Shibani Dalal

Is There a Link Between DNA Polymerase Beta and Cancer

Recent small-scale studies have shown that 30 % of human tumors examined to date express DNA polymerase beta variant proteins. One of the DNA polymerase beta colon cancer-associated mutants, K289M, has been shown to synthesize DNA with a lower fidelity than wild-type Pol beta. Thus, the K289M protein could confer a mutator phenotype to the cell, resulting in genomic instability. Another DNA polymerase beta variant identified in colon carcinoma interferes with base excision repair in cells. This may result in unfilled gaps which can serve as substrates for recombination and result in genomic instability. DNA polymerase beta has also been shown to be overexpressed in a variety of tumors. In some cases, overexpression of polymerase beta in cells confers a transformed phenotype to the cells. In other cases, overexpression results in telomere fusions. Thus, mutant forms or aberrant quantities of polymerase beta confer a mutator phenotype to cells. Combined with the small-scale tumor studies, these mechanistic studies implicate variant forms of DNA polymerase beta in the etiology of human cancer.

The E295K DNA polymerase beta gastric cancer-associated variant interferes with base excision repair and induces cellular transformation.

ABSTRACT Approximately 30% of human tumors examined for mutations in polymerase beta (pol β) appear to express pol β variant proteins (D. Starcevic, S. Dalal, and J. B. Sweasy, Cell Cycle 3:998-1001, 2004). Many of these variants result from a single amino acid substitution. We have previously shown that the K289M and I260M colon and prostate cancer variants, respectively, induce cellular transformation most likely due to sequence-specific mutator activity (S. Dalal et al., Biochemistry 44:15664-15673, 2005; T. Lang et al., Proc. Natl. Acad. Sci. USA 101:6074-6079, 2004; J. B. Sweasy et al., Proc. Natl. Acad. Sci. USA 102:14350-14355, 2005). In the work described here, we show that the E295K gastric carcinoma pol β variant acts in a dominant-negative manner by interfering with base excision repair. This leads to an increase in sister chromatid exchanges. Expression of the E295K variant also induces cellular transformation. Our data suggest that unfilled gaps are channeled into a homology-directed repair pathway that could lead to genomic instability. The results indicate that base excision repair is critical for maintaining genome stability and could therefore be a tumor suppressor mechanism.

The Leu22Pro tumor-associated variant of DNA polymerase beta is dRP lyase deficient

Approximately 30% of human tumors characterized to date express DNA polymerase beta (pol β) variant proteins. Two of the polymerase beta cancer-associated variants are sequence-specific mutators, and one of them binds to DNA but has no polymerase activity. The Leu22Pro (L22P) DNA polymerase beta variant was identified in a gastric carcinoma. Leu22 resides within the 8 kDa amino terminal domain of DNA polymerase beta, which exhibits dRP lyase activity. This domain catalyzes the removal of deoxyribose phosphate during short patch base excision repair. We show that this cancer-associated variant has very little dRP lyase activity but retains its polymerase activity. Although residue 22 has no direct contact with the DNA, we report here that the L22P variant has reduced DNA-binding affinity. The L22P variant protein is deficient in base excision repair. Molecular dynamics calculations suggest that alteration of Leu22 to Pro changes the local packing, the loop connecting helices 1 and 2 and the overall juxtaposition of the helices within the N-terminal domain. This in turn affects the shape of the binding pocket that is required for efficient dRP lyase catalysis.

Targeting Cancer with a Lupus Autoantibody

A cell-penetrating lupus anti-DNA antibody inhibits DNA repair, sensitizes cancer cells to DNA-damaging therapy in vitro and in vivo, and is synthetically lethal to BRCA2-deficient human cancer cells. Taming the Big Bad Wolf Just like the wolves for which lupus is named, the antibodies involved in its pathogenesis can attack almost any part of a patient, causing widespread damage. Now, Hansen et al. show that these biological wolves can sometimes be tamed and their ferociousness put to use in treating another deadly disease. Lupus is an autoimmune disease associated with antibodies that target host DNA, wreaking havoc on patients’ cells throughout the body. Recently, cancer researchers have tried to co-opt some of these antibodies, particularly those that can penetrate human cells, for use as vehicles for therapeutic agents. While using lupus antibodies to deliver proteins to protect normal cells from therapeutic ionizing radiation delivered to a tumor, researchers discovered that one antibody, 3E10, could itself sensitize cancer cells to radiation treatment. The authors then characterized this observed effect in malignant cells and determined its mechanism. They found that 3E10 bound single-stranded DNA and interfered with its repair, making the cells more susceptible to DNA-damaging agents such as doxorubicin and radiation. In addition, 3E10 alone was toxic to cancer cells with deficient DNA repair pathways, such as those that harbor BRCA2 mutations. Further research is necessary to identify other pathways that make tumor cells susceptible to 3E10 and to analyze the pharmacokinetics and other characteristics of this treatment. However, 3E10 was already shown to be safe in a previous phase 1 trial in lupus patients and should now be able to transition into clinical trials for cancer patients as well. Although researchers have not yet discovered a cure for lupus, the big bad wolf’s offspring may potentially tame another life-threatening illness. Systemic lupus erythematosus (SLE) is distinct among autoimmune diseases because of its association with circulating autoantibodies reactive against host DNA. The precise role that anti-DNA antibodies play in SLE pathophysiology remains to be elucidated, and potential applications of lupus autoantibodies in cancer therapy have not previously been explored. We report the unexpected finding that a cell-penetrating lupus autoantibody, 3E10, has potential as a targeted therapy for DNA repair–deficient malignancies. We find that 3E10 preferentially binds DNA single-strand tails, inhibits key steps in DNA single-strand and double-strand break repair, and sensitizes cultured tumor cells and human tumor xenografts to DNA-damaging therapy, including doxorubicin and radiation. Moreover, we demonstrate that 3E10 alone is synthetically lethal to BRCA2-deficient human cancer cells and selectively sensitizes such cells to low-dose doxorubicin. Our results establish an approach to cancer therapy that we expect will be particularly applicable to BRCA2-related malignancies such as breast, ovarian, and prostate cancers. In addition, our findings raise the possibility that lupus autoantibodies may be partly responsible for the intrinsic deficiencies in DNA repair and the unexpectedly low rates of breast, ovarian, and prostate cancers observed in SLE patients. In summary, this study provides the basis for the potential use of a lupus anti-DNA antibody in cancer therapy and identifies lupus autoantibodies as a potentially rich source of therapeutic agents.

The D246V Mutant of DNA Polymerase β Misincorporates Nucleotides EVIDENCE FOR A ROLE FOR THE FLEXIBLE LOOP IN DNA POSITIONING WITHIN THE ACTIVE SITE

DNA polymerase β, a member of the X family of DNA polymerases, is known to be involved in base excision repair. A key to determining the biochemical properties of this DNA polymerase is structure-function studies of site-specific mutants that result in substitution of particular amino acids at critical sites. In a previous genetic screen, we identified three 3′-azido-2′,3′-dideoxythymidine 5′-triphosphate-resistant mutants, namely E249K, D246V, and R253M, of polymerase β in the flexible loop of the palm domain. In this work, we perform an extensive kinetic analysis to investigate the role of the D246V mutant on polymerase fidelity. We find that D246V misincorporates T opposite template bases G and C. The mechanistic basis of misincorporation appears to be altered DNA positioning within the active site. We provide evidence that the fidelity of D246V is greatly affected by the base that is 5′ of the templating base. We propose that the Asp residue at position 246 helps to maintain the proper positioning of the DNA within the polymerase active site and maintains the fidelity of polymerase β. Altogether, the results suggest that the flexible loop domain of polymerase β plays a major role in its fidelity.

Y265C DNA polymerase beta knockin mice survive past birth and accumulate base excision repair intermediate substrates

DNA is susceptible to damage by a wide variety of chemical agents that are generated either as byproducts of cellular metabolism or exposure to man-made and harmful environments. Therefore, to maintain genomic integrity, having reliable DNA repair systems is important. DNA polymerase β is known to be a key player in the base excision repair pathway, and mice devoid of DNA polymerase beta do not live beyond a few hours after birth. In this study, we characterized mice harboring an impaired pol β variant. This Y265C pol β variant exhibits slow DNA polymerase activity but WT lyase activity and has been shown to be a mutator polymerase. Mice expressing Y265C pol β are born at normal Mendelian ratios. However, they are small, and 60% die within a few hours after birth. Slow proliferation and significantly increased levels of cell death are observed in many organs of the E14 homozygous embryos compared with WT littermates. Mouse embryo fibroblasts prepared from the Y265C pol β embryos proliferate at a rate slower than WT cells and exhibit a gap-filling deficiency during base excision repair. As a result of this, chromosomal aberrations and single- and double-strand breaks are present at significantly higher levels in the homozygous mutant versus WT mouse embryo fibroblasts. This is study in mice is unique in that two enzymatic activities of pol β have been separated; the data clearly demonstrate that the DNA polymerase activity of pol β is essential for survival and genome stability.

Substrate-dependent millisecond domain motions in DNA polymerase β.

DNA polymerase β (Pol β) is a 39-kDa enzyme that performs the vital cellular function of repairing damaged DNA. Mutations in Pol β have been linked to various cancers, and these mutations are further correlated with altered Pol β enzymatic activity. The fidelity of correct nucleotide incorporation into damaged DNA is essential for Pol β repair function, and several studies have implicated conformational changes in Pol β as a determinant of this repair fidelity. In this work, the rate constants for domain motions in Pol β have been determined by solution NMR relaxation dispersion for the apo and substrate-bound, binary forms of Pol β. In apo Pol β, molecular motions, primarily isolated to the DNA lyase domain, are observed to occur at 1400 s(-1). Additional analysis suggests that these motions allow apo Pol β to sample a conformation similar to the gapped DNA-substrate-bound form. Upon binding DNA, these lyase domain motions are significantly quenched, whereas evidence for conformational motions in the polymerase domain becomes apparent. These NMR studies suggest an alteration in the dynamic landscape of Pol β due to substrate binding. Moreover, a number of the flexible residues identified in this work are also the location of residues, which upon mutation lead to cancer phenotypes in vivo, which may be due to the intimate role of protein motions in Pol β fidelity.

Fluorescence Resonance Energy Transfer Studies of DNA Polymerase β: THE CRITICAL ROLE OF FINGERS DOMAIN MOVEMENTS AND A NOVEL NON-COVALENT STEP DURING NUCLEOTIDE SELECTION*

Background: DNA Pol β participates in base excision repair by choosing correct dNTP to fill single-nucleotide gaps in DNA. Results: Pol β experiences a non-covalent step with correct dNTP selection. Conclusion: Correct and incorrect dNTP incorporation by Pol β are different. Significance: FRET-based system of Pol β elucidates a mechanism of substrate choice necessary for understanding the molecular basis of human disease. During DNA repair, DNA polymerase β (Pol β) is a highly dynamic enzyme that is able to select the correct nucleotide opposite a templating base from a pool of four different deoxynucleoside triphosphates (dNTPs). To gain insight into nucleotide selection, we use a fluorescence resonance energy transfer (FRET)-based system to monitor movement of the Pol β fingers domain during catalysis in the presence of either correct or incorrect dNTPs. By labeling the fingers domain with ((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS) and the DNA substrate with Dabcyl, we are able to observe rapid fingers closing in the presence of correct dNTPs as the IAEDANS comes into contact with a Dabcyl-labeled, one-base gapped DNA. Our findings show that not only do the fingers close after binding to the correct dNTP, but that there is a second conformational change associated with a non-covalent step not previously reported for Pol β. Further analyses suggest that this conformational change corresponds to the binding of the catalytic metal into the polymerase active site. FRET studies with incorrect dNTP result in no changes in fluorescence, indicating that the fingers do not close in the presence of incorrect dNTP. Together, our results show that nucleotide selection initially occurs in an open fingers conformation and that the catalytic pathways of correct and incorrect dNTPs differ from each other. Overall, this study provides new insight into the mechanism of substrate choice by a polymerase that plays a critical role in maintaining genome stability.

DNA Polymerase Beta is Critical for Genomic Stability of Sperm Cells

Maintaining genome integrity in germ cells is important, given that the germ cells produce the next generation of offspring. Base excision repair is a DNA repair pathway that is responsible for the repair of most endogenous DNA damage. A key enzyme that functions in this repair pathway is DNA polymerase beta (Pol β). We previously used conditional gene targeting to engineer mice with sperm deleted of the Pol B gene, which encodes Pol β. We characterized mutagenesis in the sperm of these mice and compared it to wild-type and mice heterozygous for the Pol B gene. We found that sperm obtained that were heterozygously or homozygously deleted of the Pol B gene exhibited increased mutation frequencies compared to wild-type sperm. We identified an increase in transition mutations in both heterozygously and homozygously deleted sperm, and the types of mutations induced suggest that a polymerase other than Pol β functions in its absence. Interestingly, most of the transversions we observed were induced only in heterozygous, compared with wild-type sperm. Our results suggest that haploinsufficiency of Pol β leads to increased frequencies and varieties of mutations. Our study also shows that Pol β is critical for genome stability in the germline.

The I260Q variant of DNA polymerase beta extends mispaired primer termini due to its increased affinity for deoxynucleotide triphosphate substrates.

DNA polymerase beta plays a key role in base excision repair. We have previously shown that the hydrophobic hinge region of polymerase beta, which is distant from its active site, plays a critical role in the fidelity of DNA synthesis by this enzyme. The I260Q hinge variant of polymerase beta misincorporates nucleotides with a significantly higher catalytic efficiency than the wild-type enzyme. In the study described here, we show that I260Q extends mispaired primer termini. The kinetic basis for extension of mispairs is defective discrimination by I260Q at the level of ground-state binding of the dNTP substrate. Our results suggest that the hydrophobic hinge region influences the geometry of the dNTP binding pocket exclusively. Because the DNA forms part of the binding pocket, our data are also consistent with the interpretation that the mispaired primer terminus affects the geometry of the dNTP binding pocket such that the I260Q variant has a higher affinity for the incoming dNTP than wild-type polymerase beta.