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BackgroundPapillary renal-cell carcinoma, which accounts for 15 to 20% of renal-cell carcinomas, is a heterogeneous disease that consists of various types of renal cancer, including tumors with indolent, multifocal presentation and solitary tumors with an aggressive, highly lethal phenotype. Little is known about the genetic basis of sporadic papillary renal-cell carcinoma, and no effective forms of therapy for advanced disease exist.MethodsWe performed comprehensive molecular characterization of 161 primary papillary renal-cell carcinomas, using whole-exome sequencing, copy-number analysis, messenger RNA and microRNA sequencing, DNA-methylation analysis, and proteomic analysis.ResultsType 1 and type 2 papillary renal-cell carcinomas were shown to be different types of renal cancer characterized by specific genetic alterations, with type 2 further classified into three individual subgroups on the basis of molecular differences associated with patient survival. Type 1 tumors were associated with MET alterations, whereas type 2 tumors were characterized by CDKN2A silencing, SETD2 mutations, TFE3 fusions, and increased expression of the NRF2-antioxidant response element (ARE) pathway. A CpG island methylator phenotype (CIMP) was observed in a distinct subgroup of type 2 papillary renal-cell carcinomas that was characterized by poor survival and mutation of the gene encoding fumarate hydratase (FH).ConclusionsType 1 and type 2 papillary renal-cell carcinomas were shown to be clinically and biologically distinct. Alterations in the MET pathway were associated with type 1, and activation of the NRF2-ARE pathway was associated with type 2; CDKN2A loss and CIMP in type 2 conveyed a poor prognosis. Furthermore, type 2 papillary renal-cell carcinoma consisted of at least three subtypes based on molecular and phenotypic features. (Funded by the National Institutes of Health.).

Abstract: Escherichia coli DNA polymerase II (pol-II) is a highly conserved protein that appears to have a role in replication restart, as well as in translesion synthesis across specific DNA adducts under some conditions. Here, we have investigated the effects of elevated expression of pol-II (without concomitant SOS induction) on translesion DNA synthesis and mutagenesis at 3,N 4-ethenocytosine (ɛC), a highly mutagenic DNA lesion induced by oxidative stress as well as by exposure to industrial chemicals such as vinyl chloride. In normal cells, survival of transfected M13 single-stranded DNA bearing a single ɛC residue (ɛC-ssDNA) is about 20% of that of control DNA, with about 5% of the progeny phage bearing a mutation at the lesion site. Most mutations are C→A and C→T, with a slight predominance of transversions over transitions. In contrast, in cells expressing elevated levels of pol-II, survival of ɛC-ssDNA is close to 100%, with a concomitant mutation frequency of almost 99% suggesting highly efficient translesion DNA synthesis. Furthermore, an overwhelming majority of mutations at ɛC are C→T transitions. Purified pol-II efficiently catalyzes translesion synthesis at ɛC in vitro, accompanied by high levels of mutagenesis with the same specificity. These results suggest that the observed in vivo effects in pol-II over-expressing cells are due to pol-II-mediated DNA synthesis. Introduction of mutations in the carboxy terminus region (β interaction domain) of polB eliminates in vivo translesion synthesis at ɛC, suggesting that the ability of pol-II to compete with pol-III requires interaction with the β processivity subunit of pol-III. Thus, pol-II can compete with pol-III for translesion synthesis. [Copyright &y& Elsevier]

UVM is an SOS-independent inducible response characterized by elevated mutagenesis at a sitespecific 3, N[sup 4]-ethenocytosine (εC) residue borne on M13 single-stranded DNA transfected into Escherichia coli cells pretreated with DNA-damaging agents. By constructing and using E. coli strain AM124 (polA polB umuDC dinB lexA1[Ind-]), we show here that the UVM response is manifested in cells deficient for SOS induction, as well as for all four of the 'nonreplicative' DNA polymerases, namely DNA polymerase I (polA), II (polB), IV (dinB) and V (umuDC). These results confirm that UVM represents a novel, previously unidentified cellular response to DNAdamaging agents. To address the question as to whether the UVM response is accompanied by an error-prone DNA replication activity, we applied a newly developed in vitro replication assay coupled to an in vitro mutation analysis system. In the assay, circular M13 single-stranded DNA bearing a sitespecific lesion is converted to circular doublestranded replicative-form DNA in the presence of cell extracts and nucleotide precursors under conditions that closely mimic M13 replication in viva. The newly synthesized (minus) DNA strand is selectively amplified by ligation-mediated polymerase chain reaction (LM-PCR), followed by a multiplex sequence analysis to determine the frequency and specificity of mutations. Replication of DNA bearing a site-specific εC lesion by cell extracts from uninduced E. coli AM124 cells results in a mutation frequency of about 13%. Mutation frequency is elevated fivefold (to 58%) in cell extracts from UVM-induced AM124 cells, with C → A mutations predominating over C → T mutations, a specificity similar to that observed in viva. These results, together with previously reported data, suggest that the UVM response is mediated through the induction of a transient error-prone DNA replication activity and that a modification of DNA polymerase III or the expression of a pre... [ABSTRACT FROM AUTHOR]

A base substitution mutation (mutA) in the Escherichia coli glyV tRNA gene potentiates asp → gly mistranslation and confers a strong mutator phenotype that is SOS independent, but requires recA, recB and recC genes. Here, we demonstrate that mutA cells express an error-prone DNA polymerase by using an in vitro experimental system based on the conversion of phage M13 single-stranded viral DNA bearing a model mutagenic lesion to the double-stranded replicative form. Amplification of the newly synthesized strand followed by multiplex DNA sequence analysis revealed that mutation fixation at 3,N4-ethenocytosine (ℇC) was ≈3% when the DNA was replicated by normal cell extracts, ≈48% when replicated by mutA cell extracts and ≈3% when replicated by mutA recA double mutant cell extracts, in complete agreement with previous in vivo results. Mutagenesis at undamaged DNA sites was significantly elevated by mutA cell-free extracts in the M13 lacZ(α) forward mutagenesis system. Neither polA (DNA polymerase I) nor polB (DNA polymerase II) genes are required for the mutA phenotype, suggesting that the phenotype is mediated through a modification of DNA polymerase III or the activation of a previously unidentified DNA polymerase. These findings define the major features of a novel mutagenic pathway and imply the existence of previously unrecognized links between translation, recombination and replication. [ABSTRACT FROM AUTHOR]

The mapping of mutA and mutC mutator alleles to the glyV and glyW glycine tRNA genes, respectively, and the subsequent discovery that the mutA phenotype is abolished in a ΔrecA strain raise the possibility that asp → gly misinsertion may induce a novel mutagenic pathway. The recA requirement suggests three possibilities: (i) the SOS mutagenesis pathway is activated in mutA cells; (ii) loss of recA function interferes with mutA-promoted asp → gly misinsertion; or (iii) a hitherto unrecognized recA-dependent mutagenic pathway is activated by translational stress. By assaying the expression levels of a reporter plasmid bearing a umuC ::lacZ fusion, we show that the SOS regulon is not in a derepressed state in mutA cells. Neither overexpression of the lexA gene through a multicopy plasmid nor replacement of the wild-type lexA allele with the lexA1[Ind-] allele interferes with the expression of the mutA phenotype. The mutA phenotype is unaffected in cells defective for dinB, as shown here, and is unaffected in cells defective for umuD and umuC genes, as shown previously. We show that mutA-promoted asp → gly misinsertion occurs in recA[sup -] cells and, therefore, the requirement for recA is ‘downstream’ of mistranslation. Finally, we show that the mutA phenotype is abolished in cells deficient for recB, suggesting that cellular recombination functions may be required for the expression of the mutator phenotype. We propose that translational stress induces a previously unrecognized mutagenic pathway in Escherichia coli. [ABSTRACT FROM AUTHOR]