Crystallographic snapshots of a replicative DNA polymerase encountering an abasic site

Abstract

Abasic sites are common DNA lesions, which are strong blocks to replicative polymerases and are potentially mutagenic when bypassed. We report here the 2.8 Å structure of the bacteriophage RB69 replicative DNA polymerase attempting to process an abasic site analog. Four different complexes were captured in the crystal asymmetric unit: two have DNA in the polymerase active site whereas the other two molecules are in the exonuclease mode. When compared to complexes with undamaged DNA, the DNA surrounding the abasic site reveals distinct changes suggesting why the lesion is so poorly bypassed: the DNA in the polymerase active site has not translocated and is therefore stalled, precluding extension. All four molecules exhibit conformations that differ from the previously published structures. The polymerase incorporates dAMP across the lesion under crystallization conditions, indicating that the different conformations observed in the crystal may be part of the active site switching reaction pathway.

A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative thymine lesion, thymine glycol

“…The Tg lesion is intrahelical and engages in a regular Watson-Crick base pair with the incorporated A. In contrast to our previously published structure with the abasic site analog furan (23), translocation takes place after incorporation opposite Tg. Because the methyl group of the oxidized thymine protrudes axially from the pyrimidine ring, it prevents the adjacent 5Ј template base from stacking against Tg, thereby impeding extension, in agreement with the computational predictions (20,21) and the biochemical (10-13) and biological (9,(15)(16)(17)(18) studies.…”

“…6]. In the F⅐dAMP structure, we observed a unique conformation of the ␤-hairpin loop, where it swung down toward the polymerase active site and contacted the single-stranded 5Ј template (23) (Fig. 6a).…”

“…) Position of the ␤-Hairpin Loop. A ␤-hairpin located in the exonuclease domain (residues 248-265 in RB69) and conserved in polymerases of the B family has been proposed to play a role in strand separation (29) and in active site switching (23,29,30) [supporting information (SI) Fig. 6].…”

“…The acyATP originally was used for screening for ternary complexes of RB69 gp43; although incorporation of a chain terminator at the 3Ј end of the primer was not necessary to obtain a binary complex, acyATP was used because crystals grew more readily in the presence of acyATP as compared with dATP or dideoxyATP. Diffraction data were collected to 2.65-Å resolution at the Advanced Photon Source at Argonne National Laboratory (Argonne, IL) and phased by using the structure of the previously solved binary complex of RB69 gp43 with DNA containing an abasic site analog (23). The structure was refined to R work and R free values of 22.8% and 28.2%, respectively.…”

One‐step purification of Taq DNA polymerase using nucleotide‐mimetic affinity chromatography

“…The thermostable Taq Pol has been the key factor in transforming the initial PCR method of Kary Mullis [6] into one with an vast impact in molecular biology and biotechnology. Available crystal structures for most known polymerase families, including the Pol-I (of which Taq Pol is a member) and Pol-II families of DNA polymerases, have confirmed that such enzymes exhibit striking similarities in their overall architecture, the catalytic site, and the mechanism of nucleotidyl transfer [15,[25][26][27][28][29]. Therefore, a structure-biased combinatorial library of dNTP-mimetic ligands could serve as a useful pool for selecting ligands suitable for a targeted DNA polymerase.…”