DNA Polymerase Beta Activity Research Articles

Enhanced expression and activity of DNA polymerase beta in human ovarian tumor cells: impact on sensitivity towards antitumor agents.

DNA polymerase beta, one of the most inaccurate DNA synthesizing enzymes, has been shown to confer genetic instability when up-regulated in cells, a situation found in several human cancers. Here, we demonstrated that enhanced activity and expression of this enzyme occur in the human ovarian tumor 2008/C13*5.25 cells, which are resistant to the antitumor agent cisplatin and hypersensitive to 6-thioguanine. We found that translesion synthesis across platinated DNA crosslinks as well as increased incorporation into DNA of 6-thioguanine took place in the 2008/C13*5.25 cells compared to the parental 2008 cells. Such features being molecular signatures of DNA polymerase beta, these findings suggest that deregulation of its expression in cancer cells may contribute to the modulation of the response to antitumor treatments and therefore to tumor progression.

Identification of an Intrinsic 5′-Deoxyribose-5-phosphate Lyase Activity in Human DNA Polymerase λ: 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.

DNA synthesis and dRPase activities of polymerase beta are both essential for single-nucleotide patch base excision repair in mammalian cell extracts.

In mammalian cells the majority of altered bases in DNA are processed through a single-nucleotide patch base excision repair mechanism. Base excision repair is initiated by a DNA glycosylase that removes a damaged base and generates an abasic site (AP site). This AP site is further processed by an AP endonuclease activity that incises the phosphodiester bond adjacent to the AP site and generates a strand break containing 3'-OH and 5'-sugar phosphate ends. In mammalian cells, the 5'-sugar phosphate is removed by the AP lyase activity of DNA polymerase beta (Pol beta). The same enzyme also fills the gap, and the DNA ends are finally rejoined by DNA ligase. We measured repair of oligonucleotide substrates containing a single AP site in cell extracts prepared from normal and Pol beta-null mouse cells and show that the reduced repair in Pol beta-null extracts can be complemented by addition of purified Pol beta. Using this complementation assay, we demonstrate that mutated Pol beta without dRPase activity is able to stimulate long patch BER. Mutant Pol beta deficient in DNA synthesis, but with normal dRPase activity, does not stimulate repair in Pol beta-null cells. However, under conditions where we measure base excision repair accomplished exclusively through a single-nucleotide patch BER, neither dRPase nor DNA synthesis mutants of Pol beta alone, or the two together, were able to complement the repair defect. These data suggest that the dRPase and DNA synthesis activities of Pol beta are coupled and that both of these Pol beta functions are essential during short patch BER and cannot be efficiently substituted by other cellular enzymes.

Modulation of base excision repair by low density lipoprotein, oxidized low density lipoprotein and antioxidants in mouse monocytes.

In the present study, we found that oxidized low density lipoprotein, but not low density lipoprotein, down-regulated base excision repair activity in extracts of mouse monocyte cell line PU5-1.8. An enzyme required in this pathway, DNA polymerase beta, was also down-regulated. In contrast, treatment of monocytes with a combination of ascorbate and alpha-tocopherol up-regulated base excision repair activity and expression of DNA polymerase beta. Co-treatment of monocytes with antioxidants plus oxidized low density lipoprotein prevented down-regulation by oxidized low density lipoprotein. Oxidative DNA damage, as measured by 8-hydroxyguanine accumulation in genomic DNA, was found in cells treated with oxidized low density lipoprotein; 8-hydroxyguanine was not found in the cells treated with low density lipoprotein, antioxidants or oxidized low density lipoprotein plus antioxidants. These results establish a linkage between the DNA base excision repair pathway, oxidative DNA damage and oxidized low density lipoprotein treatment in mouse monocytes. Since oxidized low density lipoprotein is implicated in chronic disease conditions such as atherogenesis, these findings facilitate understanding of genetic toxicology mechanisms related to human health and disease.

Damage to DNA and activity of nuclear DNA repair and replicative enzymes following N-nitrosodiethylamine treatment to rats.

Continuous administration in the drinking water of hepatocarcinogen N-nitrosodiethylamine (NDEA) to male rats (200 mg/L) for 60 days resulted in DNA damage in the form of single strand breaks. The damage, which is measured as a shift in the sedimentation of DNA in alkaline sucrose density gradients, was found to be maximum at the fourth week of treatment, and the sedimentation pattern of DNA was found to return to near normal size by the seventh week of NDEA treatment. Simultaneously, there were perturbations in the nuclear enzymes involved in DNA replication and repair. Activities of DNA polymerase beta, DNA ligase, and topoisomerase were found to increase in as early as the first week of NDEA treatment and reached the maximum at the fourth week, and thereafter declined to normal level by the eighth week of treatment. Concomitantly, the activities of DNA polymerase alpha, DNA primase, and RNA polymerase which were unaltered in the initial period of carcinogen treatment recorded a marked increase after sixth week of NDEA treatment. Results suggest that administration of NDEA inflicts DNA damage, which is manifested as increase in DNA repair enzymes in the initial period and activated DNA replicative enzymes at a later period, indicating the active proliferation of transformed cells.

Exogenous gangliosides. How do they cross the blood-brain barrier and how do they inhibit cell proliferation.

Gangliosides have been used to treat specific central nervous system lesions and to inhibit proliferation of neuroblastoma cells in vitro. However, the mechanisms by which they (1) cross the blood-brain barrier and (2) inhibit cell proliferation have not been clearly defined. Evidence is presented in support of the hypotheses that (1) serum albumin functions in the transport of gangliosides across the blood-brain barrier, and (2) when gangliosides inhibit cell proliferation, they do so by inhibiting the activity of DNA polymerases alpha and beta.

Shift of DNA Polymerase α Nuclear Distribution and Activity in Apoptotic Human Leukaemia Cells

The changes in the distribution of DNA polymerase alpha in nuclei from HL-60 cells treated with Methotrexate (MTX) for up to 15 hr. were checked by means of both confocal analysis and electron microscopy analysis. The results provided evidence that, relative to controls, in the MTX treated cells the enzyme undergoes a topographical rearrangement throughout the nucleus, showing a pattern of distribution which calls to mind the nuclear matrix structure. The "in vitro" analysis of DNA polymerases alpha, beta, and gamma activities revealed that, in nuclei from control cells, DNA polymerase alpha was the principal DNA polymerase driving this "in vitro" system, while after 15 hr. of MTX treatment its activity was largely decreased and replaced by DNA polymerase beta, which is believed to be associated with DNA repair. Taken together, these results suggest that among the intracellular processes elicited by MTX-induced apoptosis in HL60 cells, the redistribution of DNA polymerase alpha and the stimulation of DNA polymerase beta activity might represent an extreme attempt of the cell to preserve the replicative machinery during fragmentation and chromatin margination.

Regulation of DNA metabolic enzymes upon induction of preB cell development and V(D)J recombination: up-regulation of DNA polymerase delta.

Withdrawal of interleukin-7 from cultured murine preB lymphocytes induces cell differentiation including V(D)J immunoglobulin gene rearrangements and cell cycle arrest. Advanced steps of the V(D)J recombination reaction involve processing of coding ends by several largely unidentified DNA metabolic enzymes. We have analyzed expression and activity of DNA polymerases alpha, beta, delta and epsilon, proliferating cell nuclear antigen (PCNA), topoisomerases I and II, terminal deoxynucleotidyl transferase (TdT) and DNA ligases I, III and IV upon induction of preB cell differentiation. Despite the immediate arrest of cell proliferation, DNA polymerase delta protein levels remained unchanged for approximately 2 days and its activity was up-regulated several-fold, while PCNA was continuously present. Activity of DNA polymerases alpha,beta and epsilon decreased. Expression and activity of DNA ligase I were drastically reduced, while those of DNA ligases III and IV remained virtually constant. No changes in DNA topoisomerases I or II expression and activity occurred and TdT expression was moderately increased early after induction. Our results render DNA polymerase delta a likely candidate acting in DNA synthesis related to V(D)J recombination in lymphocytes.

Lack of the DNA repair protein O6-methylguanine-DNA methyltransferase in histologically normal brain adjacent to primary human brain tumors.

Exposure to exogenous alkylating agents, particularly N-nitroso compounds, has been associated with increased incidence of primary human brain tumors, while intrinsic risk factors are currently unknown. The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is a major defense against the carcinogenicity of N-nitroso compounds and other alkylators. We report here that in 55% (64/117) of cases, histologically normal brain tissue adjacent to primary human brain tumors lacked detectable MGMT activity [methyl excision repair-defective (Mer-) status]. The incidence of Mer- status in normal brain tissue from brain tumor patients was age-dependent, increasing from 21% in children 0.25-19 years of age to 75% in adults over 50. In contrast, Mer- status was found in 12% (5/43) of normal brain specimens from patients operated for conditions other than primary brain tumors and was not age-dependent. The 4.6-fold elevation in incidence of Mer- status in brain tumor patients is highly significant (chi2 = 24; p < or = 0.001). MGMT activity was independent of age in the lymphocytes of brain tumor patients and was present in lymphocytes from six of nine tumor patients whose normal brain specimen was Mer-. DNA polymerase beta, apurinic/apyrimidinic endonuclease, and lactate dehydrogenase activities were present in all specimens tested, including Mer- specimens from brain tumor patients. Our data are consistent with a model of carcinogenesis in human brain in which epigenetically regulated lack of MGMT is a predisposing factor and alkylation-related mutagenesis is a driving force.

Tokishakuyakusan Effect on DNA Polymerase α Activity in Relationship to DNA Synthesis before and/or after the LH/FSH Surge in Rats

DNA polymerase alpha activity in ovaries of mature cycling rats during the normal estrous cycle changed in a cyclic manner with a peak at 1800 h in proestrus. Tokishakuyakusan (TS) in vivo did not affect the changes in DNA polymerase alpha and beta activities during the estrous cycle. LH and FSH at 1000 or 1700 h in proestrus increased DNA polymerase alpha activity, but the DNA polymerase alpha activity induced by LH or FSH was not significantly affected by the addition of TS. DNA polymerase beta activity did not change with LH, FSH or TS. In PMS-treated or -untreated immature rats, TS enhanced ovarian DNA polymerase alpha activity but had no significant effect on LH or FSH action. In ovaries, incubated in vitro, in untreated mature or immature rats, TS enhanced ovarian DNA polymerase alpha activity but had no significant effect on LH or FSH action. These results suggest that TS stimulates ovarian DNA polymerase alpha activity in relationship to DNA synthesis and does not affect the effect of LH or FSH on the activity by preovulatory follicle before and/or after the LH/FSH surge.

Changes in the activities of DNA polymerases in growing rat lungs.

We hypothesized that cellular proliferation and the capacity to repair DNA damage in the lung might differ during the pre- and postnatal periods, because the lung is exposed to higher oxygen concentrations and/or various mutagens after birth. In order to test this hypothesis, changes in DNA content and the activities of DNA polymerase alpha and beta were studied in the lungs of 1-day prenatal to 42-day postnatal rats. Total DNA polymerase activity reached its highest level at 1 day prenatal and 1 day after birth. The activity decreased exponentially by 28% up to 14 days of age, a change inversely related to the change in DNA content. The change in total DNA polymerase activity agreed closely with the change in DNA polymerase alpha activity, but not the activity of the beta form, although small elevations in both DNA polymerase alpha and beta were observed on day 3, possibly reflecting the mechanical effect of delivery. The activity of DNA polymerase beta remained relatively constant from 1 day before birth to 21 days after birth, varying by only about 5%. From these results, it is concluded that: (1) cellular proliferation in the lung is most active during the first 2 weeks after birth as supported by the increases in DNA polymerase alpha activity and DNA content, and (2) anticipating the oxygen enriched atmosphere after birth, the level of DNA polymerase beta, involved in the DNA repair system, is already elevated during the prenatal period and remains constant throughout the postnatal period.

Enhanced repair of a cisplatin-damaged reporter chloramphenicol-O-acetyltransferase gene and altered activities of DNA polymerases alpha and beta, and DNA ligase in cells of a human malignant glioma following in vivo cisplatin therapy.

Current evidence suggest an important role for increased repair of drug-induced DNA damage as one of the major mechanisms involved in tumor cell resistance to cis-DDP. In this study, we examined the DNA repair capacity and the activities of three DNA repair related proteins, namely, DNA polymerases alpha and beta, and total DNA ligase in cells of a malignant oligodendroglioma obtained from a patient before therapy and compared it with those of a specimen of the tumor acquired after the patient had failed cis-DDP therapy. DNA repair capacity was quantitated as the extent of reactivation of the chloramphenicol-O-acetyltransferase (CAT) gene in a eukaryotic expression vector that had been damaged and inactivated by prior treatment with cis-DDP and then transfected into the tumor cells. The extent of DNA-platinum adduct formation in the expression vector was determined by flameless atomic absorption spectrometry. The level of cis-DDP resistance of cells of the two tumors was determined with the capillary tumor stem cell assay. We observed a 2.8-fold increased capacity to repair Pt-DNA adducts and reactivate the CAT gene in cells of the tumor obtained after cis-DDP therapy, compared to cells of the untreated tumor. This was associated with increases of 9.4-fold and a 2.3-fold, respectively, in DNA polymerase beta and total DNA ligase activities in cells of the treated tumor. At 5 microM cis-DDP, there was a 5.9-fold increase in the in vitro cis-DDP resistance of post-therapy tumor cells relative to cells of the untreated tumor.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of DNA Polymerase β Activity in the Pituitary Gland by Hormonal Feedback

The activity of DNA polymerase beta (beta-enzyme) in bovine and rat pituitary glands was remarkably higher in the anterior than in the posterior lobe. In sucrose gradient centrifugation, the activity in the anterior lobe of the bovine pituitary gland appeared at the sedimentation constant, 3.3 S, while that of rat pituitary gland appeared at 3.3 S, 7.3 S and 12 S, respectively. The activity in each fraction increased in the anterior lobes of rat pituitaries that were stimulated to release TSH by decreased thyroid hormone levels induced by an antithyroid drug. These results suggest that the activity of the beta-enzyme is high in the cells of the anterior lobe which are hyperfunctioned to produce pituitary thyreotrophic hormone and may be controlled by hormonal feedback regulation.

Poly(ADP-ribose) polymerase stimulates DNA polymerase alpha by physical association.

The direct effect of the eukaryotic nuclear DNA-binding protein poly(ADP-ribose) polymerase on the activity of DNA polymerase alpha was investigated. Homogenously purified poly(ADP-ribose) polymerase (5 to 10 micrograms/ml) stimulated the activity of immunoaffinity-purified calf or human DNA polymerase alpha by about 6 to 60-fold in a dose-dependent manner. It had no effect on the activities of DNA polymerase beta, DNA polymerase gamma, and primase, indicating that its effect is specific for DNA polymerase alpha. Apparently, poly(ADP-ribosyl)ation of DNA polymerase alpha was not necessary for the stimulation. The stimulatory activity is due to poly(ADP-ribose) polymerase itself since it was immunoprecipitated with a monoclonal antibody directed against poly(ADP-ribose) polymerase. Kinetic analysis showed that, in the presence of poly(ADP-ribose) polymerase, the saturation curve for DNA template primer became sigmoidal; at very low concentrations of DNA, it rather inhibited the reaction in competition with template DNA, while, at higher DNA doses, it greatly stimulated the reaction by increasing the Vmax of the reaction. By the automodification of poly(ADP-ribose) polymerase, however, both the inhibition at low DNA concentration and the stimulation at high DNA doses were largely lost. Furthermore, stimulation by poly(ADP-ribose) polymerase could not be attributed to its DNA-binding function alone since its fragment, containing only the DNA-binding domain, could not exert full stimulatory effect on DNA polymerase, as of the intact enzyme. Poly(ADP-ribose) polymerase is co-immunoprecipitated with DNA polymerase alpha, using anti-DNA polymerase alpha antibody, clearly showing that poly(ADP-ribose) polymerase may be physically associated with DNA polymerase alpha. In a crude extract of calf thymus, a part of poly(ADP-ribose) polymerase activity existed in a 400-kDa, as well as, a larger 700-kDa complex containing DNA polymerase alpha, suggesting the existence in vivo of a complex of these two enzymes.

Alterations in DNA synthesis and cellular constituents in mouse lung following bleomycin injections.

Changes in the DNA synthesis and cellular constituents of mouse lung following repeated bleomycin (BLM) injections were studied. ICR mice were administered BLM subdermally for 10 days. Wet lung weight was increased 1.36 times on day 5 after the final administration compared with control mice receiving an identical volume of saline only for 10 days. The total number of cells in the bronchoalveolar lavage fluid of the BLM group reached a maximum on day 14, and histologic investigation of the lungs revealed marked cellular infiltrations. The labeling index obtained by the antibromodeoxyuridine monoclonal antibody method for cells was increased from days 5 to 14 in the BLM group. By day 28, these inflammatory changes had subsided and fibrotic remodeling had occurred. DNA polymerase activity in the lung tissue reached its maximal level on day 5 and remained unchanged until day 14. This phenomenon occurred in parallel with increases in DNA content and synthesis. During this period, an increase in DNA polymerase-beta activity and new induction of DNA polymerase-alpha activity were observed by phosphocellulose column chromatography. From these observations, it is concluded that: (1) repeated injections of BLM cause DNA injury in lung cells; (2) there is a subsequent increase in the DNA repair function as supported by the finding of an increase in DNA polymerase-beta activity; and (3) these lead further to cell proliferation as supported by the increase in both DNA polymerase-alpha activity and DNA content. Thus, a close relationship between morphologic changes and altered DNA synthesis was observed in the lungs of mice after BLM injections.

Inactivation of DNA polymerase beta by in vitro phosphorylation with protein kinase C

The Mr = 38,300 polypeptide of the purified recombinant rat DNA polymerase beta served as an excellent substrate for protein kinase C (PKC) in vitro but not for the catalytic subunit of cAMP-dependent protein kinase. The phosphorylation by PKC resulted in inactivation of DNA polymerase beta activity, and recovery was achieved by dephosphorylation with alkaline phosphatase. Since the phosphorylated DNA polymerase beta was retained with use of a single-stranded DNA-cellulose column, inactivation might occur at a site different from that for the DNA binding. Amino acid sequence analysis of the phosphopeptides revealed that the phosphorylated sites were 2 serine residues at positions 44 and 55 from the NH2 terminus, either or both of which might be involved in the catalytic activity of DNA polymerase beta. Thus, the inactivation of the DNA repair enzyme, DNA polymerase beta, by PKC may be an important process in the modification of DNA metabolism in the nucleus through signal transduction processes.

Electrofocusing Analysis of DNA Polymerase β in Hypophysectomized Rat Testis

Isoelectric focusing studies indicated that three peaks of DNA polymerase beta (beta-enzyme) activity were focused at isoelectric point of 7.1, 8.2 and 9.1 in testes of sham-operated rats, whereas in testes of hypophysectomized rats only two peaks of the beta-enzyme activity appeared at pH 7.1 and 9.1 and the peak at pH 8.2 was reconstituted by the treatment of gonadotropins and testosterone. In the brain hypophysectomy did not affect the activities of isoelectrically different beta-enzymes. These facts imply that under in vivo conditions the beta-enzymes in testes focused at different isoelectric points are modified with unknown factors and the beta-enzyme focused at pH 8.2 is affected by pituitary hormones.

Active-site modification of mammalian DNA polymerase .beta. with pyridoxal 5'-phosphate: mechanism of inhibition and identification of lysine 71 in the deoxynucleoside triphosphate binding pocket

Pyridoxal 5'-phosphate is a potent inhibitor of the DNA polymerase activity of recombinant rat DNA polymerase beta. Kinetic studies indicate that the mechanism of PLP inhibition is complex. In a lower range of PLP concentration, inhibition is competitive with respect to substrate dNTP, whereas at higher levels of PLP several forms of enzyme combine with PLP and are involved in the overall inhibition, and a possible model for these interactions during the catalytic process is suggested. Reduction of the PLP-treated enzyme with sodium [3H]borohydride results in covalent incorporation of about 4 mol of PLP/mol of enzyme, and the modified enzyme is not capable of DNA polymerase activity. The presence of dNTP during the modification reaction blocks incorporation of 1 mol of PLP/mol of enzyme, and the enzyme so modified is almost fully active. This protective effect is not observed in the absence of template-primer. Tryptic peptide mapping of the PLP-modified enzyme reveals four major sites of modification. Of these four sites, only one is protected by dNTP from pyridoxylation. Sequence analysis of the tryptic peptide corresponding to the protected site reveals that it spans residues 68-80 in the amino acid sequence of the enzyme, with Lys 71 as the site of pyridoxylation. These results indicate that Lys 71 is at or near the binding pocket for the dNTP substrate.

Interaction of Hyperthermia and Radiation in Tolerant and Nontolerant HeLa S3 cells: Role of DNA Polymerase Inactivation

The activities of DNA polymerase alpha and beta were measured in tolerant and nontolerant HeLa S3 suspension cells. The heat-inactivation of the enzymes and their recovery when cells were incubated at 37 degrees C after the heat challenge was compared to the synergistic action of heat and radiation and its disappearance at the level of cell survival. Thermotolerant cells were radiosensitized by heat similarly to nontolerant cells, but the sensitization decreased more rapidly in the tolerant cells when time at 37 degrees C was allowed between the two treatments. For polymerase activities the extent of inactivation, as well as the kinetics of recovery, were similar in tolerant and nontolerant cells. The results show that the activities of DNA polymerase alpha and beta do not always correlate with the extent of heat radiosensitization. It is concluded that heat inactivation of these enzymes may not be taken as a general cause for the synergistic effect of hyperthermia and radiation. As an alternative mechanism, changes in nuclear protein binding due to cellular heating are suggested, since these correlate well with effects observed for radiosensitization under different experimental conditions, including the use of thermotolerant cells.

Comparative studies of the inhibitory properties of antibiotics on human immunodeficiency virus and avian myeloblastosis virus reverse transcriptases and cellular DNA polymerases.

The inhibition of human immunodeficiency virus (HIV) reverse transcriptase by certain antibiotics and related compounds was studied in comparison with that of avian myeloblastosis virus (AMV) reverse transcriptase and cellular DNA polymerases alpha and beta. In general, compounds that inhibited HIV reverse transcriptase also inhibited AMV reverse transcriptase. For example, 10 micrograms/ml of the isoquinoline quinones used in this study inhibited approximately 80% of the activity of reverse transcriptases of HIV and AMV, but did not inhibit the activity of DNA polymerases alpha and beta even at 50 micrograms/ml. AMV enzyme was more sensitive than HIV enzyme to colistin, enduracidins A and B, janiemycin, glysperin A, and thielavins A and B. The streptonigrin alkyl esters, however, inhibited HIV reverse transcriptase only. Sakyomicin A, luzopeptins, ellagic acid and suramine inhibited the activities of reverse transcriptases and cellular DNA polymerases.