Identification and Characterization of Thermostable Y-Family DNA Polymerases η, ι, κ and Rev1 From a Lower Eukaryote, Thermomyces lanuginosus.

Alexandra Vaisman,John P Mcdonald,Sender L Aspelund,Mallory R Smith,Thomas C Evans,Roger Woodgate

doi:10.3389/fmolb.2021.778400

Alexandra Vaisman, John P Mcdonald + Show 4 more

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https://doi.org/10.3389/fmolb.2021.778400

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Abstract

Y-family DNA polymerases (pols) consist of six phylogenetically separate subfamilies; two UmuC (polV) branches, DinB (pol IV, Dpo4, polκ), Rad30A/POLH (polη), and Rad30B/POLI (polι) and Rev1. Of these subfamilies, DinB orthologs are found in all three domains of life; eubacteria, archaea, and eukarya. UmuC orthologs are identified only in bacteria, whilst Rev1 and Rad30A/B orthologs are only detected in eukaryotes. Within eukaryotes, a wide array of evolutionary diversity exists. Humans possess all four Y-family pols (pols η, ι, κ, and Rev1), Schizosaccharomyces pombe has three Y-family pols (pols η, κ, and Rev1), and Saccharomyces cerevisiae only has polη and Rev1. Here, we report the cloning, expression, and biochemical characterization of the four Y-family pols from the lower eukaryotic thermophilic fungi, Thermomyces lanuginosus. Apart from the expected increased thermostability of the T. lanuginosus Y-family pols, their major biochemical properties are very similar to properties of their human counterparts. In particular, both Rad30B homologs (T. lanuginosus and human polɩ) exhibit remarkably low fidelity during DNA synthesis that is template sequence dependent. It was previously hypothesized that higher organisms had acquired this property during eukaryotic evolution, but these observations imply that polι originated earlier than previously known, suggesting a critical cellular function in both lower and higher eukaryotes.

Highlights

The Y-family DNA polymerases are responsible for copying damaged DNA during DNA replication in a process called translesion synthesis (TLS) (Sale et al, 2012)
Based on previous investigations and observations, it has been noted that the genomes of higher eukaryotes, such as humans and mice, possess genes for the four known eukaryotic Y-family pols, whereas, the model organism S. cerevisiae only possesses genes for two of the eukaryotic Y-family pols, RAD30 and REV1
BLAST homology searches revealed that the genomes of filamentous fungi such as Neurospora crassa and Aspergillus nidulans and even the corn smut Ustilago maydis harbor a POLI gene, indicating to us that the genetic and biochemical characteristics of translesion synthesis in some “higher” fungi could be quite akin to that in higher eukaryotes such as humans

Summary

Introduction

The Y-family DNA polymerases are responsible for copying damaged DNA during DNA replication in a process called translesion synthesis (TLS) (Sale et al, 2012). These enzymes are highly specialized in order to accommodate different structural DNA distortions caused by a wide variety of DNA lesions. Thermomyces lanuginosus Y-Family DNA Polymerases branches; Rad30A/POLH (polη); Rad30B/POLI (polι); and DinB (pol IV, Dpo, polκ); and Rev (Ohmori et al, 2001). Across the different domains of life, Y-family polymerase subfamilies are found in various combinations. One example is the thermophilic fungus, Thermomyces lanuginosus (T. lanuginosus) which possesses all four eukaryotic Y-family subfamilies much like humans, in contrast to its fungal relatives, S. cerevisiae and Schizosaccharomyces pombe (S. pombe). Is there logic in such seemingly random distribution of polι? Using phylogenetic analysis and comparing the biochemical characterization of Y-family pols from different species, we hoped to shed some light on this question

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