Andrew Collins

Initial rates of DNA incision in UV-irradiated human cells differences between normal, xeroderma pigmentosum and tumour cells

Following UV-irradiation and in the presence of inhibitors of DNA synthesis (hydroxyurea and 1-beta-D-arabinofuranosylcytosine) human cells accumulate strand breaks in their DNA--as a result of enzymic incision without subsequent rejoining. We have developed a sensitive procedure which makes stringent use of these inhibitors so as to maximize the frequency of breaks detected after low levels of UV (0.25-10 Jm-2) and to permit analysis of the kinetics of break accumulation over short intervals after irradiation (up to 90 min). Since the rate of accumulation of breaks declines quickly with time of incubation (not simply as a consequence of substrate depletion), we have calculated initial rate constants by extrapolating to zero time for a range of UV doses (i.e. different substrate concentrations). Using these constants as indices of enzymic incision, we have compared a wide range of human cell types, and have (in some cases) been able to estimate the enzymatic parameters KM and Vmax for the incision step. Assessed in this way the human cells tested fall into a number of distinct categories. Fibroblasts from normal embryos and from xeroderma pigmentosum (XP) variant and Blooms syndrome show high and uniform levels of incision readily distinguishable from XP(A), in turn distinct from XP(D). Tumour-derived cells and SV40-transformed fibroblasts also fall into a group with similar incision capacity, significantly lower than that of normal diploid cells. We discuss possible reasons for this distinction, and evaluate the use of inhibitors in repair studies.

DNA damage in ultraviolet-irradiated HeLa and CHO-Kl cells examined by alkaline lysis and hydroxyapatite chromatography

DNA damage and repair activity have been examined in cultured mammalian cells by the technique of alkaline lysis followed by analysis of the single-strand content of DNA by hydroxyapatite chromatography; the rate of unwinding of DNA in alkali is related to the number of single-strand breaks present. When HeLa cells are ultraviolet-irradiated and incubated, on subsequent alkaline lysis the DNA unwinds at a faster rate than the DNA of unirradiated cells. The rate is increased if inhibitors of DNA synthesis (hydroxyurea, deoxyadenosine or 1-beta-D-arabinofuranosyl cytosine) are present during the incubation. Similar effects are seen in CHO-Kl cells synchronised in G1 phase, though mitotic CHO-Kl cells show little effect of post-irradiation incubation or of the presence of inhibitors. It appears that agents known to block replicative DNA synthesis can also inhibit repair DNA synthesis following ultraviolet irradiation, leading to an accumulation of single-strand breaks in DNA produced by repair nuclease activity. This phenomenon is probably responsible for the retarded sedimentation of such DNA on alkaline sucrose gradients.

The Inhibition of DNA Repair

Publisher Summary This chapter focuses on the inhibition of DNA repair. It discusses the effects of the inhibition of repair on chromosome aberrations. A classic method for dissecting biochemical systems involves the inhibition of specific steps in a sequence of reactions. Such inhibition can cause accumulation of the substrate for the blocked reaction, allowing identification of intermediates in the sequence. Knowledge of the site and mode of action of an inhibitor can give information about the molecular mechanism of the reaction. Manipulation of a metabolic pathway, with added inhibitors, permits an analysis of the regulatory processes that coordinate enzymatic activity in the pathway. For the measurement of breaks in DNA, a commonly used technique is alkaline sucrose gradient sedimentation. The rate at which DNA sedimentation occurs after alkaline denaturation depends on the single-strand length. The DNA breaks that accumulate in UV-irradiated cells, incubated with DNA synthesis inhibitors, are produced by cellular endonucleases near the sites of cyclobutane pyrimidine dimers. Novobiocin inhibits replicative DNA synthesis in mammalian cells, specifically blocking initiation of synthesis of nascent DNA.

DNA Repair Under Stress

SUMMARY When the excision repair process of eukaryote cells is arrested by inhibitors of repair synthesis including hydroxyurea (HU), 1-β-d-arabinofuranosylcytosine (araC) or aphidicolin, major cellular changes follow the accumulation of repair-associated DNA breaks. These changes, each of which reflects more or less severe cellular stress, include cycle delay, chromosome behaviour, fall in NAD level, the development of double-stranded DNA breaks, rapid chromosome fragmentation and cell killing. Disruption of the repair process by agents such as araC after therapeutic DNA damage may, therefore, have some potential value in cancer treatment. The extreme cellular problems associated with the artificial arrest of repair may have their subtler counterparts elsewhere, and we discuss several systems where delays in the completion of excision repair in the absence of repair synthesis inhibitors have marked repercussions on cell viability. We also show that the average completion time of an excision repair patch varies according to the state of cell culture, and that completion time is extended after treatment with insulin or following trypsin detachment. Under certain growth conditions ultraviolet irradiation followed by mitogenic stimulation results in double-stranded DNA breakage and additional cell killing, and we discuss these data in the light of protocols that have been used successfully to transform human or rodent cells in vitro. Finally, we consider whether the rejoining of DNA breaks accumulated by repair synthesis inhibitors is a valid model system for studying ligation, and show that this protocol provides an extremely sensitive assay for most incision events and, thereby, a means for discriminating between normal human cells on the one hand, and Cockayne’s Syndrome cells and their heterozygotes on the other.

DNA repair in ultraviolet-irradiated HeLa cells is disrupted by aphidicolin: The inhibition of repair need not imply the absence of repair synthesis

Aphidicolin, a potent and specific inhibitor of eukaryotic DNA polymerase alpha, has been reported to inhibit repair DNA synthesis in ultraviolet-irradiated, normal human fibroblasts but not in HeLa cells. By the use of assays for repair other than the measurement of repair synthesis, it is shown here that repair in HeLa cells is in fact susceptible to aphidicolin. Severe inhibition of DNA repair, with failure of individual repair events to be completed, and a smaller number of lesions removed, can occur even though repair synthesis continues.

Cell cycle-dependent regulation of excision repair of UV damage

The incision step of excision repair of UV-damaged DNA, represented by the accumulation of DNA strand breaks in the presence of hydroxyurea and 1-beta-D-arabinofuranosylcytosine, was measured in cultured cells of Microtus agrestis during quiescence and at different times after release from quiescence. The UV dose-dependent kinetics of enzymic incision change rapidly through the first cell cycle after release, with a maximum potential for enzymic incision and a minimum affinity of the incision system for damage sites occurring in late G1. The potential for incision falls to a lower level as cells approach a randomly proliferating state, whilst the affinity rises. Incision activity declines rapidly after UV irradiation, leaving many damage sites unrepaired; the enhanced survival seen in cells held in quiescence after irradiation is thus partly due to some other repair mode.

Estimates of the rate of ligation during excision repair of ultraviolet-damaged DNA in mammalian cells

When ultraviolet-irradiated mammalian cells are incubated with inhibitors of repair DNA synthesis, incomplete repair sites--seen as DNA breaks--accumulate. If the inhibition is reversed, the breaks are joined. Thus the ligation step of excision repair can be investigated. With aphidicolin as inhibitor, ligation occurs at up to 15-times the rate of incision. 3-Aminobenzamide (which inhibits poly(ADPribose) synthesis) does not delay the rejoining of DNA breaks.

Replica plating of cultured human cells on polyester mesh

Replica plating, i.e., the production of multiple copies of a pattern of colonies derived from single cells, can now be applied to human cell lines using polyester mesh as a substrate. In searching for somatic cell mutants, often the only means of identifying a mutant clone is an assay that leaves the selected cells nonviable. With replica plating it is possible to retrieve that clone from the corresponding position on the replica. In addition, DNA from the colonies on the polyester replica can be transferred to nitrocellulose, facilitating the identification of clones using radioactive tracers or DNA probes.

Abnormal mutation frequencies in human repair-defective hybrid cell lines.

Two intraspecific human cell hybrids, HD2 and HD1A, produced from fusion between HeLa cells and xeroderma pigmentosum fibroblasts, express XPD-like rates of excision repair and hypersensitivity to UV-radiation. In the present paper we describe unusual patterns of UV-induced mutation in both cell lines. Though HD2 very closely resembles XPD both phenotypically and genetically, in UV-dose response it is hypomutable at the loci for ouabain and diphtheria toxin resistance. At equitoxic dose, however, it shows normal mutability, HD1A, by contrast, is hypermutable as a function either of UV dose or in terms of equitoxicity for these genes. HD1As mutator phenotype is a dominant characteristic and is not associated with grossly abnormal DNA precursor pool imbalance. The possibility remains that DNA polymerase infidelity underlies its hypermutability.

UV IRRADIATION AND THE MAMMALIAN CELL CYCLE

ABSTRACT The efficiency of cyclobutane pyrimidine dimer production by UV varies through the cell cycle as predicted by a computer model based on changes in target geometry. But changes in UV sensitivity are not entirely due to the changes in damage; other factors influence cell survival, probably including DNA precursor pool sizes.