DNA polymerase beta

Mammalian DNA polymerase beta(beta-pol) is a single polypeptide chain enzyme of 39kDa. beta-pol has enzymatic activities appropriate for roles in base excision repair and other DNA metabolism events involving gap-filling DNA synthesis. Many crystal structures of beta-pol complexed with dNTP and DNA substrates have been solved, and mouse fibroblast cell lines deleted in the beta-pol gene have been examined. These approaches have enhanced our understanding of structural and functional aspects of beta-pol's role in protecting genomic DNA.     

Neonatal lethality with abnormal neurogenesis in mice deficient in DNA polymerase beta

DNA polymerase beta (Polbeta) has been implicated in base excision repair in mammalian cells. However, the physiological significance of this enzyme in the body remains unclear. Here, we demonstrate that mice carrying a targeted disruption of the Polbeta gene showed growth retardation and died of a respiratory failure immediately after the birth. Histological examination of the embryos revealed defective neurogenesis characterized by apoptotic cell death in the developing central and peripheral nervous systems. Extensive cell death occurred in newly generated post-mitotic neuronal cells and was closely associated with the period between onset and cessation of neurogenesis. These findings indicate that Polbeta plays an essential role in neural development.     

DNA聚合酶iota在HEK-293细胞中高表达体系的建立

目的:将带有DNA聚合酶iota(DNA Polymerase iota,Polι)目的基因的真核表达质粒PCDNA3.1转入HEK-293细胞,建立DNA聚合酶iota在HEK-293细胞中的高表达体系,为进一步研究DNA聚合酶在DNA损伤修复中的生物学功能奠定了基础.方法:用脂质体2000(Lipofectamine2000)将带有目的基因的真核表达质粒PCDNA3.1转入HEK-293细胞,通过G418筛选抗性克隆,用一步法提取细胞总RNA,采用逆转录聚合酶链式反应(RT-PCR)技术检测出高表达克隆.结果:通过G418筛选筛选出了具有G418抗性的克隆,通过RT-PCR技术检测出高表达DNA聚合酶iota的HEK-293细胞系.结论:建立了高表达DNA聚合酶iota的HEK-293细胞系.为进一步研究DNA聚合酶在DNA损伤修复中的生物学功能奠定了基础.     

Evidence of finely tuned expression of DNA polymerase beta in vivo using transgenic mice

DNA polymerase (Pol) is an error-prone repair DNA polymerase that has been shown to create genetic instability and tumorigenesis when overexpressed by only 2-fold in cells, suggesting that a rigorous regulation of its expression may be essential in vivo. To address this question, we have generated mice which express a transgene (Tg) bearing the Pol cDNA under the control of the ubiquitous promoter of the mouse H-2K gene from the major histocompatibility complex. These mice express the Tg only in thymus, an organ which normally contains the most abundant endogenous Pol mRNA and protein, supporting the idea of a tight regulation of Pol in vivo. Furthermore, we found no tumor incidence, suggesting that the single Pol overexpression event is not sufficient to initiate tumorigenesis in vivo.     

Structural design of a eukaryotic DNA repair polymerase: DNA polymerase beta

DNA polymerase beta, the smallest eukaryotic DNA polymerase, is designed to synthesize DNA in short DNA gaps during DNA repair. It is composed of two specialized domains that contribute essential enzymatic activities to base excision repair (BER). Its amino-terminal domain possesses a lyase activity necessary to remove the 5'-deoxyribose phosphate (dRP) intermediate generated during BER. Removal of the dRP moiety is often the rate-limiting step during BER. Failure to remove this group may initiate alternate BER pathways. The larger polymerase domain has nucleotidyl transferase activity. This domain has a modular organization with sub-domains that bind duplex DNA, catalytic metals, and the correct nucleoside triphosphate in a template-dependent manner. X-ray crystal structures of DNA polymerase beta, with and without bound substrates, has inferred that domain, sub-domain, and substrate conformational changes occur upon ligand binding. Many of these conformational changes are distinct from those observed in structures of other DNA polymerases. This review will examine the structural aspects of DNA polymerase beta that facilitate its role in BER.     

Arginine methylation regulates DNA polymerase beta

Alterations in DNA repair lead to genomic instability and higher risk of cancer. DNA base excision repair (BER) corrects damaged bases, apurinic sites, and single-strand DNA breaks. Here, a regulatory mechanism for DNA polymerase beta (Pol beta) is described. Pol beta was found to form a complex with the protein arginine methyltransferase 6 (PRMT6) and was specifically methylated in vitro and in vivo. Methylation of Pol beta by PRMT6 strongly stimulated DNA polymerase activity by enhancing DNA binding and processivity, while single nucleotide insertion and dRP-lyase activity were not affected. Two residues, R83 and R152, were identified in Pol beta as the sites of methylation by PRMT6. Genetic complementation of Pol beta knockout cells with R83/152K mutant revealed the importance of these residues for the cellular resistance to DNA alkylating agent. Based on our findings, we propose that PRMT6 plays a role as a regulator of BER.     

RNA aptamers selected against DNA polymerase beta inhibit the polymerase activities of DNA polymerases beta and kappa

DNA polymerase β (polβ), a member of the X family of DNA polymerases, is the major polymerase in the base excision repair pathway. Using in vitro selection, we obtained RNA aptamers for polβ from a variable pool of 8 × 10¹² individual RNA sequences containing 30 random nucleotides. A total of 60 individual clones selected after seven rounds were screened for the ability to inhibit polβ activity. All of the inhibitory aptamers analyzed have a predicted tri-lobed structure. Gel mobility shift assays demonstrate that the aptamers can displace the DNA substrate from the polβ active site. Inhibition by the aptamers is not polymerase specific; inhibitors of polβ also inhibited DNA polymerase κ, a Y-family DNA polymerase. However, the RNA aptamers did not inhibit the Klenow fragment of DNA polymerase I and only had a minor effect on RB69 DNA polymerase activity. Polβ and κ, despite sharing little sequence similarity and belonging to different DNA polymerase families, have similarly open active sites and relatively few interactions with their DNA substrates. This may allow the aptamers to bind and inhibit polymerase activity. RNA aptamers with inhibitory properties may be useful in modulating DNA polymerase actvity in cells.     

Mechanism of nucleotide incorporation in DNA polymerase beta

DNA polymerases play a central role in the mechanisms of DNA replication and repair. Here, we report mechanisms of the beta-polymerase catalyzed phosphoryl transfer reactions corresponding to correct and incorrect nucleotide incorporations in the DNA. Based on energy minimizations, molecular dynamics simulations, and free energy calculations of solvated ternary complexes of pol beta and by employing a mixed quantum mechanics molecular mechanics Hamiltonian, we have uncovered the identities of transient intermediates in the phosphoryl transfer pathways. Our study has revealed that an intriguing Grotthuss hopping mechanism of proton transfer involving water and three conserved aspartate residues in pol beta's active site mediates the phosphoryl transfer in the correct as well as misincorporation of nucleotides. The significance of this catalytic step in serving as a kinetic check point of polymerase fidelity may be unique to DNA polymerase beta, and is discussed in relation to other known mechanisms of DNA polymerases.     

FEN1 stimulation of DNA polymerase beta mediates an excision step in mammalian long patch base excision repair

In mammalian cells, single-base lesions, such as uracil and abasic sites, appear to be repaired by at least two base excision repair (BER) subpathways: "single-nucleotide BER" requiring DNA synthesis of just one nucleotide and "long patch BER" requiring multi-nucleotide DNA synthesis. In single-nucleotide BER, DNA polymerase beta (beta-pol) accounts for both gap filling DNA synthesis and removal of the 5'-deoxyribose phosphate (dRP) of the abasic site, whereas the involvement of various DNA polymerases in long patch BER is less well understood. Recently, we found that beta-pol plays a role in mammalian cell extract-mediated long patch BER, in that formation of a key excision product, 5'-dRP-trinucleotide (5'-dRP-N(3)), is dependent upon beta-pol (Dianov, G. L., Prasad, R., Wilson, S. H., and Bohr, V.A. (1999) J. Biol. Chem. 274, 13741-13743). The structure-specific endonuclease flap endonuclease 1 (FEN1) has also been suggested to be involved in long patch BER excision. Here, we demonstrate by immunodepletion experiments that 5'-dRP-N(3) excision in long patch BER of uracil-DNA in a human lymphoid cell extract is, indeed, dependent upon FEN1. Next, we reconstituted the excision step of long patch BER using purified human proteins and an oligonucleotide substrate with 5'-dRP at the margin of a one-nucleotide gap. Formation of the excision product 5'-dRP-N(3) was dependent upon both strand displacement DNA synthesis by beta-pol and FEN1 excision. FEN1 stimulated strand displacement DNA synthesis of beta-pol. FEN1 acting either alone, or without DNA synthesis by beta-pol, produced a two-nucleotide excision product, 5'-dRP-N(1), but not 5'-dRP-N(3). These results demonstrate that human FEN1 and beta-pol can cooperate in long patch BER excision and specify the predominant excision product seen with a cell extract.     

DNA polymerase beta can substitute for DNA polymerase I in the initiation of plasmid DNA replication.

We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair. We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E. coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication. Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E. coli polA mutant in the presence of mammalian DNA polymerase beta. Our results suggest that mammalian DNA polymerase beta can substitute for E. coli DNA polymerase I by initiating DNA replication of this plasmid from the 3' OH terminus of the RNA-DNA hybrid at the origin of replication.     

Involvement of DNA polymerase beta in DNA replication and mutagenic consequences

Overexpression in mammalian cells of the error-prone DNA polymerase beta (Pol beta) has been found to increase the spontaneous mutagenesis. Here, we investigated a possible mechanism used by Pol beta to be a genetic instability enhancer: its interference in replicative DNA synthesis, which is normally catalysed by the DNA polymerases alpha, delta and epsilon. By taking advantage of the ability to incorporate efficiently into DNA the chain terminator ddCTP as well as the oxidised nucleotide 8-oxo-dGTP, we show here that purified Pol beta can compete with the replicative DNA polymerases during replication in vitro of duplex DNA when added to human cell extracts. We found that involvement of Pol beta lowers replication fidelity and results in a modified error-specificity. Furthermore, we demonstrated that involvement of Pol beta occurred during synthesis of the lagging strand. These in vitro data provide one possible explanation of how overexpression of the enzyme could perturb the genetic instability in mammalian cells. We discuss these findings within the scope of the up-regulation of Pol beta in many cancer cells.     

Mammalian DNA polymerase beta can substitute for DNA polymerase I during DNA replication in Escherichia coli.

Mammalian DNA polymerase beta is the smallest known eukaryotic polymerase and is expressed as an active protein in Escherichia coli harboring a plasmid containing its cDNA. Since some catalytic functions of DNA polymerase beta and E. coli DNA polymerase I are similar, we wished to determine if DNA polymerase beta could substitute for DNA polymerase I in bacteria. We found that the expression of mammalian DNA polymerase beta in E. coli restored growth in a DNA polymerase I-defective bacterial mutant. Sucrose density gradient analysis revealed that DNA polymerase beta complements the replication defect in the mutant by increasing the rate of joining of Okazaki fragments. These findings demonstrate that DNA polymerase beta, believed to function in DNA repair in mammalian cells, can also function in DNA replication. Moreover, this complementation system will permit study of the in vivo function of altered species of DNA polymerase beta, an analysis currently precluded by the difficulty in isolating mutants in mammalian cells.     

DNA repair synthesis in mouse spermatogenesis involves DNA polymerase beta activity

The role of DNA polymerase alpha-DNA primase complex and DNA polymerase beta in DNA replication and ultraviolet-induced DNA repair synthesis has been analyzed in mouse spermatogenesis. Autoradiographic experiments with germ cells in culture, indicating an involvement of DNA polymerase alpha and/or delta in DNA replication, and of DNA polymerase beta in DNA repair synthesis, have been confirmed by studying partially purified enzymes. These findings support the idea that, different from other biological systems, in meiotic and post meiotic male mouse germ cells DNA polymerase beta is the main DNA polymerase form needed for DNA repair.     

Steady-state kinetics of mouse DNA polymerase beta.

DNA polymerase beta from mouse myeloma has been purified to near homogeneity, and its properties have been examined. The enzyme did not catalyze a detectable level of dNTP turnover, pyrophosphate exchange, pyrophosphorolysis, 3'-exonuclease degradation, or 5'-exonuclease degradation. Steady-state kinetic studies point to an ordered bibi mechanism for the polymerization reaction. Metal activation, which is required for polymerization, did not alter the Km for either the dNTP or the template--primer.     

Comparison of functional properties of mammalian DNA polymerase lambda and DNA polymerase beta in reactions of DNA synthesis related to DNA repair

DNA polymerase lambda (Pol lambda) is a novel enzyme of the family X of DNA polymerases. Pol lambda has some properties in common with DNA polymerase beta (Pol beta). The substrate properties of Pol lambda were compared to Pol beta using DNAs mimicking short-patch (SP) and long-patch (LP) base excision repair (BER) intermediates as well as recessed template primers. In the present work, the influence of several BER proteins such as flap-endonuclease-1 (FEN1), PCNA, and apurinic/apyrimidinic endonuclease-1 (APE1) on the activity of Pol lambda was investigated. Pol lambda is unable to catalyze strand displacement synthesis using nicked DNA, although this enzyme efficiently incorporates a dNMP into a one-nucleotide gap. FEN1 and PCNA stimulate the strand displacement activity of Pol lambda. FEN1 processes nicked DNA, thus removing a barrier to Pol lambda DNA synthesis. It results in a one-nucleotide gapped DNA molecule that is a favorite substrate of Pol lambda. Photocrosslinking and functional assay show that Pol lambda is less efficient than Pol beta in binding to nicked DNA. APE1 has no influence on the strand displacement activity of Pol lambda though it stimulates strand displacement synthesis catalyzed with Pol beta. It is suggested that Pol lambda plays a role in the SP BER rather than contributes to the LP BER pathway.