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. 2000 Apr 11;97(8):3832-7.
doi: 10.1073/pnas.070471597.

VSports最新版本 - Removal of oxygen free-radical-induced 5',8-purine cyclodeoxynucleosides from DNA by the nucleotide excision-repair pathway in human cells

I Kuraoka  1 , C Bender, A Romieu, J Cadet, R D Wood, T Lindahl
Affiliations

Removal of oxygen free-radical-induced 5',8-purine cyclodeoxynucleosides from DNA by the nucleotide excision-repair pathway in human cells

I Kuraoka et al. Proc Natl Acad Sci U S A. .

V体育安卓版 - Abstract

Exposure of cellular DNA to reactive oxygen species generates several classes of base lesions, many of which are removed by the base excision-repair pathway. However, the lesions include purine cyclodeoxynucleoside formation by intramolecular crosslinking between the C-8 position of adenine or guanine and the 5' position of 2-deoxyribose. This distorting form of DNA damage, in which the purine is attached by two covalent bonds to the sugar-phosphate backbone, occurs as distinct diastereoisomers. It was observed here that both diastereoisomers block primer extension by mammalian and microbial replicative DNA polymerases, using DNA with a site-specific purine cyclodeoxynucleoside residue as template, and consequently appear to be cytotoxic lesions. Plasmid DNA containing either the 5'R or 5'S form of 5',8-cyclo-2-deoxyadenosine was a substrate for the human nucleotide excision-repair enzyme complex. The R diastereoisomer was more efficiently repaired than the S isomer. No correction of the lesion by direct damage reversal or base excision repair was detected. Dual incision around the lesion depended on the core nucleotide excision-repair protein XPA. In contrast to several other types of oxidative DNA damage, purine cyclodeoxynucleosides are chemically stable and would be expected to accumulate at a slow rate over many years in the DNA of nonregenerating cells from xeroderma pigmentosum patients. High levels of this form of DNA damage might explain the progressive neurodegeneration seen in XPA individuals VSports手机版. .

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Figures

Figure 1
Figure 1
The 5′R and 5′S diastereoisomers of 5′,8-cyclo-2′-deoxyadenosine in DNA damaged by hydroxyl radicals. In these unusual lesions, there are two covalent linkages of the purine base to the sugar-phosphate backbone.
Figure 2
Figure 2
Inhibition of primer extension by T7 DNA polymerase and mammalian DNA Pol δ by cyPu. Autoradiograph after denaturing 14% PAGE showing the inhibition of primer extension by 5′R- and 5′S-cyclodeoxyadenosine. A 32P end-labeled primer was annealed to cyPu-containing plasmid DNA predigested with PvuI and XhoI, then extension reactions were performed with either T7 DNA polymerase or purified recombinant human DNA Pol δ with PCNA. Lanes 1–3, T7 DNA polymerase; Lane 1, control plasmid without cyclo-dA; lane 2, 5′R-cyclo-dA; lane 3, 5′S-cyclo-dA. Lanes 4–6, Pol δ/PCNA. Lane 4, control without cyclo-dA; lane 5, 5′R-cyclo-dA; lane 6, 5′S-cyclo-dA. M, size markers (3′-32P-labeled MspI digest of pBR322).
Figure 3
Figure 3
Search for a DNA glycosylase or lesion reversal enzyme acting on cyclopurine deoxynucleosides. (a) DNA glycosylase assay. Lanes 1 and 2, duplex oligonucleotide containing a uracil instead of cyPu at the same position; lanes 3 and 4, control oligonucleotide without any lesion; lanes 5 and 6, 5′R-cyclodeoxyadenosine-containing oligonucleotide; lanes 7 and 8, 5′S-cyclodeoxyadenosine-containing oligonucleotide. These oligonucleotides were incubated with (lanes 2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) HeLa whole cell extract (WCE) and then treated with 1 M piperidine at 90°C. The arrows mark the positions of intact oligonucleotide (I), and product (P) after DNA glycosylase action followed by piperidine treatment. (b) Lesion reversal assay. Lanes 1 and 4, control oligonucleotide without any lesion; lanes 2 and 5, 5′R-cyclodeoxyadenosine-containing oligonucleotide; lanes 3 and 6, 5′S-cyclodeoxyadenosine-containing oligonucleotide. The duplex oligonucleotides were incubated with (lanes 4–6) or without (lanes 1–3) HeLa cell extract and then digested with Sau3AI. The arrows mark the positions of intact oligonucleotide (I), and product of Sau3AI cleavage (S). Only partial restriction enzyme cleavage of the short control oligonucleotide was observed, even at high Sau3AI concentrations. (a and b) The relevant, lesion-containing strand had been 5′-32P-labeled, and 14% polyacrylamide gels are shown.
Figure 4
Figure 4
DNA repair synthesis in response to a cyPu lesion. (a) The plasmid M13mp18 cyclo-dA is shown diagrammatically. Eight BstNI restriction sites and the three AlwI restriction sites are indicated. One AlwI site (underlined) overlaps the region containing the cyPu residue. Unique PvuI, HindIII, and XhoI restriction enzyme sites are also indicated. (b) Autoradiograph after denaturing 14% PAGE, demonstrating DNA repair synthesis in the region containing the cyPu lesion. Plasmid DNA was incubated with HeLa cell extracts and radioactively labeled deoxynucleoside triphosphates (see Materials and Methods) and subsequently digested with BstNI before electrophoresis. Lanes 1–4, Pt-GTG substrate as positive control; lanes 5–8, control DNA without any lesion; lanes 9–12, 5′R-cyclo-dA substrate; lanes 13–16, 5′S-cyclo-dA substrate. M, size markers as in Fig. 2.
Figure 5
Figure 5
Initiation of nucleotide excision-repair by dual incision at cyPu lesions in DNA by human cell extracts. (a) HeLa whole cell extract (WCE) was incubated with plasmid DNA containing a 5′,8-cyclo-2′-deoxyadenosine residue in either the R or S stereoisomeric form, as indicated (lanes 1 and 4). Excision products are indicated by the bracket. Addition of 2 μl neutralizing XPA antiserum to the 50 μl reaction mixture was used in lanes 2, 3, 5, and 6. In addition, lanes 3 and 6 show reaction mixtures supplemented with 450 ng purified XPA protein to reverse the inhibitory effect of the antiserum. The M1 and M2 markers are oligonucleotides of 22 residues containing a single cyPu residue in the 5′R or 5′S form, respectively. An autoradiograph of a 16% polyacrylamide gel is shown after denaturing gel electrophoresis. (b) Quantification of the excision products in the 20–28 nt range by phosphorimager analysis.
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