors, confirming that the two pathways differ epigenetically in addition to genetically. We also found that 12% of the loci were hypermethylated in apparently normal urothelium from bladders with cancer, indicating an epigenetic field defect. X-chromosome inactivation analysis indicated that this field defect did not result in clonal expansion but occurred independently across the urothelium of bladders with cancer. The hypomethylation present in noninvasive tumors may counterintuitively provide a biological explanation for the failure of these tumors to become invasive. In addition, an epithelium-wide epigenetic defect in bladders with cancer might contribute to a loss of epithelial integrity and create a permissible environment for tumors to arise. Cancer Res; 70(20); 8169-78. ©2010 AACR.

We compared the distribution of mutations in rpoB that lead to rifampin resistance in strains with differing levels of polymerase IV (Pol IV), including strains with deletions of the Pol IV-encoding dinB gene, strains with a chromosomal copy of dinB, strains with the F128 plasmid, and strains with plasmid amplification of either the dinB operon (dinB-yafNOP) or the dinB gene alone. This analysis identifies several hot spots specific to Pol IV which are virtually absent from the normal spontaneous spectrum, indicating that Pol IV does not contribute significantly to mutations occurring during exponential growth in liquid culture.Damage-inducible polymerases (20, 22; for reviews, see references 9 and 16), such as the SOS-induced polymerase IV (Pol IV) and Pol V in Escherichia coli, not only bypass certain noncoding lesions but also increase replication errors across from normal bases (19,20,23). Their discovery has led to the suggestion that a significant fraction of spontaneous mutations in growing cells under normal conditions might be due to errors caused by basal levels of error-prone polymerases (18). The dinB-encoded Pol IV is the leading candidate, since the overexpression of dinB on high-copy plasmids leads to increases in base substitutions and frameshifts, particularly Ϫ1 frameshifts (11,12,23). Moreover, several studies have shown an approximately twofold decrease in spontaneous mutations in strains with an inactivating allele of dinB that also reduces the expression of three genes downstream of dinB-yafNOP (14, 18), although this effect is not present if only dinB is inactivated (14). The expression of dinB and yafNOP is increased after SOS induction by DNA-damaging agents (4), and these four genes have been shown to be part of an operon (14).We decided to examine the spectra of base substitution mutations in strains with differing levels of dinB expression, since a comparison of detailed genetic fingerprints of these strains might reveal patterns specific to processes involving and not involving Pol IV. We recently characterized a system using mutations in the rpoB gene that yield the rifampin resistance (Rif r ) phenotype at 37°C in order to analyze the base substitution profiles of mutagens and mutators (10). We have now characterized 77 mutations in rpoB. Each of the six base substitutions is monitored with a set of 9 to 17 sites. In the study reported here, we looked at cells that carry a single copy of the dinB operon on the chromosome and compared the mutational spectrum of these cells with those of strains with deletions of the dinB gene, strains that carry a second copy of the dinB operon on an FЈ plasmid, and strains that carry a multicopy plasmid with an insert containing the dinB operon in one case and just the dinB gene in another case. We showed that some mutational hot spots are specific for the overexpression of the dinB operon and that others are found in the spectrum of wild-type strains but not after the amplification of the dinB operon. A comparison of the different spectra leads us...