Repair Of Ultraviolet-induced Damage Research Articles

Analysis of the structure and spatial distribution of ultraviolet-induced DNA repair patches in human cells made in the presence of inhibitors of replicative synthesis

The repair of ultraviolet-induced damage in the presence of hydroxyurea or hydroxyurea and arabinosylcytosine was investigated in confluent human fibroblasts at the level of DNA loops attached to the nuclear matrix. Estimation of single-strand break frequencies based on the release of DNA from the DNA-nuclear matrix complex after incubation with nuclease S1 revealed the occurrence of multiple incision events per DNA loop in the presence of inhibitors. When both inhibitors were employed, over 90% of the repair-labelled DNA was not ligated within 2 h post-incubation. In the absence of ligation of repair patches, we observed a preferential release of repair-labelled DNA from the nuclear matrix by nuclease S1 compared to prelabelled DNA, regardless of the period of post-UV incubation. The results suggest that repair events are clustered to some extent in a certain area of a DNA loop. However, the position of these clusters relative to the attachment sites of DNA loops at the nuclear matrix is random. The data are discussed in terms of denaturation of a putative repair complex in the presence of hydroxyurea resulting in an excess of incisions over repaired sites.

Effects of nucleotide pool imbalances on the excision repair of ultraviolet-induced damage in the DNA of human diploid fibroblasts.

Recent studies in our laboratory and others have demonstrated that DNA polymerase inhibitors such as the ara nucleosides, aphicolin and dideoxythymidine are potent inhibitors of the DNA excision repair process in confluent human fibroblasts as evidenced by the agent-dependent accumulation of single-strand interruptions in the DNA of UV-irradiated, but not in unirradiated, cellular DNA. In rapidly cycling cells, on the other hand, these agents are weak inhibitors at best but when used in combination with the ribonucleotide reductase inhibitor, hydroxyurea, a significant enhancement of inhibitory capacity is seen. In an attempt to better understand the mechanism of repair inhibition by DNA polymerase inhibitors, and the nature of this hydroxyurea enhancement, experiments were initiated in which the effects of a series of ribonucleotide reductase inhibitors on dNTP pools and on the DNA repair process were determined in both quiescent cultures and log-phase cultures of human fibroblasts. It was determined that hydroxyurea, deoxyadenosine, pyridine-2-carboxaldehyde thiosemicarbazone (TSC), pyrozoloimidazole (IMPY), 3,5-diamino-1,2,4-triazole (guanazole), 3,4,5-trihydroxy benzohydroxamic acid (THBA) and 3,4-dihydroxy benzohydroxamic acid (DHBA) are all effective inhibitors of the DNA repair process in confluent cells but not in log-phase cells. Moreover, the effects of these inhibitors can be reversed by the addition of certain combinations of deoxynucleosides. These reversal studies and the direct analysis of dNTP pool modulation by these compounds in log phase and confluent cultures support the notion that specific pool depletions rather than general imbalance of pools gives rise to the inhibition of the DNA excision repair process.

DNA-binding proteins in xeroderma pigmentosum fibroblasts

DNA-binding proteins in human fibroblasts were examined by chromatography on DNA-cellulose columns. By successive chromatography on columns containing native, denatured, and UV-irradiated DNA-cellulose respectively the proteins binding to different types of DNA could be studied. Elution of the columns with sodium chloride followed by polyacrylamide gel electrophoresis allowed several DNA-binding proteins to be identified. All of the major DNA-binding proteins were present in strains of xeroderma pigmentosum cells respectively deficient in excision-repair and post-replication repair of ultraviolet-induced damage.

Synergistic effect of caffeine on the cytotoxicity of ultraviolet irradiation and of hydrocarbon epoxides in strains of Xeroderma pigmentosum.

XERODERMA pigmentosum (XP) is an inherited human disease clinically characterised by abnormal pigmentation patterns and the development of multiple skin carcinoma on areas exposed to the Sun1. Cells from most XP patients are defective in excision repair of ultraviolet-induced lesions in DNA1–3, but there is a class of patients—XP variants—who have all the clinical manifestations of the disease but who, until recently, appeared to have normal DNA repair. Cleaver4 and others5,6 have shown that variants have normal rates of excision repair of damage caused by ultraviolet light as measured by ultraviolet-stimulated uptake of labelled thymidine or bromodeoxyuridine. Cleaver also reported that variants XP4BE (his patient 13) and XP13BE (his patient 14) have normal sensitivity to ultraviolet light as measured by survival of colony-forming ability4. His survival data, however, consisted of only two points for each strain. As Fig. 1 shows, we find that the percentage survival of the colony-forming ability of variants XP4BE and XP13BE as well as variant XP7TA and XP30RO is more sensitive to ultraviolet irradiation than that of normal cells. We also showed that the survival of the cloning ability of XP variants is more sensitive than normal to active derivatives of four aromatic amide carcinogens7 and to the K-region epoxides of three polycyclic hydrocarbons8. These two classes of carcinogens bind covalently to DNA9,10 and the bound adducts seem to be recognised by the same repair enzymes which function in the repair of ultraviolet-induced damage in these human cells7–11.

Accurate repair of ultraviolet-induced damage in Micrococcus radiodurans

Accurate repair of ultraviolet-induced damage in Micrococcus radiodurans

Involvement of DNA polymerase 3 in excision repair after ultraviolet irradiation.

THE dark repair of ultraviolet-induced damage in Escherichia coli K-12 involves at least two processes1–3:(1)post-replicational repair of gaps produced in daughter strands of the DNA by replication past ultraviolet-induced lesions and (2) excision repair of these lesions. This report is concerned with only the latter repair process.