Biochemical properties of Saccharomyces cerevisiae DNA polymerase IV

Although mammals encode multiple family X DNA polymerases implicated in DNA repair, Saccharomyces cerevisiae has only one, DNA polymerase IV (pol IV). To better understand the repair functions of pol IV, here we characterize its biochemical properties. Like mammalian pol beta and pol lambda, but not pol mu, pol IV has intrinsic 5'-2-deoxyribose-5-phosphate lyase activity. Pol IV has low processivity and can fill short gaps in DNA. Unlike the case with pol beta and pol lambda, the gap-filling activity of pol IV is not enhanced by a 5'-phosphate on the downstream primer but is stimulated by a 5'-terminal synthetic abasic site. Pol IV incorporates rNTPs into DNA with an unusually high efficiency relative to dNTPs, a property in common with pol mu but not pol beta or pol lambda. Finally, pol IV is highly inaccurate, with an unusual error specificity indicating the ability to extend primer termini with limited homology. These properties are consistent with a possible role for pol IV in base excision repair and with its known role in non-homologous end joining of double strand breaks, perhaps including those with damaged ends.     

Single-turnover kinetic analysis of the mutagenic potential of 8-oxo-7,8-dihydro-2'-deoxyguanosine during gap-filling synthesis catalyzed by human DNA polymerases lambda and beta

In the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) damage, many DNA polymerases exhibit a dual coding potential which facilitates efficient incorporation of matched dCTP or mismatched dATP. This also holds true for the insertion of 8-oxodGTP opposite template bases dC and dA. Employing single-turnover kinetic methods, we examined human DNA polymerase beta and its novel X-family homolog, human DNA polymerase lambda, to determine which nucleotide and template base was preferred when encountering 8-oxodG and 8-oxodGTP, respectively. While DNA polymerase beta preferentially incorporated dCTP over dATP, DNA polymerase lambda did not modulate a preference for either dCTP or dATP when opposite 8-oxodG in single-nucleotide gapped DNA, as incorporation proceeded with essentially equal efficiency and probability. Moreover, DNA polymerase lambda is more efficient than DNA polymerase beta to fill this oxidized single-nucleotide gap. Insertion of 8-oxodGTP by both DNA polymerases lambda and beta occurred predominantly against template dA, thereby reiterating how the asymmetrical design of the polymerase active site differentially accommodated the anti and syn conformations of 8-oxodG and 8-oxodGTP. Although the electronegative oxygen at the C8 position of 8-oxodG may induce DNA structural perturbations, human DNA ligase I was found to effectively ligate the incorporated 8-oxodGMP to a downstream strand, which sealed the nicked DNA. Consequently, the erroneous nucleotide incorporations catalyzed by DNA polymerases lambda and beta as well as the subsequent ligation catalyzed by a DNA ligase during base excision repair are a threat to genomic integrity.     

A structural solution for the DNA polymerase lambda-dependent repair of DNA gaps with minimal homology

Human DNA polymerase lambda (Pol lambda) is a family X member with low frameshift fidelity that has been suggested to perform gap-filling DNA synthesis during base excision repair and during repair of broken ends with limited homology. Here, we present a 2.1 A crystal structure of the catalytic core of Pol lambda in complex with DNA containing a two nucleotide gap. Pol lambda makes limited contacts with the template strand at the polymerase active site, and superimposition with Pol beta in a ternary complex suggests a shift in the position of the DNA at the active site that is reminiscent of a deletion intermediate. Surprisingly, Pol lambda can adopt a closed conformation, even in the absence of dNTP binding. These observations have implications for the catalytic mechanism and putative DNA repair functions of Pol lambda.     

Mode of inhibition of short-patch base excision repair by thymine glycol within clustered DNA lesions

Clustered DNA damage, where two or more lesions are located proximally to each other, is frequently induced by ionizing radiation. Individual base lesions within a cluster are repaired by base excision repair. In this study we addressed the question of how thymine glycol (Tg) within a cluster would affect the repair of opposing lesions by human cell extracts. We have found that Tg located opposite to an abasic site does not affect cleavage of this site by apurinic/apyrimidinic (AP) endonuclease. However, Tg significantly compromised the next step of the repair. Although purified DNA polymerase beta was able to incorporate the correct nucleotide (dAMP) opposite to Tg, the rate of incorporation was reduced by 3-fold. Tg does not affect 5'-sugar phosphate removal by the 2-deoxyribose-5-phosphate (dRP) lyase activity of DNA polymerase beta, but further processing of the strand break by purified DNA ligase III was slightly diminished. In agreement with these findings, although an AP site located opposite to Tg was efficiently incised in human cell extract, only a limited amount of fully repaired product was observed, suggesting that such clustered DNA lesions may have a significantly increased lifetime in human cells compared with similar single-standing lesions.     

DNase VII of human placenta. Mechanism studies

The mechanism of the human placental DNase VII, described previously (Hollis, G. F., and Grossman, L. (1981) J. Biol. Chem. 256, 8074-8079) has been investigated in further detail. The enzyme initiates exonucleolytic hydrolysis from the 3'-end of DNA in a nonprocessive, or distributive, manner, regardless of whether the carbohydrate moiety associated with the 3'-terminal nucleotide contains H or OH at its 2' and 3' positions. DNase VII does not have associated RNase H activity; however, it is capable of removing 3'-terminal ribonucleotides. The enzyme also can hydrolyze DNA containing a terminal nucleotide lacking a purine or pyrimidine as well as termini containing noncomplementary nucleotides. DNase VII activity is product-inhibited by deoxynucleoside 5'-monophosphates. From kinetic studies, the mononucleotide deoxyadenylic acid is a noncompetitive inhibitor with a Ki = 0.3 mM. The resemblance of DNase VII to the 3'----5' exonuclease activity of Escherichia coli DNA polymerase I and its possible role in excision repair and proofreading are discussed.     

Contributions of an endonuclease IV homologue to DNA repair in the African swine fever virus

We recently demonstrated that African swine fever virus DNA polymerase X (Pol X) is extremely error-prone during single-nucleotide gap-filling and that the downstream ASFV DNA ligase seals 3' mismatched nicks with high efficiency. To further assess the credence of our hypothesis that these proteins may promote viral diversification by functioning within the context of an aberrant DNA repair pathway, herein we characterize the third protein expected to function in this system, a putative AP endonuclease (APE). Assays of the purified protein using oligonucleotide substrates unequivocally establish canonical APE activity, 3'-phosphatase and 3'-phosphodiesterase activities (in the context of a single-nucleotide gap), 3' --> 5' exonuclease activity (in the context of a nick), and nucleotide incision repair activity against 5,6-dihydrothymine. The 3' --> 5' exonuclease activity is shown to be highly dependent upon the identity of the nascent 3' base pair and to be inhibited when 2-deoxyribose-5-phosphate, rather than phosphate, constitutes the 5' moiety of the nick. ASFV APE retains activity when assayed in the presence of EDTA but is inactivated by incubation with 1,10-phenanthroline in the absence of a substrate, suggesting that it is an endonuclease IV homologue possessing intrinsic metal cofactors. The activities of ASFV APE, when considered alongside those of Pol X and ASFV DNA ligase, provide an enhanced understanding of (i) the types of damage that are likely to be sustained by the viral genome and (ii) the mechanisms by which the minimalist ASFV DNA repair pathway, consisting of just these three proteins, contributes to the fitness of the virus.     

Murine DNA polymerase alpha fills gaps to completion in a direct assay. Altered kinetics of de novo DNA synthesis at single nucleotide gaps

DNA polymerase alpha was studied in a direct gap-filling assay. Using a defined template, DNA synthesis was primed from the M13 17-mer universal primer and blocked by an oligonucleotide hybridized 56 nucleotides downstream of the primer. DNA polymerase alpha filled this gap to completion. A time course of the reaction showed that in 50% of the substrate molecules, gaps were filled to completion within 10 min. In another 35% of the molecules the final nucleotide was lacking after 10 min. This nucleotide was added at a reduced rate, and was not incorporated into all of the molecules even after 6 h. The reduced rate of incorporation of the final nucleotide is reflected in an increased Km for de novo incorporation of one nucleotide at a single nucleotide gap (0.7 microM), as opposed to the Km for de novo incorporation of one nucleotide into singly primed M13 DNA (0.18 microM). DNA polymerase alpha purified from murine cells infected with the parvovirus minute virus of mice, and HeLa cell DNA polymerase alpha 2, exhibited the same kinetics of gap filling as did DNA polymerase alpha purified from uninfected Ehrlich ascites murine tumor cells. T4 DNA polymerase filled gaps to completion in this assay. Escherichia coli DNA polymerase I Klenow fragment quantitatively displaced the downstream oligonucleotide, and extended nascent DNA chains for an additional 100 nucleotides. Nicks and single-nucleotide gaps produced in gap-filling reactions by murine DNA polymerase alpha and T4 DNA polymerase were sealed by T4 DNA ligase.     

Structure-function studies of DNA polymerase lambda

DNA polymerase lambda is a member of the X family of polymerases that is implicated in non-homologous end-joining of double-strand breaks in DNA and in base excision repair of DNA damage. To better understand the roles of DNA polymerase lambda in these repair pathways, here we review its structure and biochemical properties, with emphasis on its gap-filling polymerization activity, its dRP lyase activity and its unusual DNA synthetic (in)fidelity.     

Kinetic investigation of the inhibitory effect of gemcitabine on DNA polymerization catalyzed by human mitochondrial DNA polymerase

Gemcitabine, 2'-deoxy-2', 2'-difluorocytidine (dFdC), is a drug approved for use against various solid tumors. Clinically, this moderately toxic nucleoside analog causes peripheral neuropathy, hematological dysfunction, and pulmonary toxicity in cancer patients. Although these side effects closely mimic symptoms of mitochondrial dysfunction, there is no direct evidence to show gemcitabine interferes with mitochondrial DNA replication catalyzed by human DNA polymerase gamma. Here we employed presteady state kinetic methods to directly investigate the incorporation of the 5'-triphosphorylated form of gemcitabine (dFdCTP), the excision of the incorporated monophosphorylated form (dFdCMP), and the bypass of template base dFdC catalyzed by human DNA polymerase gamma. Opposite template base dG, dFdCTP was incorporated with a 432-fold lower efficiency than dCTP. Although dFdC is not a chain terminator, the incorporated dFdCMP decreased the incorporation efficiency of the next 2 correct nucleotides by 214- and 7-fold, respectively. Moreover, the primer 3'-dFdCMP was excised with a 50-fold slower rate than the matched 3'-dCMP. When dFdC was encountered as a template base, DNA polymerase gamma paused at the lesion and one downstream position but eventually elongated the primer to full-length product. These pauses were because of a 1,000-fold decrease in nucleotide incorporation efficiency. Interestingly, the polymerase fidelity at these pause sites decreased by 2 orders of magnitude. Thus, our pre-steady state kinetic studies provide direct evidence demonstrating the inhibitory effect of gemcitabine on the activity of human mitochondrial DNA polymerase.     

An intrinsic 5'-deoxyribose-5-phosphate lyase activity in DNA polymerase beta from Leishmania infantum supports a role in DNA repair

Leishmania infantum is a parasitic protozoan which infects humans. This paper reports the expression in Escherichia coli and purification of the L. infantum gene product (AF182167), as well as its characterization as a DNA polymerase beta (Polbeta)-like, template-dependent DNA repair enzyme, with a metal preference for Mn2+ over Mg2+. As is the case with mammalian Polbeta and DNA polymerase lambda (Pollambda), L. infantum DNA polymerase beta (Li Polbeta) prefers gapped-DNA substrates having a 5'-phosphate end, in agreement with its role in DNA repair reactions. Purified Li Polbeta also displayed a 5'-deoxyribose-5-phosphate (dRP) lyase activity, consistent with a beta-elimination mechanism. The concerted action of dRP lyase and DNA polymerization activities of Li Polbeta on a uracil-containing DNA suggests its participation in "single-nucleotide" base excision repair (BER). Analysis of Li Polbeta DNA polymerization activity at different stages of the L. infantum infective cycle supports a role for Li Polbeta in nuclear DNA repair after the oxidative damage occurring inside the macrophage.     

DNA polymerase lambda can elongate on DNA substrates mimicking non-homologous end joining and interact with XRCC4-ligase IV complex

Non-homologous end joining (NHEJ) is one of two pathways responsible for the repair of double-strand breaks in eukaryotic cells. The mechanism involves the alignment of broken DNA ends with minimal homology, fill in of short gaps by DNA polymerase(s), and ligation by XRCC4-DNA ligase IV complex. The gap-filling polymerase has not yet been positively identified, but recent biochemical studies have implicated DNA polymerase lambda (pol lambda), a novel DNA polymerase that has been assigned to the pol X family, in this process. Here we demonstrate that purified pol lambda can efficiently catalyze gap-filling synthesis on DNA substrates mimicking NHEJ. By designing two truncated forms of pol lambda, we also show that the unique proline-rich region in pol lambda plays a role in limiting strand displacement synthesis, a feature that may help its participation in in vivo NHEJ. Moreover, pol lambda interacts with XRCC4-DNA ligase IV via its N-terminal BRCT domain and the interaction stimulates the DNA synthesis activity of pol lambda. Taken together, these data strongly support that pol lambda functions in DNA polymerization events during NHEJ.     

Removal of 3'-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1.

A recombinant human AP endonuclease, HAP1, was constructed and characterized with respect to its ability to recognize and act upon a model double-stranded 39-mer oligodeoxyribonucleotide substrate containing a strand break site with 3'-phosphoglycolate and 5'-phosphate end-group chemistries. This oligodeoxyribonucleotide substrate exactly duplicates the chemistry and configuration of a major DNA lesion produced by ionizing radiation. HAP1 was found to recognize the strand break, and catalyze the release of the 3'-phosphoglycolate as free phosphoglycolic acid. The enzyme had a Vmax of 0.1 fmole/min/pg of HAP1 protein, and a Km of 0.05 microM for the 3'-phosphoglycolate strand break lesion. The mechanism of catalysis was hydrolysis of the phosphate ester bond between the 3'-phosphoglycolate moiety and the 3'-carbon of the adjacent dGMP moiety within the oligonucleotide. The resulting DNA contained a 3'-hydroxyl which supported nucleotide incorporation by E. coli DNA polymerase I large fragment. AP endonucleolytic activity of HAP1 was examined using an analogous double-stranded 39-mer oligodeoxyribonucleotide substrate, in which the strand break site was replaced by an apyrimidinic site. The Vmax and Km for the AP endonuclease reaction were 68 fmole/min/pg of HAP1 protein and 0.23 microM, respectively.     

Efficiency and fidelity of human DNA polymerases λ and β during gap-filling DNA synthesis

The base excision repair (BER) pathway coordinates the replacement of 1-10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase λ (Pol λ), an enzyme proposed to support the known BER DNA polymerase β (Pol β), as it filled 1-10-nucleotide gaps at 1-nucleotide intervals. Pol λ incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol λ, Pol β did not have an absolute threshold on gap size as the catalytic efficiency for a correct dNTP gradually decreased as the gap size increased from 2 to 10 nucleotides and then recovered for non-gapped DNA. Surprisingly, an increase in gap size resulted in lower polymerase fidelity for Pol λ, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol λ was up to 160-fold more error-prone than Pol β, thereby suggesting Pol λ would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol λ and its N-terminal domain truncation mutants. This nucleotide preference was shown to be dependent upon the identity of the adjacent 5'-template base. Our results suggested that both Pol λ and Pol β would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol λ, like Pol β, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol λ may play a role in long-patch BER.     

The base substitution fidelity of DNA polymerase beta-dependent single nucleotide base excision repair

Damaged DNA bases are removed from mammalian genomes by base excision repair (BER). Single nucleotide BER requires several enzymatic activities, including DNA polymerase and 5',2'-deoxyribose-5-phosphate lyase. Both activities are intrinsic to four human DNA polymerases whose base substitution error rate during gap-filling DNA synthesis varies by more than 10,000-fold. This suggests that BER fidelity could vary over a wide range in an enzyme dependent manner. To investigate this possibility, here we describe an assay to measure the fidelity of BER reactions reconstituted with purified enzymes. When human uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and DNA ligase 1 replace uracil opposite template A or G, base substitution error rates are 相似文献   

Sequence requirements for protein-primed initiation and elongation of phage O29 DNA replication

The double-stranded linear DNA of Bacillus subtilis phage O29 is replicated by a mechanism in which a terminal protein (TP) acts as a primer. The second 3'-terminal nucleotide of the template directs the incorporation of the 5'-terminal nucleotide into the TP, giving rise to the initiation complex TP-dAMP. Elongation then proceeds by a sliding-back mechanism in which the dAMP covalently linked to the TP pairs to the 3'-terminal nucleotide of the template strand to recover full-length DNA. We have studied the sequence requirements for efficient initiation of replication using mutated TP-free double-stranded DNA fragments. Efficient initiation only requires the terminal repetition 5'-AA. The 3'-terminal T, although not used as template, increases the affinity of DNA polymerase for the initiator nucleotide; in addition, although to a minor extent, the third 3'-terminal position also directs the formation of the initiation complex and modulates the initiation rate at the second position. Efficient elongation requires a previous sliding-back, demanding again a repetition of two nucleotides at the 3' end; if the sliding-back is prevented, a residual elongation can proceed directly from the second position or after jumping back from the third to the first position.     

Modulation of the 5'-deoxyribose-5-phosphate lyase and DNA synthesis activities of mammalian DNA polymerase beta by apurinic/apyrimidinic endonuclease 1

The Ape1 protein initiates the repair of apurinic/apyrimidinic sites during mammalian base excision repair (BER) of DNA. Ape1 catalyzes hydrolysis of the 5'-phosphodiester bond of abasic DNA to create nicks flanked by 3'-hydroxyl and 5'-deoxyribose 5-phosphate (dRP) termini. DNA polymerase (pol) beta catalyzes both DNA synthesis at the 3'-hydroxyl terminus and excision of the 5'-dRP moiety prior to completion of BER by DNA ligase. During BER, Ape1 recruits pol beta to the incised apurinic/apyrimidinic site and stimulates 5'-dRP excision by pol beta. The activities of these two enzymes are thus coordinated during BER. To examine further the coordination of BER, we investigated the ability of Ape1 to modulate the deoxynucleotidyltransferase and 5'-dRP lyase activities of pol beta. We report here that Ape1 stimulates 5'-dRP excision by a mechanism independent of its apurinic/apyrimidinic endonuclease activity. We also demonstrate a second mechanism, independent of Ape1, in which conditions that support DNA synthesis by pol beta also enhance 5'-dRP excision. Ape1 modulates the gap-filling activity of pol beta by specifically inhibiting synthesis on an incised abasic substrate but not on single-nucleotide gapped DNA. In contrast to the wild-type Ape1 protein, a catalytically impaired mutant form of Ape1 did not affect DNA synthesis by pol beta. However, this mutant protein retained the ability to stimulate 5'-dRP excision by pol beta. Simultaneous monitoring of 5'-dRP excision and DNA synthesis by pol beta demonstrated that the 5'-dRP lyase activity lags behind the polymerase activity despite the coordination of these two steps by Ape1 during BER.     

Human DNA polymerase lambda possesses terminal deoxyribonucleotidyl transferase activity and can elongate RNA primers: implications for novel functions

DNA polymerase lambda is a novel enzyme of the family X of DNA polymerases. The recent demonstration of an intrinsic 5'-deoxyribose-5'-phosphate lyase activity, a template/primer dependent polymerase activity, a distributive manner of DNA synthesis and sequence similarity to DNA polymerase beta suggested a novel beta-like enzyme. All these properties support a role of DNA polymerase lambda in base excision repair. On the other hand, the biochemical properties of the polymerisation activity of DNA polymerase lambda are still largely unknown. Here we give evidence that human DNA polymerase lambda has an intrinsic terminal deoxyribonucleotidyl transferase activity that preferentially adds pyrimidines onto 3'OH ends of DNA oligonucleotides. Furthermore, human DNA polymerase lambda efficiently elongates an RNA primer hybridized to a DNA template. These two novel properties of human DNA polymerase lambda might suggest additional roles for this enzyme in DNA replication and repair processes.