A recurrent cancer-associated substitution in DNA polymerase \u03b5 produces a hyperactive enzyme

Xuanxuan Xing,Sushma Sharma,Polina V Shcherbakova,Chelsea R Bulock,Elizabeth A Moore,Daniel P Kane,Andrei Chabes

doi:10.1038/s41467-018-08145-2

Abstract

Alterations in the exonuclease domain of DNA polymerase ε (Polε) cause ultramutated tumors. Severe mutator effects of the most common variant, Polε-P286R, modeled in yeast suggested that its pathogenicity involves yet unknown mechanisms beyond simple proofreading deficiency. We show that, despite producing a catastrophic amount of replication errors in vivo, the yeast Polε-P286R analog retains partial exonuclease activity and is more accurate than exonuclease-dead Polε. The major consequence of the arginine substitution is a dramatically increased DNA polymerase activity. This is manifested as a superior ability to copy synthetic and natural templates, extend mismatched primer termini, and bypass secondary DNA structures. We discuss a model wherein the cancer-associated substitution limits access of the 3’-terminus to the exonuclease site and promotes binding at the polymerase site, thus stimulating polymerization. We propose that the ultramutator effect results from increased polymerase activity amplifying the contribution of Polε errors to the genomic mutation rate.

Highlights

Alterations in the exonuclease domain of DNA polymerase ε (Polε) cause ultramutated tumors
PolεP301R was clearly capable of hydrolyzing 3ʹ-termini, it was severely impaired in comparison to the wild-type enzyme
The exceptionally strong mutator effect of the human Polε-P286R variant modeled in yeast suggested functional alterations beyond a simple loss of proofreading, but the nature of these additional alterations remained elusive

Summary

Introduction

Alterations in the exonuclease domain of DNA polymerase ε (Polε) cause ultramutated tumors. Modeling of the P286R variant in yeast produced an exceptionally strong mutator phenotype exceeding that of an exonuclease-dead Polε mutant by two orders of magnitude[11] Mirroring these observations, PoleP286R mice are dramatically more cancer-prone than Pole exonuclease-deficient mice[12]. The mutator effects of many other, less common, Polε variants exceed the effects of exonuclease deficiency[13] These observations strongly argue that the development of an ultramutated phenotype requires some functional changes in the protein distinct from a loss of proofreading. The inability to position the 3ʹ-terminus in the exonuclease site makes Polε a more efficient DNA polymerase, a consequence that is not achieved by simple elimination of catalytic residues These findings provide insight into the molecular mechanisms that drive the development of ultramutated cancers, and have implications for understanding the normal physiological role of Polε in DNA replication and mutation avoidance

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