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Comparative Study
. 2000 Jun 15;19(12):3100-9.
doi: 10.1093/emboj/19.12.3100.

Mechanisms of accurate translesion synthesis by human DNA polymerase eta

C Masutani  1 , R Kusumoto, S Iwai, F Hanaoka
Affiliations
Comparative Study

Mechanisms of accurate translesion synthesis by human DNA polymerase eta

C Masutani (VSports最新版本) et al. EMBO J. .

V体育官网 - Abstract

The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta (pol eta), which is involved in the replication of damaged DNA. Pol eta catalyzes efficient and accurate translesion synthesis past cis-syn cyclobutane di-thymine lesions. Here we show that human pol eta can catalyze translesion synthesis past an abasic (AP) site analog, N-2-acetylaminofluorene (AAF)-modified guanine, and a cisplatin-induced intrastrand cross-link between two guanines. Pol eta preferentially incorporated dAMP and dGMP opposite AP, and dCMP opposite AAF-G and cisplatin-GG, but other nucleotides were also incorporated opposite these lesions. However, after incorporating an incorrect nucleotide opposite a lesion, pol eta could not continue chain elongation. In contrast, after incorporating the correct nucleotide opposite a lesion, pol eta could continue chain elongation, whereas pol alpha could not VSports手机版. Thus, the fidelity of translesion synthesis by human pol eta relies not only on the ability of this enzyme to incorporate the correct nucleotide opposite a lesion, but also on its ability to elongate only DNA chains that have a correctly incorporated nucleotide opposite a lesion. .

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"VSports在线直播" Figures

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Fig. 1. Processivity of human pol η. (A) Enzyme titration. Decreasing amounts of pol η (3.2, 1.6, 0.8, 0.4 and 0.2 fmol in lanes 1, 2, 3, 4 and 5, respectively) or pol α (0.3, 0.15, 0.07, 0.04 and 0.02 fmol in lanes 7, 8, 9, 10 and 11, respectively) were incubated with 500 fmol of the single-stranded phagemid template annealed to 5′-32P-labeled primer for 15 min in the reaction mixture. Lanes 6 and 12 contained no enzyme. The products were subjected to polyacrylamide gel electrophoresis under denaturing conditions. An autoradiogram of the gel is shown. (B) Time course. Pol η (0.8 fmol in lanes 1–5) or pol α (0.15 fmol in lanes 6–10) was incubated with 500 fmol of 5′-32P- labeled primer–templates for the indicated time periods. An autoradiogram of the gel is shown.
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Fig. 2. Translesion synthesis by human pol η. Increasing amounts of pol α (4.7 and 23.6 fmol in lanes 2 and 3, respectively) or pol η (0.25, 1.4 and 7.1 fmol in lanes 5, 6 and 7, respectively) were incubated with the 5′-32P-labeled primer–templates indicated beside each panel at 37°C for 15 min in the standard reaction mixture. Lanes 1 and 4 contained no enzyme. The products were subjected to polyacrylamide gel electrophoresis under denaturing conditions. The autoradiograms of the gels are shown. (A) Undamaged control for CPD and (6–4) photoproduct [(6–4)PP]. (B) CPD at the bridged TT. (C) The (6–4) photoproduct at the bridged TT. (D) The abasic analog at X. (E) Undamaged control for AAF. (F) AAF-modified guanine at the indicated site. (G) Undamaged control for cisplatin. (H) Intrastrand cross-link of two guanines by cisplatin at the indicated site.
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Fig. 3. Nucleotide selectivity of pol η incorporation opposite lesions. Pol η (0.25 fmol in lanes 2–7) was incubated with 30mer DNA annealed to a 5′-32P-labeled 16mer (A–F, I and J) or 17mer (G, H, K and L) DNA in the presence of all four dNTPs (lane 3) or with one of the indicated dNTPs (lanes 4–7), or in the absence of dNTPs (lane 2). The autoradiograms of the gels are shown. (A) Undamaged control for the CPD and (6–4)PP templates with a 16mer primer. (B) CPD at the bridged TT with a 16mer primer. (C) The (6–4) photoproduct at the bridged TT with a 16mer primer. (D) The AP analog at X with a 16mer primer. (E) Undamaged control for the AAF-G template with a 16mer primer. (F) AAF-G at the indicated site with a 16mer primer. (G) Undamaged control for AAF-G template with a 17mer primer. (H) AAF-G at the indicated site with a 17mer primer. (I) Undamaged control for the cisplatin-GG template with a 16mer primer. (J) Cisplatin-GG at the indicated site with a 16mer primer. (K) Undamaged control for the cisplatin-GG template with a 17mer primer. (L) Cisplatin-GG at the indicated site with a 17mer primer.
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Fig. 4. Ability of pol η to elongate DNA chains past lesions. Sets of 5′-32P-labeled 17mer (A, B, E, F, G, J and K) or 18mer (C, D, H, I, L and M) primers, which contained different sequences at their 3′ ends (indicated by N, where N was A, C, G and T in lanes 1–4, 5–8, 9–12 and 13–16, respectively), were annealed to 30mer templates. Increasing amounts of pol η or pol α were incubated with these primed templates. The auto-radiograms of the gels are shown. The amounts of pol η were 0.12 fmol in lanes 2, 6, 10 and 14, 0.7 fmol in lanes 3, 7, 11 and 15 (A–D, F, H, J and L), 0.25 fmol in lanes 2, 6, 10 and 14, and 1.4 fmol in lanes 3, 7, 11 and 15 (E, G, I, K and M). Pol α (lanes 4, 8, 12 and 16) was present at 4.7 fmol. (A) Undamaged control for the CPD template with a 17mer primer. (B) CPD at the bridged TT with a 17mer primer. (C) Undamaged control for the CPD template with an 18mer primer. (D) CPD at the bridged TT with an 18mer primer. (E) The AP analog at X with a 17mer primer. (F) Undamaged control for the AAF-G template with a 17mer primer. (G) AAF-G at the indicated site with a 17mer primer. (H) Undamaged control for the AAF-G template with an 18mer primer. (I) AAF-G at the indicated site with an 18mer primer. (J) Undamaged control for the cisplatin-GG template with a 17mer primer. (K) Cisplatin-GG at the indicated site with a 17mer primer. (L) Undamaged control for the cisplatin-GG template with an 18mer primer. (M) Cisplatin-GG at the indicated site with an 18mer primer.
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Fig. 4. Ability of pol η to elongate DNA chains past lesions. Sets of 5′-32P-labeled 17mer (A, B, E, F, G, J and K) or 18mer (C, D, H, I, L and M) primers, which contained different sequences at their 3′ ends (indicated by N, where N was A, C, G and T in lanes 1–4, 5–8, 9–12 and 13–16, respectively), were annealed to 30mer templates. Increasing amounts of pol η or pol α were incubated with these primed templates. The auto-radiograms of the gels are shown. The amounts of pol η were 0.12 fmol in lanes 2, 6, 10 and 14, 0.7 fmol in lanes 3, 7, 11 and 15 (A–D, F, H, J and L), 0.25 fmol in lanes 2, 6, 10 and 14, and 1.4 fmol in lanes 3, 7, 11 and 15 (E, G, I, K and M). Pol α (lanes 4, 8, 12 and 16) was present at 4.7 fmol. (A) Undamaged control for the CPD template with a 17mer primer. (B) CPD at the bridged TT with a 17mer primer. (C) Undamaged control for the CPD template with an 18mer primer. (D) CPD at the bridged TT with an 18mer primer. (E) The AP analog at X with a 17mer primer. (F) Undamaged control for the AAF-G template with a 17mer primer. (G) AAF-G at the indicated site with a 17mer primer. (H) Undamaged control for the AAF-G template with an 18mer primer. (I) AAF-G at the indicated site with an 18mer primer. (J) Undamaged control for the cisplatin-GG template with a 17mer primer. (K) Cisplatin-GG at the indicated site with a 17mer primer. (L) Undamaged control for the cisplatin-GG template with an 18mer primer. (M) Cisplatin-GG at the indicated site with an 18mer primer.
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