Specific amino acid residues in the beta sliding clamp establish a DNA polymerase usage hierarchy in Escherichia coli.

Escherichia coli dnaN159 strains encode a mutant form of the beta sliding clamp (beta159), causing them to display altered DNA polymerase (pol) usage. In order to better understand mechanisms of pol selection/switching in E. coli, we have further characterized pol usage in the dnaN159 strain. The dnaN159 allele contains two amino acid substitutions: G66E (glycine-66 to glutamic acid) and G174A (glycine-174 to alanine). Our results indicated that the G174A substitution impaired interaction of the beta clamp with the alpha catalytic subunit of pol III. In light of this finding, we designed two additional dnaN alleles. One of these dnaN alleles contained a G174A substitution (beta-G174A), while the other contained D173A, G174A and H175A substitutions (beta-173-175). Examination of strains bearing these different dnaN alleles indicated that each conferred a distinct UV sensitive phenotype that was dependent upon a unique combination of Delta polB (pol II), Delta dinB (pol IV) and/or Delta umuDC (pol V) alleles. Taken together, these findings indicate that mutations in the beta clamp differentially affect the functions of these three pols, and suggest that pol II, pol IV and pol V are capable of influencing each others' abilities to gain access to the replication fork. These findings are discussed in terms of a model whereby amino acid residues in the vicinity of those mutated in beta159 (G66 and G174) help to define a DNA polymerase usage hierarchy in E. coli following UV irradiation.

DNA polymerase epsilon associates with the elongating form of RNA polymerase II and nascent transcripts.

DNA polymerase epsilon co-operates with polymerases alpha and delta in the replicative DNA synthesis of eukaryotic cells. We describe here a specific physical interaction between DNA polymerase epsilon and RNA polymerase II, evidenced by reciprocal immunoprecipitation experiments. The interacting RNA polymerase II was the hyperphosphorylated IIO form implicated in transcriptional elongation, as inferred from (a) its reduced electrophoretic mobility that was lost upon phosphatase treatment, (b) correlation of the interaction with phosphorylation of Ser5 of the C-terminal domain heptapeptide repeat, and (c) the ability of C-terminal domain kinase inhibitors to abolish it. Polymerase epsilon was also shown to UV crosslink specifically alpha-amanitin-sensitive transcripts, unlike DNA polymerase alpha that crosslinked only to RNA-primed nascent DNA. Immunofluorescence microscopy revealed partial colocalization of RNA polymerase IIO and DNA polymerase epsilon, and immunoelectron microscopy revealed RNA polymerase IIO and DNA polymerase epsilon in defined nuclear clusters at various cell cycle stages. The RNA polymerase IIO-DNA polymerase epsilon complex did not relocalize to specific sites of DNA damage after focal UV damage. Their interaction was also independent of active DNA synthesis or defined cell cycle stage.

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.

[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 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.

DNA polymerase alpha, beta and gamma activities in ultraviolet irradiated CV-1 monkey cells.

To determine the possible role of DNA polymerase alpha, beta and gamma during the repair period following ultraviolet (lambda max : 254 nm) irradiation of monkey CV-1 cells, we measured the three enzymatic activities by using specific tests, either in crude extracts or after fractionation by sucrose gradient (5--20%) centrifugation at high salt concentration. When compared to the unirradiated control, we could not detect any significant variation in the levels of activity of DNA polymerases alpha, beta and gamma at any time (0, 12 to 48 h) after ultraviolet irradiation of the cells with doses ranging from 9 to 52.5 J.m-2.