An altered apurinic DNA endonuclease activity in group A and group D xeroderma pigmentosum fibroblasts.

Abstract

Endonuclease activity upon depurinated DNA was measured in extracts of cultured fibrobrasis from xeroderma pigmentosum patients. Cell lines from complementation groups A, B, C, E, and the XP-variant -had slightly reduced levels of activity, but cell lines from complementation group D had one-sixth of the normal activity. An Cell lines developed from skin biopsies of these individuals have been reported to be defective in excision repair of DNA damage introduced by ultraviolet light or by various chemical agents (2,3). This defect is inferred from a reduction in the overall rate of DNA repair synthesis, and it remains unclear at which step the repair pathway is affected. A few XP-cell lines have been tested for defects in early steps of excision repair: two were shown to be unable to excise pyrimidine dimers from DNA (4), and another one was reported to be defective in the first step of repair, the endonucleolytic cleavage presumably near UV lesions of the DNA (5). Somatic cell hybridization studies have recently shown that mutations which lead to the XP phenotype fall into five or six complementation groups (6, 7). This number exceeds the number of steps postulated for excision repair, suggesting that the repair pathway is regulated in a very complex manner or that there exist multiple, or branched pathways of repair.Several enzyme activities believed to be involved in excision repair have been measured in extracts of XP-cell lines. Pedrini et al. (8) found no significant differences in the levels of polynucleotide ligase activity, DNA polymerase, single-strand exonuclease and double-strand endonuclease. Bacchetti et al. (9) compared endonucleolytic activity specific for UV-irradiated DNA in normal and two XP-cell lines and detected no difference. Under their assay conditions the major UV-specific endonuclease activity in fibroblast extracts did not cleave the DNA near pyrimidine dimers, but at other undetermined lesions. In an attempt to Abbreviation: XP, xeroderma pigmentosum. identify excision repair endonucleases we have also found that extracts from a normal cell line and from the XP-cell lines CRL 1157 and CRL 1160 (see Materials and Methods) contained equal levels of an endonuclease activity which preferentially cleaves UV-irradiated DNA at sites which are about 100 times less frequent than the number of pyrimidine dimers in the DNA. However, these studies led us also to compare endonuclease activity with depurinated DNA as a substrate in extracts from normal and XP fibroblasts. Such an activity has been previously reported in many microorganisms, plants and mammals (10-13). MATERIALS AND METHODSGrowth of Cells and Preparation of Extracts. Fibroblast cell lines from skin biopsies were obtained from the American Type Culture collection (ATCC). Strain numbers, complementation groups and relevant properties are summarized irk Table 1. Cells were grown at 370 in 32 ounce prescription bottles with 50 ml of Dulbecco's modified Eagle's medium (Gibco), supplemented with 15 mM N-2-hydroxyethylpiperazine...

Differences in removal of acetylaminofluorene and pyrimidine dimers from the DNA of cultured mammalian cells.

“…32), or (ii) the two lesions are removed by different processes, and the lesion in xeroderma pigmentosum affects the expression of a number of related repair processes. Consistent with the latter hypothesis are the data of Kuhnlein et al (33) and Sutherland et al (34). The behavior of mouse cells is similarly of interest.…”

Human uracil DNA N-glycosidase: studies in normal and repair defective cultured fibroblasts

“…The activities of the cells of XP complementation groups A and D appeared to be near normal. (These were the groups shown to have altered apurinic/apyrimidinic endonuclease in a previous study (7).) The AT cell line also had normal activity, but two XP variant cell lines had consistently lower levels of activity.…”

“…We have previously analyzed apurinic/apyrimidinic endonuclease activity in extracts from normal cells and from XP cells (7). This enzyme is thought to be involved in the 123 excision of apurinic or apyrimidinic sites from the DNA, some of which are probably generated by the removal of uracil from DNA by uracil DNA N-glycosidase (22,23).…”

“…On the assumption that a defect in the glycosidase might result in a propensity toward transition mutations, and in the case of mammals toward carcinogenesis, we have initiated a study of this enzyme in cells cultured from individuals with so-called repair deficiency diseases. By a similar approach we had shown that certain lines from persons with the disease xeroderma pigmentosum (XP) have apurinic/apyrimidinic endonuclease activity with an abnormal substrate affinity (7), and that this defect is due to the absence of one of two isozymic species of the enzyme (Kuhnlein, U., Lee, B., Penhoet, E.E., and Linn, S., unpublished)…”

Role of active site tyrosines in dynamic aspects of DNA binding by AP endonuclease☆

“…Mammalian AP endo, initially isolated from human tissue (19,26,34) and cell lines (4,21,22), was confirmed as the E. coli equivalent because of its enzymatic activity and because the gene complemented E. coli deficient in exonuclease III for resistance to alkylating agents (9,40,42). AP endo binds abasic (AP) site-containing DNA in the absence of divalent cation with remarkable affinity as shown using an electrophoretic mobility shift assay (EMSA) (11,28,29,43,47) and single turnover (ST) kinetics (43).…”