Colon Cancer-associated DNA Polymerase β Variant Induces Genomic Instability and Cellular Transformation

Nemec, Antonia A.; Donigan, Katherine A.; Murphy, Drew L.; Jaeger, Joachim; Sweasy, Joann B.

doi:10.1074/jbc.m112.362111

Cited by 41 publications

(57 citation statements)

References 22 publications

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“…C127 cells are a nontransformed clonal line derived from a mammary tumor of a RIII mouse (10). C127 cells were grown in DMEM supplemented with 10% FBS, 1% penicillinstreptomycin (Invitrogen), 1600 g/ml G418, and 180 g/ml hygromycin B (for selection of exogenous Pol ␤) at 37°C in a 5% CO 2 humidified incubator as described previously (8).…”

Section: Methodsmentioning

confidence: 99%

“…Transfection, Infection, and Expression Analysis-To obtain high titer retrovirus expressing WT and S229L Pol ␤, the GP2-293 packaging cell line was transfected with pRVY Pol ␤ and pVSVG plasmids (11) using calcium phosphate as described previously (8). Western blots of Pol ␤ confirmed exogenous expression in these cell lines.…”

Section: Methodsmentioning

confidence: 99%

“…Preparation of MEF Extract-Extracts from Pol ␤ Ϫ/Ϫ MEFs were collected as described previously (8). The extracts were aliquoted and flash frozen in liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

“…Reconstituted Base Excision Repair Assay-The base excision repair assay was performed with radiolabeled LPSD DNA and incubated with Pol ␤ Ϫ/Ϫ extract containing purified WT or S229L protein as described previously (8). Equal amounts of active WT and S229L protein were used as determined by electrophoretic mobility shift assay (EMSA).…”

Section: ј-Gcctcgcagccgtccaaccaac Caacctcgatccaatgccgtcc-3јmentioning

confidence: 99%

“…Some of these nonsynonymous mutations are likely a significant contributor to cancer progression because the mutations are selected for with many of the mutations being identified in late stage tumors, and there were no corresponding mutations identified in the normal matched tissues (6). In fact, two variants identified in this screen were found to induce cellular transformation (7,8).…”

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confidence: 95%

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The S229L Colon Tumor-associated Variant of DNA Polymerase β Induces Cellular Transformation as a Result of Decreased Polymerization Efficiency

Nemec

Murphy

Donigan

et al. 2014

Journal of Biological Chemistry

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Background:The POLB gene is mutated in 40% of human colorectal tumors. Results: The S229L variant is a slow DNA polymerase that leads to the accumulation of BER intermediates and induces cellular transformation. Conclusion: The S229L variant transforms cells by inducing genomic instability. Significance: Tumor-associated variants of DNA polymerase ␤ can affect DNA repair efficiency and drive cancer.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Preparation of MEF Extract-Extracts from Pol ␤ Ϫ/Ϫ MEFs were collected as described previously (8). The extracts were aliquoted and flash frozen in liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

Section: ј-Gcctcgcagccgtccaaccaac Caacctcgatccaatgccgtcc-3јmentioning

confidence: 99%

mentioning

confidence: 95%

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The S229L Colon Tumor-associated Variant of DNA Polymerase β Induces Cellular Transformation as a Result of Decreased Polymerization Efficiency

Nemec

Murphy

Donigan

et al. 2014

Journal of Biological Chemistry

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Sequence context modulation of polycyclic aromatic hydrocarbon‐induced mutagenesis

Chary

Stone

Lloyd

2013

Environ and Mol Mutagen

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DNA structural perturbations that are induced by site specifically and stereospecifically defined benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) adducts are directly correlated with mutagenesis, leading to cellular transformation. Although previous investigations had established that replication of DNAs containing N6-BPDE dA adducts at the second position in the N-ras codon 61(CAA) (612) resulted exclusively in A to G transitions, NMR analyses not only established the structural basis for this transition mutation, but also predicted that if the adduct were positioned at the third position in the same codon, an expanded spectra of mutations was possible. To test this prediction, replication of DNAs containing C10S-BPDE and C10R-BPDE lesions linked through the N6 position of adenine in the sequence context N-ras codon 61, position 3 (C10S-BPDE and C10R-BPDE at 613) was carried out in Escherichia coli, and these data revealed a wide mutation spectrum. In addition to A to G transitions produced by replication of both lesions, replication of the C10S-BPDE and C10R-BPDE adducts also yielded A to C and A to T transversions, respectively. Analyses of single nucleotide incorporation using Sequenase 2.0 and exonuclease-deficient E. coli Klenow fragment and pol II not only revealed high fidelity synthesis, but also demonstrated the same hierarchy of preference opposite a particular lesion, independent of the sequence context. Primer extension assays with the two lesions at N-ras 613 resulted in truncated products, with the C10S-BPDE adducts being more blocking than C10R-BPDE lesions, and termination of synthesis was more pronounced at position 613 than at 612 for each of the lesions.

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Mouse models to explore the biological and organismic role of DNA polymerase beta

Sobol

2024

Environ and Mol Mutagen

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Gene knock‐out (KO) mouse models for DNA polymerase beta (Polβ) revealed that loss of Polβ leads to neonatal lethality, highlighting the critical organismic role for this DNA polymerase. While biochemical analysis and gene KO cell lines have confirmed its biochemical role in base excision repair and in TET‐mediated demethylation, more long‐lived mouse models continue to be developed to further define its organismic role. The Polb‐KO mouse was the first of the Cre‐mediated tissue‐specific KO mouse models. This technology was exploited to investigate roles for Polβ in V(D)J recombination (variable‐diversity‐joining rearrangement), DNA demethylation, gene complementation, SPO11‐induced DNA double‐strand break repair, germ cell genome stability, as well as neuronal differentiation, susceptibility to genotoxin‐induced DNA damage, and cancer onset. The revolution in knock‐in (KI) mouse models was made possible by CRISPR/cas9‐mediated gene editing directly in C57BL/6 zygotes. This technology has helped identify phenotypes associated with germline or somatic mutants of Polβ. Such KI mouse models have helped uncover the importance of key Polβ active site residues or specific Polβ enzyme activities, such as the PolbY265C mouse that develops lupus symptoms. More recently, we have used this KI technology to mutate the Polb gene with two codon changes, yielding the PolbL301R/V303R mouse. In this KI mouse model, the expressed Polβ protein cannot bind to its obligate heterodimer partner, Xrcc1. Although the expressed mutant Polβ protein is proteolytically unstable and defective in recruitment to sites of DNA damage, the homozygous PolbL301R/V303R mouse is viable and fertile, yet small in stature. We expect that this and additional targeted mouse models under development are poised to reveal new biological and organismic roles for Polβ.

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Colon Cancer-associated DNA Polymerase β Variant Induces Genomic Instability and Cellular Transformation

Cited by 41 publications

References 22 publications

The S229L Colon Tumor-associated Variant of DNA Polymerase β Induces Cellular Transformation as a Result of Decreased Polymerization Efficiency

The S229L Colon Tumor-associated Variant of DNA Polymerase β Induces Cellular Transformation as a Result of Decreased Polymerization Efficiency

Sequence context modulation of polycyclic aromatic hydrocarbon‐induced mutagenesis

Mouse models to explore the biological and organismic role of DNA polymerase beta

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