Ranga N. Venkatesan

Bio: Ranga N. Venkatesan is an academic researcher from University of Washington. The author has contributed to research in topics: DNA polymerase & DNA replication. The author has an hindex of 5, co-authored 7 publications receiving 263 citations.

Mutation at the Polymerase Active Site of Mouse DNA Polymerase δ Increases Genomic Instability and Accelerates Tumorigenesis

Abstract: Eukaryotic DNA polymerase δ (Pol δ) is an essential, highly conserved enzyme that participates in DNA replication, DNA repair, and genetic recombination. Pol δ is believed to replicate a large portion of the genome, synthesizing most of the lagging strand and perhaps contributing to leading-strand synthesis as well (14, 22, 43) The 125-kDa catalytic subunit, encoded at the mouse Pold1 locus, contains a polymerase domain near the carboxyl terminus that catalyzes DNA synthesis and an exonuclease domain near the amino terminus that catalyzes 3′→5′ exonucleolytic proofreading (5, 18). Pol δ is highly processive in association with PCNA and synthesizes DNA with great accuracy, catalyzing about one error in 10−5 to 10−6 nucleotides polymerized (11, 46, 61). Discrimination between correct and incorrect base pairs at the polymerase active site confers most of the fidelity (error rate, ca. 1 × 10−5), while proofreading increases accuracy approximately 10- to 60-fold (11). Partitioning of the newly formed primer terminus between the polymerase and exonuclease active sites is an important determinant of accuracy as well (11, 47). In cells, mismatch repair adds an additional correction mechanism for errors that escape proofreading, reducing the overall error rate to about 1 × 10−9 bp (19, 35, 36, 55). Accurate DNA replication is essential for the maintenance of genomic stability and suppression of carcinogenesis. Mutants of prokaryotic and eukaryotic DNA polymerases that harbor amino acid substitutions in the polymerase domain or that lack proofreading exhibit increased mutation frequencies in vitro and in vivo (2, 8, 34, 42, 44, 47, 60). In the case of Pol δ, inactivation of the 3′→5′ exonuclease confers a mutator phenotype, increasing mutation rates in haploid yeast and in homozygous mouse embryo fibroblasts (MEFs) (7, 16, 17, 58). The homozygous mutant mice display elevated cancer incidence, suggesting that unrepaired Pol δ errors may contribute to tumorigenesis (16, 17). Given the essentiality of Pol δ and its central role in eukaryotic DNA replication, it is important to assess the nature and severity of the consequences of polymerase active site mutations in mammals. For this study, we created mice harboring mutations at a conserved Leu residue, L604, in motif A of the Pol δ polymerase domain. We elected to study L604 mutants because the effects on accuracy of mutation at homologous residues in other DNA polymerases have been studied extensively. For example, our laboratory has shown that substitution at the homologous Ile in both Escherichia coli Pol I and Taq Pol I creates mutator mutants (40, 41, 57). In phage T4 DNA polymerase, substitution for the homologous Leu can generate either mutator or antimutator mutants (47). In yeast and human DNA polymerase α (Pol α), substitution for the orthologous Leu generates mutators of various strengths (38). Most recently, we and others have shown that replacement of L612 in Saccharomyces cerevisiae Pol δ with each of eight different amino acids increases spontaneous mutation rates up to 37-fold (28, 62). The L612G and L612K yeast Pol δ alleles, which are homologous to the L604G and L604K mutations we studied here, elevated the mutation rate 17- and 13-fold, respectively (62). The present work represents the first description of mammals harboring a mutation in the polymerase domain of a major replicative DNA polymerase. Given the significant phenotypic defects we observed in haploid yeast (62) and the central role of Pol δ in replication, it was not certain that the murine L604G and L604K alleles would yield viable animals. However, when heterozygous, these mutator mutations are compatible with mammalian development and reproduction and do not produce a significant overall increase in end-of-life pathology, indicative of extensive compensation for the defective mutant polymerases. Interestingly, both alleles result in a mutator phenotype, and a single Pold1L604K allele, but not a Pold1L604G allele, accelerates tumorigenesis and reduces life span. We discuss the implications of our results for the ongoing search for Pol δ mutations in human cancers.

Patterns of Somatic Mutation in Human Cancer Genomes

Abstract: AACR Centennial Conference: Translational Cancer Medicine-- Nov 4-8, 2007; Singapore PL02-05 All cancers are due to abnormalities in DNA. The availability of the human genome sequence has led to the proposal that resequencing of cancer genomes will reveal the full complement of somatic mutations and hence all the cancer genes. To explore the nature of the information that will be derived from cancer genome sequencing we have sequenced the coding exons of the family of 518 protein kinases, ~1.3Mb DNA per cancer sample, in 210 cancers of diverse histological types. Despite the screen being directed toward the coding regions of a gene family that has previously been strongly implicated in oncogenesis, the results indicate that the majority of somatic mutations detected are “passengers”. There is considerable variation in the number and pattern of these mutations between individual cancers, indicating substantial diversity of processes of molecular evolution between cancers. The imprints of exogenous mutagenic exposures, mutagenic treatment regimes and DNA repair defects can all be seen in the distinctive mutational signatures of individual cancers. This systematic mutation screen and others have previously yielded a number of cancer genes that are frequently mutated in one or more cancer types and which are now anticancer drug targets (for example BRAF , PIK3CA , and EGFR ). However, detailed analyses of the data from our screen additionally suggest that there exist a large number of additional “driver” mutations which are distributed across a substantial number of genes. It therefore appears that cells may be able to utilise mutations in a large repertoire of potential cancer genes to acquire the neoplastic phenotype. However, many of these genes are employed only infrequently. These findings may have implications for future anticancer drug development.

Case series: acute tumor lysis syndrome in mutator mice with disseminated lymphoblastic lymphoma.

Abstract: Acute tumor lysis syndrome (ATLS) is characterized by severe metabolic abnormalities and organ dysfunction resulting from rapid destruction of neoplastic cells. Metabolic disturbances are thought to be the primary cause of clinical ATLS symptoms, which include renal dysfunction, seizures, and cardiac arrhythmias. The histopathologic lesions associated with organ dysfunction are largely unknown because of the low rate of mortality of ATLS in humans and the few cases of ATLS identified in laboratory animals. Here, we describe histologic, immunohistochemical, and electron microscopic analyses of thirty-one ATLS cases from a cohort of 499 mice that are prone to spontaneous lymphoblastic lymphoma owing to genetic defects in DNA replication fidelity. Seventy-three percent of our cohort died with lymphoblastic lymphoma, and 8% of affected mice died with diffuse microthromboemboli consistent with ATLS. Mice with ATLS had a high spontaneous mortality rate (>50%), a large tumor burden with disseminated disease, and evidence of leukemia. Blood vessels in the lung, kidney, and other organs were occluded by microthromboemboli composed of chromatin, cellular debris, fibrin, platelets, and entrapped erythrocytes and malignant cells. This case series suggests that ATLS can occur at high frequency in mice with disseminated lymphoblastic lymphoma and leads to a high rate of spontaneous death from microthromboemboli.

An Introduction to Medical Statistics

Abstract: QUITE apart from the academic consideration that vital and medical statistics now form an obligatory part of the education of students seeking the University of London's diploma in public health, the demand for information about the methods of vital and medical statistics is increasing. The most casual reader of the newspapers is now aware that population problems are of serious practical importance and that the publications of the General Register Office cannot be ignored. It is scarcely an exaggeration to say that the recently issued preliminary report on the census of 1931 is one of the most sensational documents which has appeared for years, and that he who reads it intelligently will understand what is meant by saying that civilisation is in the melting pot. An Introduction to Medical Statistics. By Hilda M. Woods William T. Russell. Pp. x + 125. (London: P. S. King and Son, Ltd., 1931.) 7s. 6d.

Detection of ultra-rare mutations by next-generation sequencing

Abstract: Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of ∼1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, we have developed a method termed Duplex Sequencing. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be discounted as technical error. We determine that Duplex Sequencing has a theoretical background error rate of less than one artifactual mutation per billion nucleotides sequenced. In addition, we establish that detection of mutations present in only one of the two strands of duplex DNA can be used to identify sites of DNA damage. We apply the method to directly assess the frequency and pattern of random mutations in mitochondrial DNA from human cells.

Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas

Abstract: Many individuals with multiple or large colorectal adenomas or early-onset colorectal cancer (CRC) have no detectable germline mutations in the known cancer predisposition genes. Using whole-genome sequencing, supplemented by linkage and association analysis, we identified specific heterozygous POLE or POLD1 germline variants in several multiple-adenoma and/or CRC cases but in no controls. The variants associated with susceptibility, POLE p.Leu424Val and POLD1 p.Ser478Asn, have high penetrance, and POLD1 mutation was also associated with endometrial cancer predisposition. The mutations map to equivalent sites in the proofreading (exonuclease) domain of DNA polymerases ɛ and δ and are predicted to cause a defect in the correction of mispaired bases inserted during DNA replication. In agreement with this prediction, the tumors from mutation carriers were microsatellite stable but tended to acquire base substitution mutations, as confirmed by yeast functional assays. Further analysis of published data showed that the recently described group of hypermutant, microsatellite-stable CRCs is likely to be caused by somatic POLE mutations affecting the exonuclease domain.