APE1-dependent repair of DNA single-strand breaks containing 3'-end 8-oxoguanine

Abstract

DNA single-strand breaks containing 3'-8-oxoguanine (3'-8-oxoG) ends can arise as a consequence of ionizing radiation and as a result of DNA polymerase infidelity by misincorporation of 8-oxodGMP. In this study we examined the mechanism of repair of 3'-8-oxoG within a single-strand break using purified base excision repair enzymes and human whole cell extracts. We find that 3'-8-oxoG inhibits ligation by DNA ligase IIIalpha or DNA ligase I, inhibits extension by DNA polymerase beta and that the lesion is resistant to excision by DNA glycosylases involved in the repair of oxidative lesions in human cells. However, we find that purified human AP-endonuclease 1 (APE1) is able to remove 3'-8-oxoG lesions. By fractionation of human whole cell extracts and immunoprecipitation of fractions containing 3'-8-oxoG excision activity, we further demonstrate that APE1 is the major activity involved in the repair of 3'-8-oxoG lesions in human cells and finally we reconstituted repair of the 3'-8-oxoG-containing oligonucleotide duplex with purified human enzymes including APE1, DNA polymerase beta and DNA ligase IIIalpha.

Figure 1

The human DNA glycosylases OGG1, NEIL1 and NEIL2 are unable to excise 3′-8-oxoG lesions. (A) Schematic representation of the 3′-8-oxoG containing oligonucleotide substrate within a SSB. Underlined bold G stands for 8-oxodG. (B) The 20mer 3′-8-oxoG containing oligonucleotide substrate (20-8oxoG, 0.25 pmol) was incubated with OGG1, NEIL1 or NEIL2 (2.6, 2.6 and 2.3 pmol, respectively) for 20 min at 37°C prior to the addition of formamide loading dye with (lanes 1, 3 and 5) or without (lanes 2, 4 and 6) 100 mM NaOH. Samples were heated to 95°C for 5 min prior to analysis by 20% denaturing PAGE and phosphorimaging.

Figure 2

An oligonucleotide containing 3′-8-oxoG within a SSB cannot be ligated by DNA ligase IIIα or DNA ligase I. 250 fmol of the 3′-8-oxoG containing (left panel) or a control duplex oligonucleotide substrate (right panel) were incubated with DNA ligase IIIα (A) or DNA ligase I (B) for 20 min at 37°C prior to the addition of formamide loading dye. An aliquot was analysed by 20% denaturing PAGE and phosphorimaging.

Figure 3

An oligonucleotide containing 3′-8-oxoG ends is resistant to primer extension by DNA polymerase β. 250 fmol of the 3′-8-oxoG containing (left panel) and a control duplex oligonucleotide substrate (right panel) were incubated with DNA polymerase β (0–400 fmol) for 20 min at 37°C prior to the addition of formamide loading dye. An aliquot was analysed by 20% denaturing PAGE and phosphorimaging.

Figure 4

APE1 excises 3′-8-oxoG lesions present within a SSB. 250 fmol of the duplex oligonucleotide substrate containing 3′-8-oxoG opposite to C (3′-8-oxoG/C; A, right panel) or A (3′-8-oxoG/A; B, right panel) and a control duplex containing 3′-G/C base pair (A, left panel) or 3′-G/A mismatch (B, left panel) were incubated with APE1 (0–600 fmol) for 20 min at 37°C prior to the addition of formamide loading dye. An aliquot was analysed by 20% denaturing PAGE and phosphorimaging.

Figure 5

Partial purification of 3′-8-oxoG end processing activity from human cell extracts using Phosphocellulose and gel filtration chromatography. (A) Repair of the duplex oligonucleotide substrate containing 3′-8-oxoG by HeLa WCE. (B) HeLa WCE was loaded onto a Phosphocellulose column and fractions were step eluted using 0.15 M (PC-FI) and 1 M (PC-FII) KCl. 250 fmol of the 3′-8-oxoG nick containing duplex oligonucleotide substrate was incubated with WCE or Phosphocellulose fractions (2 and 5 μg) in the absence of dNTPs for 20 min at 37°C. Formamide loading dye was added and an aliquot analysed by 20% denaturing PAGE and phosphorimaging. (C) Proteins from PC-FII were further separated by gel filtration on a Superdex-75 column and the fractions obtained were analysed for 3′-8-oxoG activity using 250 fmol of the 3′-8-oxoG duplex oligonucleotide substrate by 20% denaturing PAGE and phosphorimaging. (D) Aliquots of the fractions were also analysed by SDS–PAGE and western blotting using APE1 antibodies.

Figure 6

Immunoprecipitation of APE1 from gel filtration fractions containing 3′-8-oxoG end processing activity. (A) Fractions from gel filtration chromatography containing 3′-8-oxoG excision activity (fractions 20 and 21) were mock-immunodepleted and immunodepleted of APE1 using APE1 specific antibodies. Samples were analysed by SDS–PAGE and western blotting using antibodies against APE1. (B) The original fractions, mock immunodepleted and immunodepleted fractions 20 and 21 were tested for 3′-8-oxoG activity using 250 fmol of the 3′-8-oxoG containing duplex oligonucleotide. Samples were incubated for 10 min at 37°C prior to the addition of formamide loading dye and analysis by 20% denaturing PAGE and phosphorimaging.

Figure 7

Reconstitution of the repair pathway for DNA SSBs containing 3′-8-oxoG. 250 fmol of the 3′-8-oxoG containing duplex oligonucleotide substrate was incubated with 2 pmol of APE1, 100 fmol of Pol β and 100 fmol of DNA ligase IIIα at the indicated combinations for 20 min at 37°C prior to the addition of formamide loading dye. An aliquot was analysed by 20% denaturing PAGE and phosphorimaging.