Katherine A. Donigan

Human POLB Gene Is Mutated in High Percentage of Colorectal Tumors

Background: Previous small scale studies indicate that DNA polymerase β variants are present in 30% of human tumors. Results: 40% of samples in a large human colorectal tumor collection harbor coding region variants, many of which exhibit altered function. Conclusion: Aberrant activity or fidelity phenotypes exhibited by variants may contribute to tumorigenesis. Significance: Expression of variants in human tumors plays a role in driving carcinogenesis. Previous small scale sequencing studies have indicated that DNA polymerase β (pol β) variants are present on average in 30% of human tumors of varying tissue origin. Many of these variants have been shown to have aberrant enzyme function in vitro and to induce cellular transformation and/or genomic instability in vivo, suggesting that their presence is associated with tumorigenesis or its progression. In this study, the human POLB gene was sequenced in a collection of 134 human colorectal tumors and was found to contain coding region mutations in 40% of the samples. The variants map to many different sites of the pol β protein and are not clustered. Many variants are nonsynonymous amino acid substitutions predicted to affect enzyme function. A subset of these variants was found to have reduced enzyme activity in vitro and failed to fully rescue pol β-deficient cells from methylmethane sulfonate-induced cytotoxicity. Tumors harboring variants with reduced enzyme activity may have compromised base excision repair function, as evidenced by our methylmethane sulfonate sensitivity studies. Such compromised base excision repair may drive tumorigenesis by leading to an increase in mutagenesis or genomic instability.

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

Background: Mutations in the POLB gene are present in 40% of human colorectal tumors. Results: The G231D variant is a slow polymerase that induces genomic instability and cellular transformation. Conclusion: The slow G231D variant induces cellular transformation due to its inability to fill in single nucleotide gaps. Significance: Slow pol β variants may drive tumorigenesis. Rapidly advancing technology has resulted in the generation of the genomic sequences of several human tumors. We have identified several mutations of the DNA polymerase β (pol β) gene in human colorectal cancer. We have demonstrated that the expression of the pol β G231D variant increased chromosomal aberrations and induced cellular transformation. The transformed phenotype persisted in the cells even once the expression of G231D was extinguished, suggesting that it resulted as a consequence of genomic instability. Biochemical analysis revealed that its catalytic rate was 140-fold slower than WT pol β, and this was a result of the decreased binding affinity of nucleotides by G231D. Residue 231 of pol β lies in close proximity to the template strand of the DNA. Molecular modeling demonstrated that the change from a small and nonpolar glycine to a negatively charged aspartate resulted in a repulsion between the template and residue 231 leading to the distortion of the dNTP binding pocket. In addition, expression of G231D was insufficient to rescue pol β-deficient cells treated with chemotherapeutic agents suggesting that these agents may be effectively used to treat tumors harboring this mutation. More importantly, this suggests that the G231D variant has impaired base excision repair. Together, these data indicate that the G231D variant plays a role in driving cancer.

The human gastric cancer-associated DNA polymerase β variant D160N is a mutator that induces cellular transformation.

Approximately 30% of human tumors sequenced to date harbor mutations in the POLB gene that are not present in matched normal tissue. Many mutations give rise to enzymes that contain non-synonymous single amino acid substitutions, several of which have been found to have aberrant activity or fidelity and transform cells when expressed. The DNA Polymerase β (Pol β) variant Asp160Asn (D160N) was first identified in a gastric tumor. Expression of D160N in cells induces cellular transformation as measured by hyperproliferation, focus formation, anchorage-independent growth and invasion. Here, we show that D160N is an active mutator polymerase that induces complex mutations. Our data support the interpretation that complex mutagenesis is the underlying mechanism of the observed cellular phenotypes, all of which are linked to tumorigenesis or tumor progression.

Sequence context‐specific mutagenesis and base excision repair

Base excision repair (BER) is critical for the maintenance of genome stability because it repairs at least 20,000 endogenously generated DNA lesions/cell/d. Several enzymes within the BER pathway exhibit sequence context dependency during the excision and DNA synthesis steps of repair. New evidence is emerging that germ line and tumor‐associated variants of enzymes in this repair pathway exhibit sequence context dependence that is different from their ancestral counterparts. We review what is known about the ancestral and variant BER proteins within various sequence contexts. We suggest that altering the sequence context preferences of BER proteins could give rise to rare cellular variants that might have a selective advantage in response to environmental exposure or to the tumor microenvironment.

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

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 β can affect DNA repair efficiency and drive cancer. DNA polymerase β (Pol β) plays a key role in base excision repair (BER) by filling in small gaps that are generated after base adducts are excised from the DNA. Pol β is mutated in a large number of colorectal tumors, and these mutations may drive carcinogenesis. In the present study, we wished to determine whether the S229L somatic Pol β variant identified in a stage 3 colorectal tumor is a driver of carcinogenesis. We show that S229L does not possess any defects in binding to either DNA or nucleotides compared with the WT enzyme, but exhibits a significant loss of polymerization efficiency, largely due to an 8-fold decrease in the polymerization rate. S229L participates in BER, but due to its lower catalytic rate, does so more slowly than WT. Expression of S229L in mammalian cells induces the accumulation of BER intermediate substrates, chromosomal aberrations, and cellular transformation. Our results are consistent with the interpretation that S229L is a driver of carcinogenesis, likely as a consequence of its slow polymerization activity during BER in vivo.

Tumor-associated mutations in a conserved structural motif alter physical and biochemical properties of human RAD51 recombinase

Human RAD51 protein catalyzes DNA pairing and strand exchange reactions that are central to homologous recombination and homology-directed DNA repair. Successful recombination/repair requires the formation of a presynaptic filament of RAD51 on ssDNA. Mutations in BRCA2 and other proteins that control RAD51 activity are associated with human cancer. Here we describe a set of mutations associated with human breast tumors that occur in a common structural motif of RAD51. Tumor-associated D149N, R150Q and G151D mutations map to a Schellman loop motif located on the surface of the RecA homology domain of RAD51. All three variants are proficient in DNA strand exchange, but G151D is slightly more sensitive to salt than wild-type (WT). Both G151D and R150Q exhibit markedly lower catalytic efficiency for adenosine triphosphate hydrolysis compared to WT. All three mutations alter the physical properties of RAD51 nucleoprotein filaments, with G151D showing the most dramatic changes. G151D forms mixed nucleoprotein filaments with WT RAD51 that have intermediate properties compared to unmixed filaments. These findings raise the possibility that mutations in RAD51 itself may contribute to genome instability in tumor cells, either directly through changes in recombinase properties, or indirectly through changes in interactions with regulatory proteins.

DNA polymerase β variant Ile260Met generates global gene expression changes related to cellular transformation

Maintenance of genomic stability is essential for cellular survival. The base excision repair (BER) pathway is critical for resolution of abasic sites and damaged bases, estimated to occur 20,000 times in cells daily. DNA polymerase β (Pol β) participates in BER by filling DNA gaps that result from excision of damaged bases. Approximately 30% of human tumours express Pol β variants, many of which have altered fidelity and activity in vitro and when expressed, induce cellular transformation. The prostate tumour variant Ile260Met transforms cells and is a sequence-context-dependent mutator. To test the hypothesis that mutations induced in vivo by Ile260Met lead to cellular transformation, we characterized the genome-wide expression profile of a clone expressing Ile260Met as compared with its non-induced counterpart. Using a 1.5-fold minimum cut-off with a false discovery rate (FDR) of <0.05, 912 genes exhibit altered expression. Microarray results were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and revealed unique expression profiles in other clones. Gene Ontology (GO) clusters were analyzed using Ingenuity Pathways Analysis to identify altered gene networks and associated nodes. We determined three nodes of interest that exhibited dysfunctional regulation of downstream gene products without themselves having altered expression. One node, peroxisome proliferator-activated protein γ (PPARG), was sequenced and found to contain a coding region mutation in PPARG2 only in transformed cells. Further analysis suggests that this mutation leads to dominant negative activity of PPARG2. PPARG is a transcription factor implicated to have tumour suppressor function. This suggests that the PPARG2 mutant may have played a role in driving cellular transformation. We conclude that PPARG induces cellular transformation by a mutational mechanism.

Abstract B37: A DNA polymerase beta gene harboring the colon cancer-associated G231D variant increases the sensitivity of cells to chemotherapeutic agents and induces cellular transformation

Rapidly advancing technology has resulted in the generation of the genomic sequences of several human tumors. These studies have revealed that tumors are heterogeneous and contain many mutations, supporting the mutator phenotype hypothesis of human cancer. Drivers of carcinogenesis are thought to be mutated genes that occur at high frequencies in large numbers of human tumors. However, functional characterization of many of the somatic mutations is lacking. We have identified several mutations of the DNA polymerase beta (POLB) gene in human colorectal cancer. Pol beta is the main polymerase involved in the base excision repair (BER) pathway and aberrant expression of Pol beta has been linked to a cancerous phenotype. In this study, we characterized the homozygous G231D mutation that was identified in a Stage 3 colorectal tumor. We have demonstrated that its expression in epithelial cells induced cellular transformation and increased the rate of proliferation. The transformed phenotype persisted in the cells even once the expression of G231D was extinguished. These data indicated that the expression of G231D caused an inheritable change; this change is most likely due to an increase in genomic instability caused by unfilled gaps proceeding through replication. Expression of G231D was insufficient to rescue Pol beta-deficient cells treated with chemotherapeutic agents suggesting that these agents may be effectively used to treat tumors harboring this mutation. Biochemical analysis of G231D revealed that it was 100-fold slower than WT Pol beta as determined by pre-steady state kinetics. The slow rate was a result of the decreased binding affinity of nucleotides by G231D. Residue 231 of Pol beta lies in close proximity to the template strand of the DNA. Molecular modeling showed that the change from a glycine to a negatively charged amino acid caused a repulsion between the template and residue 231 leading to the disruption of the dNTP binding pocket. Together, these data indicate that a person harboring the G231D Pol beta mutation may have an increased risk of cancer. Moreover, the altered sensitivity to chemotherapeutic drugs in cells carrying the G231D variant may have further implications in how to better improve the therapeutic outcome in patients with this mutation and supports the need for personalized cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B37.