DNA Polymerase Activity In Extracts Research Articles

Inhibition of Mn2+-induced error-prone DNA synthesis with Cd2+ and Zn2+

Bivalent metal cations are key components in the reaction of DNA synthesis. They are necessary for all DNA polymerases, being involved as cofactors in catalytic mechanisms of nucleotide polymerization. It is also known that in the presence of Mn2+ the accuracy of DNA synthesis is considerably decreased. The findings of this work show that Cd2+ and Zn2+ selectively inhibit the Mn2+-induced error-prone DNA polymerase activity in extracts of cells from human and mouse tissues. Moreover, these cations in low concentrations also can efficiently inhibit the activity of homogeneous preparations of DNA polymerase iota (Pol ι), which is mainly responsible for the Mn2+-induced error-prone DNA polymerase activity in cell extracts. Using a primary culture of granular cells from postnatal rat cerebellum, we show that low concentrations of Cd2+ significantly increase cell survival in the presence of toxic Mn2+ doses. Thus, we have shown that in some cases low concentrations of Cd2+ can display a positive influence on cells, whereas it is widely acknowledged that this metal is not a necessary microelement and is toxic for organisms.

DNA polymerase in nuclei isolated from herpes simplex virus type-2-infected cells. Characterization of the reaction product and inhibition by substrate analogs

Nuclei isolated from herpes simplex virus (HSV) type 2-infected KB cells were examined for their capacity to serve as an in situ source of herpes DNA polymerase. In contrast to purified enzymes with added template, approx. 80% of the DNA synthesized in isolated nuclei was viral. The average size of DNA fragments labeled in vitro was 3.2·10 6 Da. Based on an increase in DNA density when nuclei were incubated in the presence of BrdUTP rather than dTTP, 16% of the nucleotides were added during the in vitro reaction. Sucrose gradient analysis of DNA polymerase activity in extracts of isolated nuclei demonstrated the nearly exclusive presence of herpes DNA polymerase. K m concentrations for the four dNTPs were from 0.14 to 0.55 μM. DNA synthesis was inhibited competitively by the 5′-triphosphates of ara-A and ara-C ( K i = 0.03 and 0.22 μM, respectively) but not by the 5′-triphosphate of dideoxythymidine. aATP also served as a substrate ( K m = 0.014 μM) for the reaction. We conclude that nuclei from HSV-infected cells have significant advantages for the detailed study of inhibitors of herpesvirus replication.

Effects of chirality in 9-(2-hydroxy-3-nonyl)adenine upon deoxyribonucleic acid synthesis in herpes simplex virus-infected cells

The antiherpes activities of erythro- and threo-9-(2-hydroxy-3-nonyl)adenines (EHNA and THNA) have been determined. All isomers inhibited the replication of herpes simplex virus (HSV) and inhibited DNA synthesis in HSV-infected cells. The two enantiomers of EHNA, (+)-EHNA and (−)-ENHA, displayed equal antiviral activities. This is in contrast to their activities as inhibitors of adenosine deaminase (ADA); (+)-EHNA is a 250-fold more potent inhibitor of ADA than (−)-EHNA [Bessodes et al. Biochem. Pharmac. 31, 879 (1982)]. The antiherpes activity of (+)-THNA was only slightly less than that of the EHNA isomers, whereas (−)-THNA was somewhat less active. The abilities of the four isomeres of EHNA and THNA to inhibit DNA synthesis in HSV-infected cells correlated with their abilities to inhibit virus multiplication. EHNA failed to inhibit HSV DNA polymerase activity in extracts from infected cells. Moreover, addition of EHNA to infected cells at 6 hr post-infection resulted in no inhibition of DNA synthesis. These results are inconsistent with a direct inhibition of macromolecular DNA synthesis by EHNA. Treatment of HSV-infected cells with EHNA produced a 2- to 4-fold decrease in levels of the four DNA precursors, deoxyribonucleoside 5'-triphosphates (dNTPs). This treatment had much less effect on dNTP levels in uninfected cells.

Inducible DNA polymerase in cultured rat fibroblasts treated with skin carcinogen mitomycin C

DNA polymerases are involved in the filling of excision [ 1,2] and postreplication [3,4] single-strand gaps in DNA in ultraviolet-irradiated bacteria. Gapfilling with the constitutive DNA polymerase is an error-free process that cannot be the source of ultraviolet-induced mutations [ 51. It has been reported that heat treatment of tif strain of E. coli causes the appearance of a mutagenic DNA polymerase responsible for the fixation of premutational lesions during heat-induced ‘SOS repair’ [6] . An inducibility has been shown for minor branch of postreplication repair in E. coli [7] , Micrococcus luteus [8] and in human fibroblasts [9] . The question remains: Is inducible repair in mammalian cells coupled with the induction of a DNA polymerase with a reduced fidelity in repair replication? Mitomycin C (MMC) studied extensively for its mutagenic [lo] and carcinogenic [ 111 properties can induce in E. cob the synthesis of X protein [ 121. This property usually correlates with the ability of a chemical to induce SOS repair [5] . In connection with the question above the observation of an increased DNA polymerase activity in extracts from MMC-treated mammalian cells [ 13,141 is of interest. We therefore investigated the origin and reaction properties of the DNA polymerase in cells affected by MMC.

Appearance of DNA polymerase activities during early development of Xenopus laevis

Extracts of large oocytes of Xenopus laevis contain high levels of one major DNA polymerase activity. After maturation into eggs, the overall level of DNA polymerase activity in extracts increases fourfold and a second major activity appears on Sephadex G-200 or DEAE cellulose columns. Although intense DNA synthesis occurs as the number of cells increase from one to over 100,000, no further increases in the level of either DNA polymerase activity are observed in cleavage, gastrula or early neurula stage embryos. In extracts of late neurulae or hatched embryos, however, a third major DNA polymerase activity appears coincident with an increase in the ability of the extracts to utilise native DNA templates in vitro.

Denervation effects on newt limb regeneration: DNA polymerase activity in vitro

DNA polymerase activity in extracts of newt ( Triturus viridescens) tissues was assayed in an effort to characterize the effect of nerve resection on the regeneration of amputated limbs. Regenerating limbs display 5–20 times more polymerase activity than nonregenerating limbs. Denervation of partially regenerated limbs, which completely prevents further regeneration in vivo does not, however, affect DNA polymerase activity assayed in vitro. These results suggest that while DNA polymerase activity is responsive to the stimulus of regeneration, denervation effects are probably not directly exerted on this enzyme activity.

Repair deficiency in a bacterial mutant defective in DNA polymerase

The effect of ultraviolet irradiation (UV) has been studied in Escherichia coli K12 strain W3110 pol+, and in its derivative, W3110 polA1, which lacks appreciable DNA-polymerase activity in extracts. The “polymeraseless” derivative appears to be deficient in some step in the excision-repair process, although not totally lacking in repair capacity, since: (1) It is more UV sensitive than the parent strain, yet less UV sensitive than an excision-deficient K12 strain, AB2500; (2) It exhibits a lower single-strand DNA molecular weight than the parent strain during the growth period following UV irradiation.