Supplementary Figure 1 Abortive ligation (adenylation) of the gapped 5'-dRP-containing BER

Transcription

1 Supplementary Figure 1 Abortive ligation (adenylation) of the gapped 5'-dRP-containing BER intermediate by human BER ligases. The ligation efficiency of DNA ligase I (a) and XRCC1/DNA ligase III complex (b). The migration positions of the DNA substrate (17 drp, Substrate 1 in Supplementary Table 1), ligation product and the adenylated DNA intermediate (17 drp +AMP) are indicated.

2 Supplementary Figure 2 The assessment of drp lyase activity of wild type and pol β mutants. (a) Uncropped image for the adenylated sugar phosphate removal by pol β from the gapped and nicked 5'-dRP and 5'-AMP-dRP-containing substrates. The final cropped image is presented in

3 Fig. 1. (b,c) The drp lyase assay for the adenylated sugar phosphate removal by the 3KΔ mutant of pol β from the gapped (b) and nicked (c) 5'-AMP-dRP-containing substrates. In both panels b and c, lane 1 is a minus enzyme control, and lane 2 is a reference reaction containing wild type pol β. The positions of the gapped (17 drp +AMP, Substrate 2 in Supplementary Table 1) and nicked (18 drp +AMP, Substrate 4 in Supplementary Table 1) DNA substrates and the pol β lyase products (17-mer in panel b, 18-mer in panel c) are indicated. The 3KΔ mutant was devoid of activity (lanes 3-6). (d,e) The drp lyase assay for the adenylated sugar phosphate removal by the K68A mutant of pol β from the gapped (d) and nicked (e) 5'-AMP-dRP-containing substrates. In both panels d and e, lane 1 is a minus enzyme control and lane 2 is a reference reaction containing wild type pol β. The positions of the gapped (17 drp +AMP, Substrate 2 in Supplementary Table 1) and nicked (18 drp +AMP, Substrate 4 in Supplementary Table 1) DNA substrates and the pol β lyase products (17-mer in panel d, 18-mer in panel e) after 5'-AMP-dRP removal by wild type pol β (lane 2), and K68A mutant (lanes 6-9) are indicated.

4 Supplementary Figure 3 The actions of APTX and pol β on different DNA substrates. The drp lyase and DNA deadenylation assays for the processing of the gapped and nicked 5'-AMP-dRP (a), the nicked 5'-AMP (b), and the gapped and nicked 5'-AMP-THF (c) containing DNA substrates. (a) Lane 1 and lane 4 are minus enzyme controls; lane 2 and lane 5 are the products of APTX 5'- AMP removal; lane 3 and lane 6 are the products of pol β lyase 5'-AMP-dRP removal. The positions of the gapped (17 drp +AMP, Substrate 2 in Supplementary Table 1) and nicked (18 drp +AMP, Substrate 4 in Supplementary Table 1) DNA substrates and the pol β lyase (17-mer or 18-mer) and APTX removal (17 drp or 18 drp ) products are indicated. (b) Lane 7 is a minus enzyme control, and lane 8 is the product of APTX 5'-AMP removal. The positions of the nicked DNA substrate (18+AMP, Substrate 5 in Supplementary Table 1), and APTX removal product (18-mer) are indicated. Pol β showed no activity with this substrate (lane 9). (c) Lane 10 and lane 13 are minus enzyme controls, and lane 11 and lane 14 are the products of APTX 5'-AMP removal. The positions for the gapped (17 THF +AMP, Substrate 6 in Supplementary Table 1) and nicked (18 THF +AMP, Substrate 7 in Supplementary Table 1) DNA substrates and APTX products (17 THF or 18 THF ) are indicated. Pol β showed no activity with these substrates (lanes 12 and 15).

5 Supplementary Figure 4 Transactions of pol β, APTX and FEN1 at the BER intermediates. (a,b,c) FEN1 excision assay for the nicked 5'-AMP-dRP (a), gapped 5'-AMP-dRP (b), the nicked 5'-AMP (c) containing DNA substrates (Supplementary Table 1). Lanes 1, 9 and 17 are minus enzyme controls, lanes 2 and 10 are reference reactions containing pol β. Lanes 3-8 in panel a, lanes in panel b, and lanes in panel c are FEN1 excision cleavage products. The positions of the DNA substrate for 18 drp +AMP (Substrate 4 in panel a), 17 drp +AMP (Substrate 2 in panel b), and 18+AMP (Substrate 5 in panel c) are indicated. The positions of the products for pol β lyase (18-mer in panel a, 17-mer in panel b), and FEN1 excision cleavage (17-, 16- or 15-mer in panel a; 16- or 15-mer in panel b; 17- or 16-mer in panel c) are indicated. Pol β showed no activity with Substrate 5 (lane 18). (d) The catalytic rates of pol β, FEN1, and APTX in the processing of 5'-AMP-dRP substrate. The rates of pol β ( ) FEN1 (u ), and APTX (n ) are as k obs of 0.05 s -1, 0.03 s -1, and 0.2 s -1, respectively. (e) A model proposing the fate of the adenylated BER

6 intermediate as mediated by pol β, FEN1, and APTX activities. The normal pol β lyase reaction illustrated at the top is biphasic and fast (>800 s -1 and 2 s -1 ).

7 Supplementary Figure 5 The fate of 5'-adenylated BER intermediate in the case of APTX and/or FEN1 deficiency. (a) The drp lyase, DNA adenylation and FEN1 excision assays with purified enzymes pol β, FEN1, and APTX. Lane 1 is a minus enzyme control, lane 2, 3, and 4 are reference reactions containing FEN1, pol β, and APTX, respectively. The migration position of the DNA substrate (17 drp +AMP, Substrate 2 in Supplementary Table 1) is indicated. (b-g) The drp lyase, DNA adenylation and FEN1 excision assays in the cell extracts prepared from the yeast strains (Supplementary Table 3). DNA substrate was not pretreated with UDG. The reaction products observed in the cell extracts from the wild-type (b, lanes 5-7), hnt3δ (c, lanes 8-10), rad27δ (d, lanes 11-13), hnt3δrad27δ (e, lanes 14-16), rad27δ::polb (f, lanes 17-19), hnt3δrad27δ::polb (g, lanes 20-22) yeast strains are indicated. In panels a-g, the positions of the products for FEN1 excision cleavage (16- or 15-mer), pol β lyase (17-mer), and APTX removal of AMP (17 drp ) are indicated. Three lanes in each set (in panels b-g) correspond to the time points 5, 15, or 30 min.

8 Supplementary Table 1 DNA substrates used in this study. FAM indicates the presence of a fluorescence tag. The abbreviations drp and THF represent a 5'-deoxyribose phosphate (5'-dRP) and synthetic abasic site analog (tetrahydrofuran), respectively. The presentations for DNA substrates 1-4 are designations after UDG treatment of the 36-mer double-stranded DNA substrates.

9 Supplementary Table 2 Data collection and refinement statistics.

10 Supplementary Table 3 Saccharomyces cerevisiae strains used in this study.

11 Supplementary Table 4 The sequences of oligonucleotides and primers used in this study. a F denotes THF, U represents a uracil base, and p stands for a phosphate group. The sequences of DNA oligonucleotides are shown in 5' to 3' orientation. b The downstream oligonucleotides include the 3'-FAM label. The downstream, upstream, and template oligonucleotides were used to construct the DNA substrates represented in Supplementary Table 1. For example, D Ugap (17-mer plus a uracil base) was used to construct either the gapped 5'-dRP-containing substrate (Substrate 1, Supplementary Table 1) or the gapped 5'-AMP-dRP-containing substrate (Substrate 2, Supplementary Table 1) after its annealing with the template b in the presence of the upstream b oligonucleotide. c Forward and reverse primers were used to construct the yeast strains used in this study. d Template, primer, and downstream oligonucleotides were used to construct the DNA substrate for crystallization of pol β-dna complex.