A Crystallographic Study of the Role of Sequence Context in Thymine Glycol Bypass by a Replicative DNA Polymerase Serendipitously Sheds Light on the Exonuclease Complex

Abstract

Thymine glycol (Tg) is the most common oxidation product of thymine and is known to be a strong block for replicative DNA polymerases. A previously solved structure of the bacteriophage RB69 DNA polymerase (RB69 gp43) in complex with Tg in the sequence context 5’-G-Tg-G shed light on how Tg blocks primer elongation: The protruding methyl group of the oxidized thymine displaces the adjacent 5’-G which can no longer serve as a template for primer elongation. [Aller, P., Rould, M.A., Hogg, M, Wallace, S.S., & Doublié S. (2007) PNAS 104, 814–818] Several studies showed that in the 5’-C-Tg-Purine sequence context Tg is more likely to be bypassed by Klenow fragment, a family A DNA polymerase. We set out to investigate the role of sequence context on Tg bypass in a B family polymerase and solved the crystal structures of the bacteriophage RB69 DNA polymerase in complex with Tg containing DNA in the three remaining sequence contexts: 5’-N-Tg-G with N=A, T, or C. A combination of several factors influence Tg bypass, including the associated exonuclease activity, the nature of the 3’and 5’ bases surrounding Tg and the cis/trans interconversion of Tg. We also visualized for the first time the structure of a well-ordered exonuclease complex, allowing us to identify and confirm the role of key residues (Phe123, Met256, and Tyr257) in strand separation and the stabilization of the primer strand in the exonuclease site.

Structural Characterization of Viral Ortholog of Human DNA Glycosylase NEIL1 Bound to Thymine Glycol or 5-Hydroxyuracil-containing DNA

“…A local conformational perturbation may also be at play here. The presence of (5R,6S)-Tg within double-stranded DNA is known to hinder proper stacking of the 5Ј base, especially when that base is a purine (45,59,60). Furthermore, the nature of the base opposite the lesion was shown to modulate repair of Tg by human NEIL1 (49); Tg occupies the wobble position when mispaired with G (61) and is excised much more rapidly than Tg:A (49).…”

Utility of the Bacteriophage RB69 Polymerase gp43 as a Surrogate Enzyme for Herpesvirus Orthologs

“…It has been suggested that DNA replication accessory proteins, particularly the sliding clamp gp45, may be involved in the process of translocating DNA from polymerase to exonuclease active site [15]. It has also been shown that a β hairpin loop between residues 251–262 is important for exonuclease function [48,49]. This loop is involved in stabilizing the frayed base pair at the exonuclease active site allowing the removal of the incorrect nucleotide.…”

“…The three unpaired bases of the primer strand are then sequestered into the exonuclease active site, which facilitates the excision. Structures of RB69gp43 poised with its primer-template in the exonuclease mode are available [47,48]. …”

Molecular Recognition of Canonical and Deaminated Bases by P. abyssi Family B DNA Polymerase

“…an "up" or "down" conformation as seen in the recent structure of RB69 phage DNA polymerase. 20 The tip of this particular motif does not contain the Y255-G256 motif of T4 PolB (Y257-G258 for RB69 PolB) that has been shown to be required for DNA replication fidelity. 21 Nevertheless, the β-hairpin loop and arginine 265 block the P/T junction from two different sides (Fig.…”