Mammalian DNA Polymerase Research Articles

Telbivudine: A Novel Nucleoside Analog for Chronic Hepatitis B

To review available literature on the pharmacology, pharmacokinetics, dosing and administration, efficacy, and safety of the antiviral nucleoside analog telbivudine. Information was obtained from searching MEDLINE (1966-December 2005), International Pharmaceutical Abstracts (1970-December 2005), and the Cochrane Database of Systematic Reviews (4th quarter 2005) using the search words telbivudine, L-dT, L-deoxythymidine, L-nucleosides, and nucleosides. Abstracts from the Annual Meeting of the American Association for the Study of Liver Diseases and European Association for the Study of the Liver were also searched, including bibliographies from the identified articles. Data from double-blind, placebo-controlled clinical trials and unpublished information were extracted. Telbivudine is a novel, orally administered nucleoside analog under development for use in the treatment of chronic hepatitis B. In contrast to other nucleoside analogs, telbivudine has not been associated with inhibition of mammalian DNA polymerase with mitochondrial toxicity. Telbivudine demonstrated potent activity against hepatitis B with significantly higher rate of response and superior viral suppression compared with lamivudine, the standard treatment. Telbivudine has been generally well tolerated, with low adverse effect profile, and at its effective dose, no dose-limiting toxicity has been observed. Telbivudine is a novel oral nucleoside analog effective in the treatment of chronic hepatitis B infection.

Inhibitory Effect of Monogalactosyldiacylglycerol, Extracted from Spinach Using Supercritical CO2, on Mammalian DNA Polymerase Activity

We investigated the effective extraction of monogalactosyldiacylglycerol (MGDG) from dried spinach (Spinacia oleracea) using supercritical fluid carbon dioxide (SC-CO(2)) with a modifier/entrainer. The yield of MGDG in the SC-CO(2) extract was not influenced by increasing temperature at a constant pressure, although the total extract yield was decreased. The total extract yield and MGDG yield in the extract from commercially purchased spinach (unknown subspecies), were greatly influenced by lower pressure. In a modifier (i.e., ethanol) concentration range of 2.5-20%, both the extract and MGDG yield increased as the ethanol concentration rose. The highest total extract yield (69.5 mg/g of spinach) and a good MGDG yield (16.3 mg/g of spinach) were obtained at 80 degrees C, 25 MPa, and 20% ethanol. The highest MGDG concentration (76.0% in the extract) was obtained at 80 degrees C, 25 MPa, and 2.5% ethanol, although the total extract yield under these conditions was low (5.2 mg/g of spinach). The optimal conditions for the extraction of MGDG were 80 degrees C, 20 MPa, and 10% ethanol. Of the 11 subspecies of spinach tested under these conditions, "Ujyou" had the highest concentration of MGDG. The total extract yield and MGDG concentration of Ujyou were 20.4 mg of the extract/g of spinach and 70.5%, respectively. The concentration of MGDG was higher in the SC-CO(2) extract than in the extract obtained using solvents such as methanol and n-hexane. The extract of Ujyou, which was the optimal subspecies for the extraction of MGDG, inhibited the activity of calf DNA polymerase alpha with IC(50) values of 145 microg/mL but was not effective against DNA polymerase beta.

Analyses of ultraviolet‐induced focus formation of hREV1 protein

Translesional DNA synthesis (TLS) is one of the DNA damage tolerance mechanisms that allow cells with DNA damage to continue DNA replication. Each of the mammalian Y-family DNA polymerases (Pol eta, Pol iota, Pol kappa, and REV1) has been shown to carry out TLS by itself or in combination with another enzyme in vitro. Recently, the C-terminal region of mammalian REV1 (the total 1251 residues in human) was found to interact with Pol eta, Pol iota, and Pol kappa, as well as with the REV7 subunit of another TLS enzyme, Pol zeta. Thus, it is proposed that REV1 plays a pivotal role in TLS in vivo. We here describe our study on the localization of human REV1 protein (hREV1) in nondamaged and ultraviolet (UV)-irradiated cells. Ectopically expressed hREV1 in mammalian cells was localized to the nucleus and exhibited dozens of tiny foci in approximately 3% of nondamaged cells. The percentage of focus-forming cells markedly increased after UV irradiation in a time- and dose-dependent manner. The focus formation was associated with UV-induced DNA damage. Interestingly, although the hREV1 foci in S-phase cells colocalized with PCNA foci, suggesting the association of hREV1 with the replication machinery, hREV1 focus formation was observed not only in the S phase but also outside S phase. Furthermore, it was found that the hREV1 focus formation after UV irradiation required a region near the C-terminal (826-1178).

5-(Hydroxymethyl)-2-furfural: A selective inhibitor of DNA polymerase λ and terminal deoxynucleotidyltransferase

5-(Hydroxymethyl)-2-furfural (HMF), a pyrolysate of carbohydrate isolated from instant coffee ( Coffea arabica L.), selectively inhibits the activities of mammalian DNA polymerase λ (pol λ) and terminal deoxynucleotidyltransferase (TdT) which are family X pols, in vitro. The compound influenced neither the activities of replicative DNA polymerases such as α, δ, and ε, nor even the activity of pol β which is from the same family and thought to have a very similar three-dimensional structure to the pol β-like region of pol λ. Since parts of HMF such as furan, furfuryl alcohol, and 2-furaldehyde did not influence the activities of any enzymes tested, the substituted form of furan with a hyroxymethyl group and a formyl group might be important for the inhibition of pol λ and TdT. The inhibitory effect of HMF on intact pol λ (i.e., residues 1–575), a truncated pol λ lacking the N-terminal BRCA1 C-terminus domain (133–575, del-1 pol λ) and another truncated pol λ lacking the N-terminal proline-rich region (245–575, del-2 pol λ) was dose-dependent, and 50% inhibition was observed at a concentration of 26.1, 10.3, and 4.6 μM, respectively. The IC 50 value of HMF for TdT was the same as that for del-2 pol λ (5.5 μM). The HMF-induced inhibition of both pol λ and TdT activities was competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, HMF was suggested to bind to the pol β-like region of pol λ and TdT.

Inhibitory effect of tocotrienol on eukaryotic DNA polymerase λ and angiogenesis

Tocotrienols, vitamin E compounds that have an unsaturated side chain with three double bonds, selectively inhibited the activity of mammalian DNA polymerase λ (pol λ) in vitro. These compounds did not influence the activities of replicative pols such as α, δ, and ε, or even the activity of pol β which is thought to have a very similar three-dimensional structure to the pol β-like region of pol λ. Since δ-tocotrienol had the strongest inhibitory effect among the four (α- to δ-) tocotrienols, the isomer’s structure might be an important factor in the inhibition of pol λ. The inhibitory effect of δ-tocotrienol on both intact pol λ (residues 1–575) and a truncated pol λ lacking the N-terminal BRCA1 C-terminus (BRCT) domain (residues 133–575, del-1 pol λ) was dose-dependent, with 50% inhibition observed at a concentration of 18.4 and 90.1 μM, respectively. However, del-2 pol λ (residues 245–575) containing the C-terminal pol β-like region was unaffected. Tocotrienols also inhibited the proliferation of and formation of tubes by bovine aortic endothelial cells, with δ-tocotrienol having the greatest effect. These results indicated that tocotrienols targeted both pol λ and angiogenesis as anti-cancer agents. The relationship between the inhibition of pol λ and anti-angiogenesis by δ-tocotrienol was discussed.

Inhibition of DNA Polymerase λ Suppresses 12-O-Tetradecanoylphorbol- 13-Acetate-Induced Inflammation

We found that compounds, which can selectively inhibit the activity of mammalian DNA polymerase λ (pol λ) in vitro, show an anti-12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammatory effect in mice. Originally, we screened selective inhibitors for each of the mammalian DNA polymerases, and found two novel pol λ-inhibitors, phenolic compounds termed petasiphenol and curcumin (diferuloylmethane). Curcumin is known as an anti-chronic inflammatory agent and structurally quite similar to petasiphenol. The IC50 values of petasiphenol and curcumin for pol λ were 7.8 μM and 7.0 μM, respectively, and neither compound influenced any other mammalian DNA polymerases. Expectedly, petasiphenol also showed an anti-TPA-induced inflammatory effect. A relationship between the pol λ-inhibition and the anti-inflammatory activity is suggested. Therefore, we investigated whether other anti-inflammatory compounds such as terpeno benzoic acids, triterpene acids and epolactaene derivatives could be pol λ-inhibitors. Although all the compounds influenced not only several different DNA polymerase species but DNA topoisomerase II, they all most efficiently inhibited the pol λ-activity. These results unexpectedly suggest that there is a physiological relationship between pol l inhibition and anti-TPA-induced inflammation. This finding may provide insight into the molecular mechanism of TPAinduced inflammation, or neoplastic promotion. Keywords: petasiphenol, curcumin, dna polymerase l, enzyme inhibitor, anti-inflammation

The influence of the DNA polymerase γ accessory subunit on base excision repair by the catalytic subunit

Mammalian DNA polymerase γ, the sole polymerase responsible for replication and repair of mitochondrial DNA, contains a large catalytic subunit and a smaller accessory subunit, pol γB. In addition to the polymerase domain, the large subunit contains a 3′–5′ editing exonuclease domain as well as a dRP lyase activity that can remove a 5′-deoxyribosephosphate (dRP) group during base excision repair. We show that the accessory subunit enhances the ability of the catalytic subunit to function in base excision repair mainly by stimulating two subreactions in the repair process. Pol γB appeared to specifically enhance the rate at which pol γ was able to locate damage in high molecular weight DNA. One pol γB point mutant known to have impaired ability to bind duplex DNA stimulated repair poorly, suggesting that duplex DNA binding through pol γB may help the catalytic subunit locate sites of DNA damage. In addition, the small subunit significantly stimulated the dRP lyase activity of pol γA, although it did not increase the rate at which the dRP group dissociated from the enzyme. The ability of DNA pol γ to process a high load of damaged DNA may be compromised by the slow release of the dRP group.

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 (Polβ)-like, template-dependent DNA repair enzyme, with a metal preference for Mn 2+ over Mg 2+. As is the case with mammalian Polβ and DNA polymerase lambda (Polλ), L. infantum DNA polymerase beta (Li Polβ) prefers gapped-DNA substrates having a 5′-phosphate end, in agreement with its role in DNA repair reactions. Purified Li Polβ also displayed a 5′-deoxyribose-5-phosphate (dRP) lyase activity, consistent with a β-elimination mechanism. The concerted action of dRP lyase and DNA polymerization activities of Li Polβ on a uracil-containing DNA suggests its participation in “single-nucleotide” base excision repair (BER). Analysis of Li Polβ DNA polymerization activity at different stages of the L. infantum infective cycle supports a role for Li Polβ in nuclear DNA repair after the oxidative damage occurring inside the macrophage.

Sulfoquinovosylmonoacylglycerol inhibitory mode analysis of rat DNA polymerase β

We have previously reported that sulfoquinovosylmonoacylglycerol (SQMG) is a potent inhibitor of mammalian DNA polymerases. DNA polymerase beta (pol beta) is one of the most important enzymes protecting the cell against DNA damage by base excision repair. In this study, we characterized the inhibitory action of SQMG against rat pol beta. SQMG competed with both the substrate and the template-primer for binding to pol beta. A gel mobility shift assay and a polymerase activity assay showed that SQMG competed with DNA for a binding site on the N-terminal 8-kDa domain of pol beta, subsequently inhibiting its catalytic activity. Fragments of SQMG such as sulfoquinovosylglycerol (SQG) and fatty acid (myristoleic acid, MA) weakly inhibited pol beta activity and the inhibitory effect of a mixture of SQG and MA was stronger than that of SQG or MA. To characterize this inhibition more precisely, we attempted to identify the interaction interface between SQMG and the 8-kDa domain by NMR chemical shift mapping. Firstly, we determined the binding site on a fragment of SQMG, the SQG moiety. We observed chemical shift changes primarily at two sites, the residues comprising the C-terminus of helix-1 and the N-terminus of helix-2, and residues in helix-4. Finally, based on our present results and our previously reported study of the interaction interface of fatty acids, we constructed two three-dimensional models of a complex between the 8-kDa domain and SQMG and evaluated them by the mutational analysis. The models show a SQMG interaction interface that is consistent with the data.

Inhibitory effects of glycolipids fraction from spinach on mammalian DNA polymerase activity and human cancer cell proliferation

We succeeded in purifying the fraction containing the major glycolipids in monogalactosyl diacylglycerol, digalactosyl diacylglycerol and sulfoquinovosyl diacylglycerol (SQDG) from dried vegetables. This glycolipids fraction was an inhibitor of DNA polymerase α (pol α) in vitro and also the proliferation of human cancer cells. In this study, eight common vegetables were investigated in terms of the glycolipids fraction, the amounts of major glycolipids, mammalian DNA polymerase inhibitory activity and antiproliferative activity toward human cancer cells. Green tea possessed the largest amount of glycolipids overall. Spinach contained the largest amount of SQDG, followed by parsley, green onion, chive, sweet pepper, green tea, carrot and garlic. Spinach had the strongest inhibitory effect on pol α activity and human cancer cell proliferation. A significant correlation was found between SQDG content and inhibition of DNA polymerase. Therefore, the inhibition of pol α activity by SQDG may lead to cell growth suppression. Of the six subspecies of spinach ( Spinacia oleracea) tested, “Anna” had the largest amount of SQDG, strongest inhibitory activity toward DNA polymerase and greatest effect on human cancer cell proliferation. Based on these results, the glycolipids fraction from spinach is potentially a source of food material for a novel anticancer activity.

Development of DNA polymerase IIIC inhibitors for the treatment of Gram-positive bacterial infections

Gram-positive (Gm+) bacteria express three distinct DNA polymerase-exonucleases. One of these, Gm+ DNA polymerase IIIC (DNA pol IIIC), is a highly conserved enzyme with little homology to mammalian DNA polymerase α and Gram-negative (Gm–) DNA polymerases. DNA pol IIIC has been shown to be essential in the replicative DNA synthesis of Gm+ bacteria and, as such, represents an attractive, hitherto unexploited target for antimicrobial drug development. This article briefly reviews claims for DNA pol IIIC inhibitors for the treatment of bacterial infections, registered during the period 1996 – 2004. Biological data are sparse in these patents and few claims are backed up with in vivo animal model data. Although DNA pol IIIC has clearly been validated as a bona fide target for antimicrobial drug development, the effectiveness of such an agent in the clinic, particularly against resistant strains of Gm+ bacteria, remains to be determined.

Selective inhibitors of terminal deoxyribonucleotidyltransferase (TdT): Baicalin and genistin

Studies of mammalian terminal deoxyribonucleotidyltransferase (TdT) are facilitated by use of inhibitors that selectively knock down the activity of the enzyme. We have screened for selective inhibitors of TdT and identified a natural compound with this property in the Japanese vegetable, Arctium lappa. The compound has little effect on the activities of mammalian DNA polymerases, such as alpha, beta, delta or lambda polymerase, and prokaryotic DNA polymerases, such as Taq DNA polymerase, T4 DNA polymerase and Klenow fragment. H1- and C13-NMR spectroscopic analyses showed the compound to be baicalin, a compound previously reported as an anti-inflammatory or antipyretic agent. The IC50 value of baicalin to TdT was 18.6 microM. We also found that genistin, a baicalin derivative known to be antimutagenic, more selectively inhibited TdT activity than baicalin, although its IC50 value was weaker (28.7 microM). Genistin and baicalin also inhibited the activity of truncated TdT (the so-called pol beta core domain) in which the BRCT motif was deleted in its N-terminal region. In kinetic analyses, inhibition by either genistin or baicalin was competitive with the primer and non-competitive with the dNTP substrate. The compounds may, therefore, bind directly to the primer-binding site of TdT and simultaneously disturb dNTP substrate incorporation into the primer. Genistin and baicalin should prove to be useful agents for studying TdT.

Inhibition of mammalian DNA polymerases by resveratrol: mechanism and structural determinants

Resveratrol, a natural compound found in many dietary plants and in red wine, plays an important role in the prevention of many human pathological processes, including inflammation, atherosclerosis and carcinogenesis. We have shown that the antiproliferative activity of resveratrol correlated with its ability to inhibit the replicative pols (DNA polymerases) alpha and delta in vitro [Stivala, Savio, Carafoli, Perucca, Bianchi, Maga, Forti, Pagnoni, Albini, Prosperi and Vannini (2001) J. Biol. Chem. 276, 22586-22594]. In this paper, we present the first detailed biochemical investigation on the mechanism of action of resveratrol towards mammalian pols. Our results suggest that specific structural determinants of the resveratrol molecule are responsible for selective inhibition of different mammalian pols, such as the family B pol alpha and the family X pol lambda. Moreover, the resveratrol derivative trans-3,5-dimethoxy-4-hydroxystilbene, which is endowed with a strong antiproliferative activity (Stivala et al., 2001), can inhibit pols alpha and lambda and also suppress the in vitro SV40 DNA replication. The potency of inhibition is similar to that of aphidicolin, an inhibitor of the three replicative pols alpha, delta and epsilon. Our findings establish the necessary background for the synthesis of resveratrol derivatives having more selective and potent antiproliferative activity.

Inhibitory effect of conjugated eicosapentaenoic acid on mammalian DNA polymerase and topoisomerase activities and human cancer cell proliferation

Conjugated eicosapentaenoic acid (cEPA) selectively inhibited the activities of mammalian DNA polymerases (pols) and human DNA topoisomerases (topos) [Yonezawa Y, Tsuzuki T, Eitsuka T, Miyazawa T, Hada T, Uryu K, et al. Inhibitory effect of conjugated eicosapentaenoic acid on human DNA topoisomerases I and II. Arch Biochem Biophys 2005;435:197–206]. In this report, we investigated the inhibitory effect of cEPA on a human promyelocytic leukemia cell line, HL-60, to determine which enzymes influence cell proliferation. cEPA inhibited the proliferation of HL-60 cells (LD 50 = 20.0 μM), and the inhibitory effect was stronger than that of non-conjugated EPA. cEPA arrested the cells at G1/S-phase, increased cyclin A and E protein levels, and prevented the incorporation of thymidine into the cells, indicating that it blocks the primary step of in vivo DNA replication by inhibiting the activity of replicative pols rather than topos. This compound also induced apoptosis of the cells. These results suggested the therapeutic potential of cEPA as a leading anti-cancer compound that poisons pols.

Comparison of functional properties of mammalian DNA polymerase λ and DNA polymerase β in reactions of DNA synthesis related to DNA repair

DNA polymerase λ (Pol λ) is a novel enzyme of the family X of DNA polymerases. Pol λ has some properties in common with DNA polymerase β (Pol β). The substrate properties of Pol λ were compared to Pol β 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 λ was investigated. Pol λ 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 λ. FEN1 processes nicked DNA, thus removing a barrier to Pol λ DNA synthesis. It results in a one-nucleotide gapped DNA molecule that is a favorite substrate of Pol λ. Photocrosslinking and functional assay show that Pol λ is less efficient than Pol β in binding to nicked DNA. APE1 has no influence on the strand displacement activity of Pol λ though it stimulates strand displacement synthesis catalyzed with Pol β. It is suggested that Pol λ plays a role in the SP BER rather than contributes to the LP BER pathway.

Structural analysis of isosteviol and related compounds as DNA polymerase and DNA topoisomerase inhibitors

Isosteviol ( ent-16-ketobeyeran-19-oic acid) is a hydrolysis product of stevioside, which is a natural sweetener produced in the leaves of Stevia rebaudiana (Bertoni) Bertoni. In this report, we prepared isosteviol and related compounds from stevioside by microbial transformation and chemical conversion and assayed the inhibitory activities toward DNA metabolic enzymes and human cancer cell growth. Among twelve compounds obtained, only isosteviol (compound 3) potently inhibited both mammalian DNA polymerases (pols) and human DNA topoisomerase II (topo II), and IC 50 value for pol α was 64.0 μM. This compound had no inhibitory effect on higher plant (cauliflower) pols, prokaryotic pols, human topo I, and DNA metabolic enzymes such as human telomerase, T7 RNA polymerase, and bovine deoxyribonuclease I. With pol α, isosteviol acted non-competitively with the DNA template-primer and nucleotide substrate. Isosteviol prevented the growth of human cancer cells, with LD 50 values of 84–167 μM, and 500 μg of the compound caused a marked reduction in TPA (12- O-tetradecanoylphorbol-13-acetate)-induced inflammation (inhibitory effect, 53.0%). The relationship between the structure of stevioside-based compounds and these activities were discussed.

Miscoding Events during DNA Synthesis Past the Nitration-Damaged Base 8-Nitroguanine

8-Nitro-2'-deoxyguanosine (8-NO(2)-dG) DNA adducts are induced by the reactive nitrogen species and may be associated with the development of cancer in inflammatory tissues. To explore the miscoding potential of 8-NO(2)-dG adduct, an oligodeoxynucleotide containing a single 8-NO(2)-dG adduct was prepared by photochemical synthesis and used as a template in primer extension reactions catalyzed by mammalian DNA polymerases (pol). Primer extension reactions catalyzed by pol alpha or beta were strongly retarded at the 8-NO(2)-dG lesion; a fraction of primers was extended past the lesion by incorporating preferentially dCMP, the correct base, opposite the lesion, accompanied by lesser amounts of dAMP and dGMP incorporation. In contrast, primer extension reactions catalyzed by pol eta or a truncated form of pol kappa (pol kappaDeltaC) readily extended past the 8-NO(2)-dG lesion. Pol eta and kappaDeltaC showed more broad miscoding spectra; direct incorporations of dCMP and dAMP were observed, along with lesser amounts of dGMP and dTMP incorporations and deletions. The miscoding frequencies induced by pol eta and kappaDeltaC were at least 8 times higher than that of pol alpha or beta. Miscoding frequency and specificity of 8-NO(2)-dG varied depending on the DNA polymerases used. These observations were supported by steady-state kinetic studies. 8-NO(2)-dG adduct may play an important role in initiating inflammation driven carcinogenesis.

Structural analysis of catechin derivatives as mammalian DNA polymerase inhibitors

The inhibitory activities against DNA polymerases (pols) of catechin derivatives (i.e., flavan-3-ols) such as (+)-catechin, (−)-epicatechin, (−)-gallocatechin, (−)-epigallocatechin, (+)-catechin gallate, (−)-epicatechin gallate, (−)-gallocatechin gallate, and (−)-epigallocatechin gallate (EGCg) were investigated. Among the eight catechins, some catechins inhibited mammalian pols, with EGCg being the strongest inhibitor of pol α and λ with IC 50 values of 5.1 and 3.8 μM, respectively. EGCg did not influence the activities of plant (cauliflower) pol α and β or prokaryotic pols, and further had no effect on the activities of DNA metabolic enzymes such as calf terminal deoxynucleotidyl transferase, T7 RNA polymerase, and bovine deoxyribonuclease I. EGCg-induced inhibition of pol α and λ was competitive with respect to the DNA template-primer and non-competitive with respect to the dNTP (2′-deoxyribonucleotide 5′-triphosphate) substrate. Tea catechins also suppressed TPA (12- O-tetradecanoylphorbol-13-acetate)-induced inflammation, and the tendency of the pol inhibitory activity was the same as that of anti-inflammation. EGCg at 250 μg was the strongest suppressor of inflammation (65.6% inhibition) among the compounds tested. The relationship between the structure of tea catechins and the inhibition of mammalian pols and inflammation was discussed.

DNA Polymerase λ 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.

734. Association of DNA Polymerase μ with Recombinant Adeno-Associated Viral Vectors

Recombinant adeno associated viral (rAAV) vectors have been successfully used for efficient delivery and stable transgene expression in animal models and clinical trials. Transduced vector genomes primarily persist as extra-chromosomal concatamers, with a low frequency of integration into host chromosomes. Investigations into possible mechanisms involved in vector genome integration and analyses of integration junctions reveal actively transcribed regions of chromosomes to be the hotspots for vector integration via a proposed nonhomologous end joining (NHEJ) mechanism of DNA repair (Nakai et.al 2003). Although the primary sensors of DNA damage, Ku, ATM and DNA-PK have been implicated in rAAV integration, there is limited information about the involvement of proteins that are directly involved in NHEJ. A recently discovered mammalian DNA polymerase |[mu]| (Pol|[mu]|) has been shown to be an important component of the NHEJ ligation complex and to have a functional interaction with Ku-DNA complex. In this study, we investigated the effect of rAAV on induction of Pol|[mu]| expression and the possible interaction with vector genomes. HEK293 cells transduced with rAAV2 at an MOI of 20,000 were incubated at 37|[deg]|C for 0, 0.5, 1, 3, 6, 12 and 24 hours. Total proteins were harvested at each time point and the lysates were analyzed by Western blotting using anti-Pol|[mu]| antibody. The results showed a nine-fold increase in Pol|[mu]| expression in cells incubated for 12h post transduction. A three fold increase in expression was observed at the 3h time point. The 0.5 and 1hr treatments did not show a significant increase over basal levels. Expression levels dropped down to 2 fold at the 24h time point.