GEN1 from a Thermophilic Fungus Is Functionally Closely Similar to Non-Eukaryotic Junction-Resolving Enzymes

Alasdair D.J Freeman,Yijin Liu,Anne-Cécile Déclais,Anton Gartner,David M.J Lilley

doi:10.1016/j.jmb.2014.10.008

Abstract

Processing of Holliday junctions is essential in recombination. We have identified the gene for the junction-resolving enzyme GEN1 from the thermophilic fungus Chaetomium thermophilum and expressed the N-terminal 487-amino-acid section. The protein is a nuclease that is highly selective for four-way DNA junctions, cleaving 1nt 3′ to the point of strand exchange on two strands symmetrically disposed about a diagonal axis. CtGEN1 binds to DNA junctions as a discrete homodimer with nanomolar affinity. Analysis of the kinetics of cruciform cleavage shows that cleavage of the second strand occurs an order of magnitude faster than the first cleavage so as to generate a productive resolution event. All these properties are closely similar to those described for bacterial, phage and mitochondrial junction-resolving enzymes. CtGEN1 is also similar in properties to the human enzyme but lacks the problems with aggregation that currently prevent detailed analysis of the latter protein. CtGEN1 is thus an excellent enzyme with which to engage in biophysical and structural analysis of eukaryotic GEN1.

Highlights

IntroductionIn mitotic cells, it provides a mechanism for high-fidelity double-strand break repair and contributes to the resolution of interstrand crosslinks and lesions arising during replication
To identify the GEN1 gene in thermophilic fungi, we extended our previous phylogenetic analysis of XPG superfamily members that covered a set of FEN1, EXO1, XPG and GEN1-like sequences from phylogenetically diverse eukaryotes [9], and we included sequences from the thermophilic fungi C. thermophilum, Myceliophthora thermophile, Talaromyces marneffei, Talaromyces stipitatus and Thielavia terrestris
The analysis confirms that XPG superfamily members from thermophilic fungi fall into four distinct classes represented by the XPG, FEN1, EXO1 and GEN1 nuclease families, allowing us to identify the putative GEN1 of C. thermophilum

Summary

Introduction

In mitotic cells, it provides a mechanism for high-fidelity double-strand break repair and contributes to the resolution of interstrand crosslinks and lesions arising during replication. The first process, called “dissolution”, requires the BLM helicase to translocate two adjacent junctions toward each other before they are unlinked by topoisomerase IIIα [2,3,4] This pathway, which does not lead to strand exchange, is probably the primary response to the presence of DNA junctions in mitotically dividing cells, and defects lead to Bloom syndrome [5] that is characterized by genomic instability [6]. Junction-resolving enzymes have been well characterized in bacteria, phage, archaea and yeast mitochondria (reviewed in Ref. [7])

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