PDBsum entry 2i9g

Transferase/DNA

PDB id: 2i9g

Contents

Protein chain

325 a.a.

DNA/RNA

Ligands

BPI

Metals

_CL ×2

_NA ×2

Waters ×429

References listed in PDB file

Key reference

Title

Structure of DNA polymerase beta with a benzo[c]phenanthrene diol epoxide-Adducted template exhibits mutagenic features.

Authors

V.K.Batra, D.D.Shock, R.Prasad, W.A.Beard, E.W.Hou, L.C.Pedersen, J.M.Sayer, H.Yagi, S.Kumar, D.M.Jerina, S.H.Wilson.

Ref.

Proc Natl Acad Sci U S A, 2006, 103, 17231-17236. [DOI no: 10.1073/pnas.0605069103]

PubMed id

17079493

Abstract

We have determined the crystal structure of the human base excision repair enzyme DNA polymerase beta (Pol beta) in complex with a 1-nt gapped DNA substrate containing a template N2-guanine adduct of the tumorigenic (-)-benzo[c]phenanthrene 4R,3S-diol 2S,1R-epoxide in the gap. Nucleotide insertion opposite this adduct favors incorrect purine nucleotides over the correct dCMP and hence can be mutagenic. The structure reveals that the phenanthrene ring system is stacked with the base pair immediately 3' to the modified guanine, thereby occluding the normal binding site for the correct incoming nucleoside triphosphate. The modified guanine base is displaced downstream and prevents the polymerase from achieving the catalytically competent closed conformation. The incoming nucleotide binding pocket is distorted, and the adducted deoxyguanosine is in a syn conformation, exposing its Hoogsteen edge, which can hydrogen-bond with dATP or dGTP. In a reconstituted base excision repair system, repair of a deaminated cytosine (i.e., uracil) opposite the adducted guanine was dramatically decreased at the Pol beta insertion step, but not blocked. The efficiency of gap-filling dCMP insertion opposite the adduct was diminished by >6 orders of magnitude compared with an unadducted templating guanine. In contrast, significant misinsertion of purine nucleotides (but not dTMP) opposite the adducted guanine was observed. Pol beta also misinserts a purine nucleotide opposite the adduct with ungapped DNA and exhibits limited bypass DNA synthesis. These results indicate that Pol beta-dependent base excision repair of uracil opposite, or replication through, this bulky DNA adduct can be mutagenic.

Figure 1.
Fig. 1. Structures of B[c]Ph, its B[c]Ph DE metabolite, and the 1S adduct derived from trans-opening of this epoxide by the exocyclic 2-amino group of deoxyguanosine. The tetrahydrobenzo ring numbering system and the absolute configuration designations for the diastereomer used in the present study are shown. Note that the absolute configuration at C1 of the epoxide inverts upon trans-adduct formation.

Figure 5.
Fig. 5. Conformation of the oligonucleotide containing a B[c]Ph DE-adducted templating guanine in the single-nucleotide gapped DNA substrate bound to Pol . (A) F[o] – F[c]-simulated annealing electron density omit map (gray) contoured at 2.5 showing density corresponding to the B[c]Ph DE–dG adduct (adduct shown in yellow). The dideoxy-terminated primer terminus (3') form Watson–Crick hydrogen bonds (orange) with its templating base. (B) The gapped DNA substrate is bent 90° at the 5' phosphate of the adducted deoxyguanosine monophosphate (G*, purple nucleotide with the B[c]Ph DE in yellow). Pol is omitted for clarity, and the 3' ends of the primer and template strands are indicated. Note that the adducted G* base is rotated out of the normal templating position (shown in green for the analogous unadducted templating guanine; Protein Data Bank ID code 1BPX). The duplex portions of the two structures are nearly identical (rmsd = 0.60 Å; not shown). (C) A detailed view of the conformation of the templating adducted (G*) and unadducted (G) guanine bases. The B[c]Ph DE moiety (yellow) bound at N^2 of G* is positioned where an unadducted guanine base would be found in the DNA binary polymerase complex. It is able to stack with the upstream duplex in this position. The adducted deoxyguanosine is in a syn-conformation and displaced outside of the coding template position.