Cellular DNA is continuously faced with a barrage of lesions generated through the action of exogenous agents such as ionizing radiation, ultra violet damage, alkylation, and oxygen radicals. Endogenous species can also adversely affect the delicate balance of cellular systems. Two proteins, Xanthine Dehydrogenase (XDH) and RB69 gp43 polymerase, contribute in their own way to directing nucleotide traffic within the cell.

Rhodobacter capsulatus XDH is an (αβ) ₂ heterotetrameric cytoplasmic enzyme that resembles eukaryotic xanthine oxidoreductases. We hypothesize that the canonical oxidative hydroxylation pathway, for hypoxanthine to xanthine conversion, is correct. In order to test this hypothesis, we solved the crystal structure of an active site variant (E232Q) bound to hypoxanthine. This complex prevented substrate turnover, while validating the proposed mechanism. In addition, the wild-type enzyme was analyzed in the presence of 1-methylxanthine, a slow substrate analog, and pterin-6-aldehyde, a potent inhibitor of R. capsulatus XDH, to highlight presumed binding differences between non-natural substrates and inhibitors and the natural substrate. We compared the hydrogen bonding and van der Waals interactions of the new substrate and inhibitor structures, with the previously solved structures of oxypurinol bound to the bacterial enzyme and TEI-6720 bound to the bovine protein. This allowed us to ascertain the tight binding and space filling features that distinguish inhibitors from the less constrained substrates, which facilitate substrate turnover and product release.

RB69 gp43 polymerase, in contrast, was studied with respect to bypass of DNA containing the common lesion 8-oxo-dG (8-oxo-7, 8-dihydro-2'-deoxy-guanosine). Though a repair pathway for this lesion exists naturally, some lesions remain and are incorporated into DNA by polymerases. We hypothesize that RB69 probes the minor groove and thus small major groove lesions pass undetected by the protein. To confirm this hypothesis, four ternary complexes of the Y567A variant of the polymerase α family gp43 from bactcriophage RB69 were constructed and their structures were solved. Control complexes of the Y567A variant with undamaged DNA and an incoming dTTP (2'-deoxythymidine 5'-triphosphate) or 8-oxo-dG containing DNA and an incoming dCTP (2'-deoxycytidine 5'-triphosphate), served to ensure that the mutant maintained proper folding and DNA orientation compared with the previously solved wild-type structures. The ternary complex of 8-oxo-dG opposite dA (2'-deoxy-adenosine) confirmed that the polymerase tolerates the mismatch via conversion of the 8-oxo-dG from anti to syn. The extension complex of polymerization past an 8-oxo-dG:dC base pair displays R1369's ability to incorporate additional bases past the lesion and its concomitant loss of sensitivity to any distortions in the double helix. Our structures confirm the ready bypass of this small minor groove lesion by a family α DNA polymerase, thus leading to stable incorporation of the lesion into newly replicated DNA.