Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

Role of polymerase β in complementing aprataxin deficiency during abasic-site base excision repair

Abstract

DNA polymerase β (pol β) lyase removal of 5′-deoxyribose phosphate (5′-dRP) from base excision repair (BER) intermediates is critical in mammalian BER involving the abasic site. We found that pol β also removes 5′-adenylated dRP from BER intermediates after abortive ligation. The crystal structure of a human pol β–DNA complex showed the 5′-AMP-dRP group positioned in the lyase active site. Pol β expression rescued methyl methanesulfonate sensitivity in aprataxin (hnt3)- and FEN1 (rad27)-deficient yeast.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Removal of adenylated sugar phosphate by pol β.
Figure 2: Position of the 5′-AMP-dRP group in the pol β lyase active site.
Figure 3: Complementation of Hnt3 (APTX) deficiency by pol β in the absence of Rad27 (FEN1).

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Kumar, A., Abbotts, J., Karawya, E.M. & Wilson, S.H. Biochemistry 29, 7156–7159 (1990).

    Article  CAS  Google Scholar 

  2. Piersen, C.E., Prasad, R., Wilson, S.H. & Lloyd, R.S. J. Biol. Chem. 271, 17811–17815 (1996).

    Article  CAS  Google Scholar 

  3. Prasad, R. et al. J. Biol. Chem. 273, 11121–11126 (1998).

    Article  CAS  Google Scholar 

  4. Prasad, R. et al. DNA Repair (Amst.) 4, 1347–1357 (2005).

    Article  CAS  Google Scholar 

  5. Klungland, A. & Lindahl, T. EMBO J. 16, 3341–3348 (1997).

    Article  CAS  Google Scholar 

  6. Daley, J.M., Wilson, T.E. & Ramotara, D. DNA Repair (Amst.) 9, 690–699 (2010).

    Article  CAS  Google Scholar 

  7. Prasad, R., Beard, W.A., Strauss, P.R. & Wilson, S.H. J. Biol. Chem. 273, 15263–15270 (1998).

    Article  CAS  Google Scholar 

  8. Rass, U., Ahel, I. & West, S.C. J. Biol. Chem. 282, 9469–9474 (2007).

    Article  CAS  Google Scholar 

  9. Reynolds, J.J. et al. Mol. Cell. Biol. 29, 1354–1362 (2009).

    Article  CAS  Google Scholar 

  10. Gueven, N. et al. Hum. Mol. Genet. 13, 1081–1093 (2004).

    Article  CAS  Google Scholar 

  11. Mosesso, P. et al. Cell. Mol. Life Sci. 62, 485–491 (2005).

    Article  CAS  Google Scholar 

  12. Gueven, N. et al. Neuroscience 145, 1418–1425 (2007).

    Article  CAS  Google Scholar 

  13. Prasad, R., Dianov, G.L., Bohr, V.A. & Wilson, S.H. J. Biol. Chem. 275, 4460–4466 (2000).

    Article  CAS  Google Scholar 

  14. Chen, X. et al. Methods Enzymol. 409, 39–52 (2006).

    Article  CAS  Google Scholar 

  15. Ellington, A. & Pollard, J.D. Curr. Protoc. Mol. Biol. 42, 2.12 (2001).

    Google Scholar 

  16. Prasad, R., Shock, D.D., Beard, W.A. & Wilson, S.H. J. Biol. Chem. 285, 40479–40488 (2010).

    Article  CAS  Google Scholar 

  17. Heacock, M., Poltoratsky, V., Prasad, R. & Wilson, S.H. PLoS ONE 7, e47945 (2012).

    Article  CAS  Google Scholar 

  18. Wang, Z., Wu, X. & Friedberg, E.C. Methods Companion Methods Enzymol. 7, 177–186 (1995).

    Article  Google Scholar 

  19. Otwinowski, Z. & Minor, W. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  20. Pettersen, E.F. et al. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J.K. Horton for critical reading of the manuscript and M. Heacock and D. Shock for helpful discussions. We thank A. Tomkinson (University of New Mexico Cancer Center) for the XRCC1–DNA ligase III complex. This work was supported by the Intramural Research Program of the US National Institutes of Health, National Institute of Environmental Health Sciences (grants Z01 ES050158 and ES050159 to S.H.W.).

Author information

Authors and Affiliations

Authors

Contributions

M.Ç. performed the biochemistry and the kinetic and yeast in vivo and in vitro experiments. V.K.B. performed the structure determination and data collection. A.S. constructed the yeast strains. M.Ç., R.P. and S.H.W. wrote the paper.

Corresponding author

Correspondence to Samuel H Wilson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

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 (17dRP, Substrate 1 in Supplementary Table 1), ligation product and the adenylated DNA intermediate (17dRP+AMP) are indicated.

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 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 (17dRP+AMP, Substrate 2 in Supplementary Table 1) and nicked (18dRP+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 (17dRP+AMP, Substrate 2 in Supplementary Table 1) and nicked (18dRP+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.

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 (17dRP+AMP, Substrate 2 in Supplementary Table 1) and nicked (18dRP+AMP, Substrate 4 in Supplementary Table 1) DNA substrates and the pol β lyase (17-mer or 18-mer) and APTX removal (17dRP or 18dRP) 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 (17THF+AMP, Substrate 6 in Supplementary Table 1) and nicked (18THF+AMP, Substrate 7 in Supplementary Table 1) DNA substrates and APTX products (17THF or 18THF) are indicated. Pol β showed no activity with these substrates (lanes 12 and 15).

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 11-16 in panel b, and lanes 19-24 in panel c are FEN1 excision cleavage products. The positions of the DNA substrate for 18dRP+AMP (Substrate 4 in panel a), 17dRP+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 (♦), and APTX (■) are as kobs 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 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).

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 (17dRP+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 (17dRP) are indicated. Three lanes in each set (in panels b-g) correspond to the time points 5, 15, or 30 min.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Tables 1–4 (PDF 4806 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Çağlayan, M., Batra, V., Sassa, A. et al. Role of polymerase β in complementing aprataxin deficiency during abasic-site base excision repair. Nat Struct Mol Biol 21, 497–499 (2014). https://doi.org/10.1038/nsmb.2818

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2818

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing