Molecular Basis of Transcriptional Mutagenesis at 8-Oxoguanine

Gerke E Damsma,Patrick Cramer

doi:10.1074/jbc.m109.022764

Gerke E Damsma, Patrick Cramer

Open Access

https://doi.org/10.1074/jbc.m109.022764

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Abstract

Structure-function analysis has revealed the mechanism of yeast RNA polymerase II transcription at 8-oxoguanine (8-oxoG), the major DNA lesion resulting from oxidative stress. When polymerase II encounters 8-oxoG in the DNA template strand, it can misincorporate adenine, which forms a Hoogsteen bp with 8-oxoG at the active center. This requires rotation of the 8-oxoG base from the standard anti- to an uncommon syn-conformation, which likely occurs during 8-oxoG loading into the active site. The misincorporated adenine escapes intrinsic proofreading, resulting in transcriptional mutagenesis that is observed directly by mass spectrometric RNA analysis.

Highlights

We investigated the molecular basis of 8-oxoG transcription by pol II
Incubation with GTP resulted in some misincorporation (Fig. 1c, lanes 4 –11), and for undamaged DNA, some misincorporation of ATP was observed, but these misincorporations are at a level expected from yeast pol II in the absence of TFIIS [8]
Adenine Misincorporation Results in Transcriptional Mutagenesis—To test whether misincorporation occurs when CTP is present and whether the misincorporated adenine remains in the RNA, we analyzed RNA products by MALDI mass spectrometry

Summary

EXPERIMENTAL PROCEDURES

Sample Preparation—S. cerevisiae pol II containing a hexahistidine-tagged Rpb subunit Elongation complexes (ECs) were obtained by incubating pol II with 2 M eq of nucleic acid scaffold in transcription buffer (20 mM HEPES (pH 7.6), 60 mM (NH4)2SO4, 8 mM MgSO4, 10 ␮M ZnCl2, 10% (v/v) glycerol, and 10 mM dithiothreitol) at 20 °C for 30 min. For RNA extension assays, different amounts of NTPs were added, and the mixture was incubated at 28 °C as described [10]. Bead-based RNA extension assays were carried out as described [12] with minor changes. For MALDI time-of-flight analysis, reactions were incubated with the indicated NTPs, stopped, and analyzed as described [10]. Structures were solved by molecular replacement with the program Phaser [16] using the structure of the complete 12-subunit pol II without nucleic acids (Protein Data Bank code 1Y1W) [9]. Refinement was monitored with the free R-factor, calculated from the same set of excluded reflections as in the refinement of the complete pol II complex [19] and the complete pol II EC [9, 10, 13]

RESULTS

DISCUSSION

Peak in anomalous difference