Studies of multifunctional DNA polymerase I from the extremely radiation resistant Deinococcus radiodurans: Recombinant expression, purification and characterization of the full-length protein and its large fragment.

Deinococcus radiodurans is a bacterium with extreme resistance to desiccation and radiation. Although the origins of this extreme resistance have not been fully elucidated, an efficient DNA repair machinery that includes the enzyme DNA polymerase I, is potentially crucial as part of a protection mechanism. Here we have cloned and performed small, medium, and large-scale expression of full-length D. radiodurans DNA polymerase I (DrPolI) as well as the large/Klenow fragment (DrKlenow). We then carried out functional characterization of 5' exonuclease, DNA strand displacement and polymerase activities of these proteins using gel-based and molecular beacon-based biochemical assays. With the same expression and purification strategy, we got higher yield in the production of DrKlenow than of the full-length protein, approximately 2.5 mg per liter of culture. Moreover, we detected a prominent 5' exonuclease activity of DrPolI in vitro. This activity and, DrKlenow strand displacement and DNA polymerase activities are preferentially stimulated at pH 8.0-8.5 and are reduced by addition of NaCl. Interestingly, both protein variants are more thermostable at pH 6.0-6.5. The characterization of DrPolI's multiple functions provides new insights into the enzyme's role in DNA repair pathways, and how the modulation of these functions is potentially used by D. radiodurans as a survival strategy.

DNA helicase activity in purified human RECQL4 protein.

Human RECQL4 protein was expressed in insect cells using a baculovirus protein expression system and it was purified to near homogeneity. The protein sedimented at a position between catalase (230 kDa) and ferritin (440 kDa) in glycerol gradient centrifugation, suggesting that it forms homo-multimers. Activity to displace annealed 17-mer oligonucleotide in the presence of ATP was co-sedimented with hRECQL4 protein. In ion-exchange chromatography, both DNA helicase activity and single-stranded DNA-dependent ATPase activity were co-eluted with hRECQL4 protein. The requirements of ATP and Mg for the helicase activity were different from those for the ATPase activity. The data suggest that the helicase migrates on single-stranded DNA in a 3'-5' direction. These results suggest that the hRECQL4 protein exhibits DNA helicase activity.

Engagement of DNA polymerases during apoptosis.

DNA replicative and repair machinery was investigated by means of different techniques, including in vitro nuclear enzymatic assays, immunoelectron microscopy and confocal microscopy, in apoptotic cell lines such as HL-60 treated with methotrexate, P815 and K562 exposed to low temperatures and Friend cells exposed to ionizing radiation. The results showed a shift of DNA polymerase alpha and beta activities. DNA polymerase alpha, which in controls was found to be the principal replicative enzyme driving DNA synthesis, underwent, upon apoptosis, a large decrease of its activity being replaced by DNA polymerase beta which is believed to be associated with DNA repair. Such a modulation was concomitant with a topographical redistribution of both DNA polymerase alpha and the incorporation of BrdUrd throughout the nucleus. Taken together, these results indicate the occurrence of a dramatic response of the DNA machinery, through a possible common or at least similar behaviour when different cell lines are triggered to apoptosis. Although this possibility requires further investigation, these findings suggest an extreme attempt of the cell undergoing apoptosis to preserve its nuclear environment by switching on a repair/defence mechanism during fragmentation and chromatin margination.

Comparison of hyperthermia radiosensitization and DNA polymerase inactivation in human normal and melanoma cell lines of different radiosensitivities.

Two human melanoma cell lines (one radiosensitive, HT144 and one radioresistant, SK Mel-3) and one normal human fibroblast (AG1522) were evaluated for thermal radiosensitization and the thermal enhancement ratios (TERs) were calculated. These were compared with residual polymerase activity to determine if this activity could be used to predict TERs. In all three cell lines, there was a good correlation between TER and residual polymerase alpha or beta activity. Polymerase beta was more sensitive than polymerase alpha as an indicator for TER. There were small cell line-dependent differences (not related to radiosensitivity) among the correlation curves, indicating that for each cell/tumor-type polymerase activity, vs. TER may have to be calibrated.

In vitro UV mutagenesis associated with nucleotide excision-repair gaps in Escherichia coli.

Using a cell-free system for UV mutagenesis we have recently shown that extracts prepared from Escherichia coli cells promote a UV mutagenesis pathway that depends on the uvrABC repair genes independent of DNA replication (type II UV mutagenesis; Cohen-Fix, O., and Livneh, Z. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3300-3304). Type II UV mutagenesis was defective also in extracts prepared from a uvrD strain. These deficiencies were complemented by adding purified UvrA, UvrB, UvrC, or UvrD proteins to the respective cell extracts. The Uvr proteins act at an early stage in the process, probably preparing a premutagenic single-stranded DNA gap, which subsequently serves as a substrate for the mutagenic reaction. Type II UV mutagenesis was not dependent on DNA polymerases I or on DNA polymerase II, but it was dependent on DNA polymerase III. Thus, similar to the in vivo situation, only DNA polymerase III is essential for UV mutagenesis. Antibodies against the beta subunit of DNA polymerase III holoenzyme inhibited DNA replication but not UV mutagenesis. Thus, the processivity subunit of the holoenzyme is not required for type II UV mutagenesis, in agreement with a mechanism involving filling-in of short single-stranded DNA gaps.

Radiation inactivation analysis of enzymes. Effect of free radical scavengers on apparent target sizes.

In most cases the apparent target size obtained by radiation inactivation analysis corresponds to the subunit size or to the size of a multimeric complex. In this report, we examined whether the larger than expected target sizes of some enzymes could be due to secondary effects of free radicals. To test this proposal we carried out radiation inactivation analysis on Escherichia coli DNA polymerase I, Torula yeast glucose-6-phosphate dehydrogenase, Chlorella vulgaris nitrate reductase, and chicken liver sulfite oxidase in the presence and absence of free radical scavengers (benzoic acid and mannitol). In the presence of free radical scavengers, inactivation curves are shifted toward higher radiation doses. Plots of scavenger concentration versus enzyme activity showed that the protective effect of benzoic acid reached a maximum at 25 mM then declined. Mannitol alone had little effect, but appeared to broaden the maximum protective range of benzoic acid relative to concentration. The apparent target size of the polymerase activity of DNA polymerase I in the presence of free radical scavengers was about 40% of that observed in the absence of these agents. This is considerably less than the minimum polypeptide size and may reflect the actual size of the polymerase functional domain. Similar effects, but of lesser magnitude, were observed for glucose-6-phosphate dehydrogenase, nitrate reductase, and sulfite oxidase. These results suggest that secondary damage due to free radicals generated in the local environment as a result of ionizing radiation can influence the apparent target size obtained by this method.

[Mechanisms of the radiation-induced disorder of DNA synthesis. The correlation of biosynthesis, DNA repair and DNA polymerase alpha and beta activity in the bone marrow of rats exposed to gamma quanta and fast neutrons]. / Mekhanizmy radiatsionnogo narusheniia biosinteza DNK. Sootnoshenie biosinteza, reparatsii DNK i aktivnosti DNK-polimeraz al'fa i beta v kostnom mozge u krys pri deistvii gamma-kvantov i bystrykh neitronov.

A study was made of DNA biosynthesis and repair and alpha- and beta-DNA-polymerase activity in rat bone marrow during the first 24 hours following whole-body irradiation with gamma-quanta and fast neutrons (up to 6 Gy). There was a correlation between the post-irradiation inhibition of DNA biosynthesis, a decrease in DNA-polymerase activity and template reparability. The data obtained permitted to consider the radiation-induced disturbance of DNA biosynthesis and the change in beta-polymerase activity as one of the possible mechanisms of formation of high relative biological effectiveness of neutrons.

Strand break repair, DNA polymerase activity and heat radiosensitization in thermotolerant cells.

HeLa S3 cells were made thermotolerant by 'chronic' (5 h at 42 degrees C) or 'acute' (15 min at 44 degrees C followed by 5 h at 37 degrees C) heat treatments. Cell survival, repair of radiation-induced DNA strand breaks, alpha and beta DNA polymerase activity and radiation sensitivity following hyperthermia were all measured in both control and thermotolerant cells. The ability to repair DNA strand breaks correlated well with cell survival following hyperthermia. Hyperthermic inhibition of strand break repair was reduced in thermotolerant relative to control cells, although the thermal tolerance ratios for repair were less than for hyperthermic cell killing. Both radiosensitization and DNA polymerase inactivation by hyperthermia were only slightly reduced in thermotolerant relative to control cells. Hence a poor correlation was found between these two parameters and hyperthermic cell survival. For all heat treatments applied, alpha and beta DNA polymerase activity correlated well with the extent of hyperthermic radiosensitization.

DNA replication of X-irradiated human lymphocytes.

Human peripheral blood lymphocytes are well-differentiated cells. Ordinarily, they do not divide and are considered to be in the G0 stage of the cell cycle. These cells can be stimulated to undergo DNA replication in culture by mitogens such as phytohemagglutinin. In the present study, we have examined cellular and biochemical events that occur after exposure of lymphocytes to X-irradiation. Irradiation with up to 100 rads, prior to stimulation with phytohemagglutinin, did not interfere with DNA replication. At later periods, DNA replication was inhibited proportionally to the amount of radiation. In comparison to DNA synthesis, the effect of X-irradiation on RNA and protein synthesis in phytohemagglutinin-stimulated lymphocytes was less marked. Furthermore, X-rays did not inhibit either the induction or the continual synthesis of DNA polymerase-alpha or -beta in response to phytohemagglutinin. Kinetic studies with different nucleotide substrates suggest that cellular pools of nucleotides are not significantly altered by X-irradiation. Thus, the inhibition of DNA synthesis in irradiated cells is likely to be due to damage to the cellular DNA template. The inhibition of DNA synthesis was accompanied by accumulation of cells in the G2 and M stages of the cell cycle, suggesting that inhibition of DNA replication by X-irradiation is a postmitotic event.

DNA polymerase alpha and beta activity in gamma-irradiated HeLa S3 cells.

The acute effects (less than 2 hours) of gamma-irradiation on DNA polymerase alpha and beta activity in HeLa S3 cells were studied. The enzyme activities were measured in sonicates of the irradiated cells, using an exogenous DNA as template. Both DNA alpha- and beta-polymerase activities decreased following irradiation of the cells. Doses as low as 100 rad significantly reduced the activities of the enzymes. While the activities of both DNA polymerases decreased as the dose received by the cells increased, the major reduction in enzyme activity occurred with doses of 100--200 rad. The reduction in DNA alpha- and beta-polymerase activities was maximal by 30 min post-irradiation and recovered to control values by 2 hours post-irradiation.