Fluorescence resonance energy transfer studies of DNA polymerase ß: the critical role of fingers domain movements and a novel non-covalent step during nucleotide selection.

During DNA repair, DNA polymerase ß (Pol ß) 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 ß 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 ß. 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.

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 ß 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 ß variant. This Y265C pol ß variant exhibits slow DNA polymerase activity but WT lyase activity and has been shown to be a mutator polymerase. Mice expressing Y265C pol ß 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 ß 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 ß have been separated; the data clearly demonstrate that the DNA polymerase activity of pol ß is essential for survival and genome stability.

Structural changes in the hydrophobic hinge region adversely affect the activity and fidelity of the I260Q mutator DNA polymerase ß.

The I260Q variant of DNA polymerase ß is an efficient mutator polymerase with fairly indiscriminate misincorporation activities opposite all template bases. Previous modeling studies have suggested that I260Q harbors structural variations in its hinge region. Here, we present the crystal structures of wild type and I260Q rat polymerase ß in the presence and absence of substrates. Both the I260Q apoenzyme structure and the closed ternary complex with double-stranded DNA and ddTTP show ordered water molecules in the hydrophobic hinge near Gln260, whereas this is not the case in the wild type polymerase. Compared to wild type polymerase ß ternary complexes, there are subtle movements around residues 260, 272, 295, and 296 in the mutant. The rearrangements in this region, coupled with side chain movements in the immediate neighborhood of the dNTP-binding pocket, namely, residues 258 and 272, provide an explanation for the altered activity and fidelity profiles observed in the I260Q mutator polymerase.

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

Approximately 30% of human tumors examined for mutations in polymerase beta (pol beta) appear to express pol beta 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 beta 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 beta) 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.

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.

Substrate-dependent millisecond domain motions in DNA polymerase ß.

DNA polymerase ß (Pol ß) is a 39-kDa enzyme that performs the vital cellular function of repairing damaged DNA. Mutations in Pol ß have been linked to various cancers, and these mutations are further correlated with altered Pol ß enzymatic activity. The fidelity of correct nucleotide incorporation into damaged DNA is essential for Pol ß repair function, and several studies have implicated conformational changes in Pol ß as a determinant of this repair fidelity. In this work, the rate constants for domain motions in Pol ß have been determined by solution NMR relaxation dispersion for the apo and substrate-bound, binary forms of Pol ß. In apo Pol ß, 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 ß 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 ß 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 ß fidelity.

Targeting cancer with a lupus autoantibody.

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.

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 ß). We previously used conditional gene targeting to engineer mice with sperm deleted of the Pol B gene, which encodes Pol ß. 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 ß 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 ß leads to increased frequencies and varieties of mutations. Our study also shows that Pol ß is critical for genome stability in the germline.

Potentiation of temozolomide cytotoxicity by inhibition of DNA polymerase beta is accentuated by BRCA2 mutation.

Base excision repair (BER) plays a critical role in the repair of bases damaged by oxidative metabolism or alkylating agents, such as those commonly used in cancer therapy. Incomplete BER generates intermediates that require activation of homology-dependent DNA repair to resolve. We investigated the effects of lithocholic acid (LCA), an inhibitor of the key BER enzyme DNA polymerase beta (pol beta), in cells deficient in expression of the homology-dependent repair factor BRCA2. In vitro studies show that LCA suppresses the DNA polymerase and 5'-deoxyribose phosphate lyase activities of DNA pol beta by preventing the formation of a stable pol beta-DNA complex, reducing BER effectiveness. Cytotoxicity assays based on colony formation revealed that LCA exhibits synergism with the alkylating agent temozolomide, which engages BER through DNA methylation, and that the degree of synergism is increased in cells lacking functional BRCA2. BRCA2-deficient cells also showed heightened susceptibility to both LCA and temozolomide individually. The potentiation of temozolomide cytotoxicity by LCA owes to the conversion of single-stranded DNA breaks generated through incomplete BER of methylated nucleotides into double-stranded breaks during DNA replication, as indicated by gammaH2AX immunofluorescence. Death seems to be induced in cotreated cells through an accumulation of persistent double-stranded DNA breaks. Mutations of the BRCA2 gene have been extensively characterized and are present in various cancers, implying that inhibition of BER may offer a means to augment tumor selectivity in the use of conventional cancer therapies.

Hinge residue Ile260 of DNA polymerase beta is important for enzyme activity and fidelity.

DNA polymerases ensure efficient insertion of the correct dNTP into the DNA substrate. They have evolved mechanisms for discriminating among very similar dNTP substrates. DNA polymerase beta is a repair polymerase that provides a model system for a direct study of insertion fidelity. In this study, we examined the role of hinge residue Ile260 of the rat Polbeta on enzyme activity and accuracy. We changed residue I260 to every other amino acid residue and used genetic screens to assess the activity and fidelity of the resulting mutants. The I260D, -E, -K, -N, and -R mutants are significantly less active than wild-type Polbeta. Interestingly, I260H and I260Q are active but exhibit mutator activity. This suggests that the nonpolar nature of residue 260 is important for maintaining the activity and fidelity of Polbeta. We employ molecular modeling as an aid in explaining the observed phenotypes and propose a mechanism whereby the positioning of the DNA substrate in the enzyme and within the surface of the hinge may be a key player in forming an optimal active site for phosphodiester bond formation between Watson-Crick base pairs.

The hydrophobic hinge region of rat DNA polymerase beta is critical for substrate binding pocket geometry.

The hydrophobic hinge of DNA polymerase beta facilitates closing and stabilization of the enzyme once the nucleotide substrate has bound. Alteration of the hydrophobic nature of the hinge by the introduction of a hydrophilic glutamine residue in place of isoleucine 260 results in an inaccurate polymerase. The kinetic basis of infidelity is lack of discrimination during the binding of substrate. The I260Q polymerase beta variant has lower affinity than wild type enzyme for the correct substrate and much higher affinity for the incorrect substrate. Our results demonstrate that the hinge is important for formation of the substrate binding pocket. Our results are also consistent with the interpretation that DNA polymerase beta discriminates the correct from incorrect substrate during the binding step.

Prostate-cancer-associated I260M variant of DNA polymerase beta is a sequence-specific mutator.

Studies show that 30% of 189 tumors sequenced to date express variants of the polymerase beta (pol beta) protein that are not present in normal tissue. This raises the possibility that variants of pol beta might be linked to the etiology of cancer. Here, we characterize the I260M prostate-cancer-associated variant of pol beta. Ile260 is a key residue of the hydrophobic hinge that is important for the closing of the polymerase. In this study, we demonstrate that the I260M variant is a sequence context-dependent mutator polymerase. Specifically, I260M is a mutator for misalignment-mediated errors in dipyrimidine sequences. I260M is also a low-fidelity polymerase with regard to the induction of transversions within specific sequence contexts. Our results suggest that the hinge influences the geometry of the DNA within the polymerase active site that is important for accurate DNA synthesis. Importantly, characterization of the I260M variant shows that it has a functional phenotype that could be linked to the etiology or malignant progression of human cancer.

Expression of DNA polymerase {beta} cancer-associated variants in mouse cells results in cellular transformation.

Thirty percent of the 189 tumors studied to date express DNA polymerase beta variants. One of these variants was identified in a prostate carcinoma and is altered from isoleucine to methionine at position 260, within the hydrophobic hinge region of the protein. Another variant was identified in a colon carcinoma and is altered at position 289 from lysine to methionine, within helix N of the protein. We have shown that the types of mutations induced by these cancer-associated variants are different from those induced by the wild-type enzyme. In this study, we show that expression of the I260M and K289M cancer-associated variants in mouse C127 cells results in a transformed phenotype in the great majority of cell clones tested, as assessed by focus formation and anchorage-independent growth. Strikingly, cellular transformation occurs after a variable number of passages in culture but, once established, does not require continuous expression of the polymerase beta variant proteins, implying that it has a mutational basis. Because DNA polymerase beta functions in base excision repair, our results suggest that mutations that arise during this process can lead to the onset or progression of cancer.

The D246V mutant of DNA polymerase beta misincorporates nucleotides: evidence for a role for the flexible loop in DNA positioning within the active site.

DNA polymerase beta, 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 beta 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 beta. Altogether, the results suggest that the flexible loop domain of polymerase beta plays a major role in its fidelity.

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.