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.     

Hydrocephalus, situs inversus, chronic sinusitis, and male infertility in DNA polymerase lambda-deficient mice: possible implication for the pathogenesis of immotile cilia syndrome

A growing number of DNA polymerases have been identified, although their physiological function and relation to human disease remain mostly unknown. DNA polymerase lambda (Pol lambda; also known as Pol beta2) has recently been identified as a member of the X family of DNA polymerases and shares 32% amino acid sequence identity with DNA Pol beta within the polymerase domain. With the use of homologous recombination, we generated Pol lambda(-/-) mice. Pol lambda(-/-) mice develop hydrocephalus with marked dilation of the lateral ventricles and exhibit a high rate of mortality after birth, although embryonic development appears normal. Pol lambda(-/-) mice also show situs inversus totalis and chronic suppurative sinusitis. The surviving male, but not female, Pol lambda(-/-) mice are sterile as a result of spermatozoal immobility. Microinjection of sperm from male Pol lambda(-/-) mice into oocytes gives rise to normal offspring, suggesting that the meiotic process is not impaired. Ultrastructural analysis reveals that inner dynein arms of cilia from both the ependymal cell layer and respiratory epithelium are defective, which may underlie the pathogenesis of hydrocephalus, situs inversus totalis, chronic sinusitis, and male infertility. Sensitivity of Pol lambda(-/-) cells to various kinds of DNA damage is indistinguishable from that of Pol lambda(+/+) cells. Collectively, Pol lambda(-/-) mice may provide a useful model for clarifying the pathogenesis of immotile cilia syndrome.     

DNA polymerase lambda mediates a back-up base excision repair activity in extracts of mouse embryonic fibroblasts

Mammalian DNA polymerase (pol) lambda is a member of the X-family of DNA polymerases and has striking enzymatic and structural similarities to mammalian DNA pol beta. Because pol beta provides two important enzymatic activities for base excision repair (BER), we examined whether pol lambda might also contribute to BER. We used extracts from mouse embryonic fibroblasts representing wild-type and null genotypes for pol beta and pol lambda. In combination with neutralizing antibodies against pol beta and pol lambda, our results show a BER deficiency in the pol lambda -/- cell extract compared with extract from isogenic wild-type cells. In addition, the pol lambda antibody strongly reduced in vitro BER in the pol beta -/- cell extract. These data indicate that pol lambda is able to contribute to BER in mouse fibroblast cell extract.     

DNA polymerase lambda (Pol lambda), a novel eukaryotic DNA polymerase with a potential role in meiosis

A new gene (POLL) encoding a novel DNA polymerase (Pol lambda) has been identified at mouse chromosome 19. Murine Pol lambda, consisting of 573 amino acid residues, has a 32% identity to Pol beta, involved in nuclear DNA repair in eukaryotic cells. It is interesting that Pol lambda contains all the critical residues involved in DNA binding, nucleotide binding and selection, and catalysis of DNA polymerization, that are conserved in Pol beta and other DNA polymerases belonging to family X. Murine Pol lambda, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity when assessed by in situ gel analysis. Pol lambda also conserves the critical residues of Pol beta required for its intrinsic deoxyribose phosphate lyase (dRPase) activity. The first 230 amino acid residues of Pol lambda, that have no counterpart in Pol beta, contain a BRCT domain, present in a variety of cell-cycle check-point control proteins responsive to DNA damage and proteins involved in DNA repair. Northern blotting, in situ hybridization analysis and immunostaining showed high levels of Pol lambda specifically expressed in testis, being developmentally regulated and mainly associated to pachytene spermatocytes. These first evidences, although indirect, suggest a potential role of Pol lambda in DNA repair synthesis associated with meiosis.     

Aphidicolin-resistant and -sensitive base excision repair in wild-type and DNA polymerase beta-defective mouse cells

Several DNA polymerases (Pols) can add complementary bases at the gap created during the base excision repair (BER). To characterize the BER resynthesis step, the repair of a single abasic site by wild-type and Pol beta-defective mouse cell extracts was analysed in the presence of aphidicolin, a specific inhibitor of replicative Pols. We show that there is a competition between distributive and processive Pols for the nucleotide addition at the primer terminus. In wild-type cell extracts, the initial nucleotide insertion involves mainly Pol beta but the elongation step is carried out by a replicative Pol. Conversely, in Pol beta-null cell extracts the synthesis step is carried out by a replicative Pol without any switching to an auxiliary polymerase. We present evidence that short-patch repair synthesis occurs even in the absence of both Pol beta and replicative Pols. Exogeneously added purified human Pol lambda was unable to stimulate this back-up synthesis.     

Identification of an intrinsic 5'-deoxyribose-5-phosphate lyase activity in human DNA polymerase lambda: a possible role in base excision repair.

Base excision repair (BER) is a major repair pathway in eukaryotic cells responsible for repair of lesions that give rise to abasic (AP) sites in DNA. Pivotal to this process is the 5'-deoxyribose-5-phosphate lyase (dRP lyase) activity of DNA polymerase beta (Pol beta). DNA polymerase lambda (Pol lambda) is a recently identified eukaryotic DNA polymerase that is homologous to Pol beta. We show here that human Pol lambda exhibits dRP lyase, but not AP lyase, activity in vitro and that this activity is consistent with a beta-elimination mechanism. Accordingly, a single amino acid substitution (K310A) eliminated more than 90% of the wild-type dRP lyase activity, thus suggesting that Lys(310) of Pol lambda is the main nucleophile involved in the reaction. The dRP lyase activity of Pol lambda, in coordination with its polymerization activity, efficiently repaired uracil-containing DNA in an in vitro reconstituted BER reaction. These results suggest that Pol lambda may participate in "single-nucleotide" base excision repair in mammalian cells.     

HMGB1 is a cofactor in mammalian base excision repair

Deoxyribose phosphate (dRP) removal by DNA polymerase beta (Pol beta) is a pivotal step in base excision repair (BER). To identify BER cofactors, especially those with dRP lyase activity, we used a Pol beta null cell extract and BER intermediate as bait for sodium borohydride crosslinking. Mass spectrometry identified the high-mobility group box 1 protein (HMGB1) as specifically interacting with the BER intermediate. Purified HMGB1 was found to have weak dRP lyase activity and to stimulate AP endonuclease and FEN1 activities on BER substrates. Coimmunoprecipitation experiments revealed interactions of HMGB1 with known BER enzymes, and GFP-tagged HMGB1 was found to accumulate at sites of oxidative DNA damage in living cells. HMGB1(-/-) mouse cells were slightly more resistant to MMS than wild-type cells, probably due to the production of fewer strand-break BER intermediates. The results suggest HMGB1 is a BER cofactor capable of modulating BER capacity in cells.     

The frameshift infidelity of human DNA polymerase lambda. Implications for function

DNA polymerase lambda (Pol lambda) is a member of the Pol X family having properties in common with several other mammalian DNA polymerases. To obtain clues to possible functions in vivo, we have determined the fidelity of DNA synthesis by human Pol lambda. The results indicate that the average single-base deletion error rate of Pol lambda is higher than those of other mammalian polymerases. In fact, unlike other DNA polymerases, Pol lambda generates single-base deletions at average rates that substantially exceed base substitution rates. Moreover, the sequence specificity for single-base deletions made by Pol lambda is different from that of other DNA polymerases and reveals that Pol lambda readily uses template-primers with limited base pair homology at the primer terminus. This ability, together with an ability to fill short gaps in DNA at low dNTP concentrations, is consistent with a role for mammalian Pol lambda in non-homologous end-joining. This may include non-homologous end-joining of strand breaks resulting from DNA damage, because Pol lambda has intrinsic 5',2'-deoxyribose-5-phosphate lyase activity.     

Human DNA polymerase lambda is a proficient extender of primer ends paired to 7,8-dihydro-8-oxoguanine

Pol lambda is a DNA repair enzyme with a high affinity for dNTPs, an intrinsic dRP lyase activity, a BRCT domain involved in interactions with NHEJ factors, and also capable to interact with the PCNA processivity factor. Based on this potential, Pol lambda could play a role in BER, V(D)J recombination, NHEJ and TLS. Here we show that human Pol lambda uses a templating 7,8-dihydro-8-oxoguanine (8oxoG) base, a common mutagenic form of oxidative damage, as efficiently as an undamaged dG, but giving rise to the alternative insertion of either dAMP or dCMP. However, Pol lambda strongly discriminated against the extension of the mutagenic 8oxoG:dAMP pair. Conversely, Pol lambda readily extended the non-mutagenic 8oxoG:dCMP pair with an efficiency that was even higher than that displayed on undamaged dG:dCMP pair. A similar capacity for non-mutagenic extension was also shown to occur in the case of O6-methylguanine (m6G), a mutagenic and cytotoxic DNA adduct. A comparison of these novel properties of human Pol lambda with those of other DNA polymerases involved in TLS will be discussed. Interestingly, when double-strand breaks are associated to base damage, modifications as 8oxoG could be eventually part of the synapsis required to join ends, and therefore, the capacity of Pol lambda either to insert opposite 8oxoG or to extend correct base pairs containing such a damage could be beneficial for its role in NHEJ.     

XRCC1 and DNA polymerase β in cellular protection against cytotoxic DNA single-strand breaks

Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1^-/- mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β （pol β） is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS- treated XRCC1^-/-, and to a lesser extent in pol β^-/- cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and polβ^-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1^-/- cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly（ADP-ribosyl）ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC 1 to sites of DNA damage.     

'Knock down' of DNA polymerase beta by RNA interference: recapitulation of null phenotype

DNA polymerase beta (pol beta) is the major DNA polymerase involved in the base excision repair (BER) pathway in mammalian cells and, as a consequence, BER is severely compromised in cells lacking pol beta. Pol beta null (-/-) mouse embryos are not viable and pol beta null cells are hypersensitive to alkylating agents. Using RNA interference (RNAi) technology in mouse cells, we have reduced the pol beta protein and mRNA to undetectable levels. Pol beta knockdown cell lines display a pattern of hypersensitivity to DNA damaging agents similar to that observed in pol beta null cells. Generation of pol beta knock down cells makes it possible to combine the pol beta null phenotype with deficiencies in other DNA repair proteins, thereby helping to elucidate the role of pol beta and its interactions with other proteins in mammalian cells.     

Human DNA polymerase beta initiates DNA synthesis during long-patch repair of reduced AP sites in DNA

Simple base damages are repaired through a short-patch base excision pathway where a single damaged nucleotide is removed and replaced. DNA polymerase beta (Pol beta) is responsible for the repair synthesis in this pathway and also removes a 5'-sugar phosphate residue by catalyzing a beta-elimination reaction. How ever, some DNA lesions that render deoxyribose resistant to beta-elimination are removed through a long-patch repair pathway that involves strand displacement synthesis and removal of the generated flap by specific endonuclease. Three human DNA polymerases (Pol beta, Pol delta and Pol epsilon) have been proposed to play a role in this pathway, however the identity of the polymerase involved and the polymerase selection mechanism are not clear. In repair reactions catalyzed by cell extracts we have used a substrate containing a reduced apurinic/apyrimidinic (AP) site resistant to beta-elimination and inhibitors that selectively affect different DNA polymerases. Using this approach we find that in human cell extracts Pol beta is the major DNA polymerase incorporating the first nucleotide during repair of reduced AP sites, thus initiating long-patch base excision repair synthesis.     

Deployment of DNA polymerases beta and lambda in single-nucleotide and multinucleotide pathways of mammalian base excision DNA repair

There exist two major base excision DNA repair (BER) pathways, namely single-nucleotide or “short-patch” (SP-BER), and “long-patch” BER (LP-BER). Both pathways appear to be involved in the repair of small base lesions such as uracil, abasic sites and oxidized bases. In addition to DNA polymerase β (Polβ) as the main BER enzyme for repair synthesis, there is evidence for a minor role for DNA polymerase lambda (Polλ) in BER. In this study we explore the potential contribution of Polλ to both SP- and LP-BER in cell-free extracts. We measured BER activity in extracts of mouse embryonic fibroblasts using substrates with either a single uracil or the chemically stable abasic site analog tetrahydrofuran residue. The addition of purified Polλ complemented the pronounced BER deficiency of POLB-null cell extracts as efficiently as did Polβ itself. We have developed a new approach for determining the relative contributions of SP- and LP-BER pathways, exploiting mass-labeled nucleotides to distinguish single- and multinucleotide repair patches. Using this method, we found that uracil repair in wild-type and in Polβ-deficient cell extracts supplemented with Polλ was ∼80% SP-BER. The results show that recombinant Polλ can contribute to both SP- and LP-BER. However, endogenous Polλ, which is present at a level ˜50% that of Polβ in mouse embryonic fibroblasts, appears to make little contribution to BER in extracts. Thus Polλ in cells appears to be under some constraint, perhaps sequestered in a complex with other proteins, or post-translationally modified in a way that limits its ability to participate effectively in BER.     

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.     

DNA Polymerases β and λ Mediate Overlapping and Independent Roles in Base Excision Repair in Mouse Embryonic Fibroblasts

Base excision repair (BER) is a DNA repair pathway designed to correct small base lesions in genomic DNA. While DNA polymerase beta (pol β) is known to be the main polymerase in the BER pathway, various studies have implicated other DNA polymerases in back-up roles. One such polymerase, DNA polymerase lambda (pol λ), was shown to be important in BER of oxidative DNA damage. To further explore roles of the X-family DNA polymerases λ and β in BER, we prepared a mouse embryonic fibroblast cell line with deletions in the genes for both pol β and pol λ. Neutral red viability assays demonstrated that pol λ and pol β double null cells were hypersensitive to alkylating and oxidizing DNA damaging agents. In vitro BER assays revealed a modest contribution of pol λ to single-nucleotide BER of base lesions. Additionally, using co-immunoprecipitation experiments with purified enzymes and whole cell extracts, we found that both pol λ and pol β interact with the upstream DNA glycosylases for repair of alkylated and oxidized DNA bases. Such interactions could be important in coordinating roles of these polymerases during BER.     

Distribution and roles of X-family DNA polymerases in eukaryotes

Four types of DNA polymerase (Pol beta, Pol lambda, Pol mu and TdT) have been identified in eukaryotes as members of the polymerase X-family. Only vertebrates have all four types of enzyme. Plants and fungi have one or two X-family polymerases, while protostomes, such as fruit flies and nematodes, do not appear to have any. It is possible that the well-known metabolic pathways in which these enzymes are involved are restricted to the vertebrate world. The distribution of the DNA polymerases involved in DNA repair across the various biological kingdoms differs from that of the DNA polymerases involved in chromosomal DNA replication. In this review, we focus on the interesting pattern of distribution of the X-family enzymes across biological kingdoms and speculate on their roles.     

Involvement of DNA polymerase beta in protection against the cytotoxicity of oxidative DNA damage

We had shown previously that DNA polymerase beta (beta-pol) null mouse fibroblasts, deficient in base excision repair (BER), are hypersensitive to monofunctional methylating agents but not to hydrogen peroxide (H2O2). This is surprising because beta-pol is thought to be involved in BER of oxidative as well as methylated DNA damage. We confirm these findings here in early-passage cells. However, with time in culture, beta-pol null cells become hypersensitive to H2O2 and other reactive oxygen species-generating agents. Analysis of in vitro BER reveals a strong deficiency in single-nucleotide BER of 8-oxoguanine (8-oxoG) by both early- and late-passage beta-pol null cell extracts. Therefore, in early-passage wild-type and beta-pol null cells, the capacity for single-nucleotide BER of 8-oxoG does not correlate with cellular sensitivity to H2O2. Expression of beta-pol protein in the late-passage null cells almost completely reverses the H2O2-hypersensitivity phenotype. Methoxyamine (MX) treatment sensitizes late-passage wild-type cells to H2O2 as expected for beta-pol-mediated single-nucleotide BER; however in beta-pol null cells, MX has no effect. The data indicate a role(s) of beta-pol-dependent repair in protection against the cytotoxicity of oxidative DNA damage in wild-type cells.     

The accumulation of MMS-induced single strand breaks in G1 phase is recombinogenic in DNA polymerase beta defective mammalian cells

DNA polymerase (Pol) β null mouse embryonic fibroblasts provide a useful cell system to investigate the effects of alterations in base excision repair (BER) on genome stability. These cells are characterized by hypersensitivity to the cytotoxic effects of methyl methanesulfonate (MMS) and by decreased repair of the MMS-induced DNA single strand breaks (SSB). Here, we show that, in the absence of Pol β, SSB accumulate in G₁ phase cells, accompanied by the formation of proliferating cell nuclear antigen foci in the nuclei. When replicating Pol β null cells are treated with MMS, a rapid phosphorylation of histone H2AX is detected in the nuclei of S phase cells, indicating that double strand breaks (DSB) are formed in response to unrepaired SSB. This is followed by relocalization within the nuclei of Rad51 protein, which is essential for homologous recombination (HR). These findings are compatible with a model where, in mammalian cells, unrepaired SSB produced during BER are substrates for the HR pathway via DSB formation. This is an example of a coordinated effort of two different repair pathways, BER and HR, to protect mammalian cells from alkylation-induced cytotoxicity.     

8-Oxoguanine DNA damage: at the crossroad of alternative repair pathways

Radical oxygen species (ROS) generate various modified DNA bases. Among them 8-oxo-7,8-dihydroguanine (8oxoG) is the most abundant and seems to play a major role in mutagenesis and in carcinogenesis. 8oxoG is removed from DNA by the specific glycosylase OGG1. An additional post-replication repair is needed to correct the 8oxoG/A mismatches that are produced by persistent 8oxoG residues. This review is focused on the mechanisms of base excision repair (BER) of this oxidized base. It is shown that, in vitro, efficient and complete repair of 8oxoG/C pairs requires a core of four proteins, namely OGG1, APE1, DNA polymerase (Pol) beta, and DNA ligase I. Repair occurs predominantly by one nucleotide replacement reactions (short-patch BER) and Pol beta is the polymerase of election for the resynthesis step. However, alternative mechanisms can act on 8oxoG residues since Pol beta-null cells are able to repair these lesions. 8oxoG/A mismatches are repaired by human cell extracts via two BER events which occur sequentially on the two strands. The removal of the mismatched adenine is followed by preferential insertion of a cytosine leading to the formation of 8oxoG/C pairs which are then corrected by OGG1-mediated BER. Both repair events are inhibited by aphidicolin, suggesting that a replicative DNA polymerase is involved in the repair synthesis step. We propose that Pol delta/epsilon-mediated BER (long-patch BER) is the mode of repair when lesions persist or are formed at replication. Finally, we address the issues of the relative contribution of the two BER pathways to oxidative damage repair in vivo and the possible role of BER gene variants as cancer susceptibility genes.     

Roles of DNA ligase III and XRCC1 in regulating the switch between short patch and long patch BER

Damaged DNA bases are repaired by base excision repair (BER), which can proceed via two pathways: short patch and long patch BER. During the latter, a stretch of several nucleotides is replaced by strand displacement DNA synthesis. We recently demonstrated that the ATP concentration may govern the decision between these BER sub-pathways. Employing a reconstituted BER complex containing among others DNA polymerase beta (Pol beta), DNA ligase III (Lig III) and XRCC1, here we show that Lig III and XRCC1 are essential mediators of this regulation. XRCC1 stimulates Pol beta strand displacement activity and releases inhibition of Pol beta by DNA-bound Lig III if ligation is prevented. XRCC1 is thus able to strongly promote strand displacement and long patch BER under conditions of ATP shortage. If sufficient ATP is available, ligation by Lig III prevents strand displacement, leading to short patch BER. Ligation-inactive mutants of Lig III do not prevent strand displacement by Pol beta under the same conditions. Consequently, the preferred use of short patch BER depends on the ligation competence of Lig III. Accordingly, lowering the levels of the XRCC1/Lig III complex in HeLa cells using siRNA decreases ligation capacity but enhances Pol beta-dependent DNA synthesis.