Yeast mitochondrial HMG proteins: DNA-binding properties of the most evolutionarily divergent component of mitochondrial nucleoids.

Jana Bakkaiova,Smaranda Willcox,Jozef Nosek,Jack D Griffith,Victoria Marini,Lumir Krejci,Lubomir Tomaska

doi:10.1042/bsr20150275

Abstract

Yeast mtDNA is compacted into nucleoprotein structures called mitochondrial nucleoids (mt-nucleoids). The principal mediators of nucleoid formation are mitochondrial high-mobility group (HMG)-box containing (mtHMG) proteins. Although these proteins are some of the fastest evolving components of mt-nucleoids, it is not known whether the divergence of mtHMG proteins on the level of their amino acid sequences is accompanied by diversification of their biochemical properties. In the present study we performed a comparative biochemical analysis of yeast mtHMG proteins from Saccharomyces cerevisiae (ScAbf2p), Yarrowia lipolytica (YlMhb1p) and Candida parapsilosis (CpGcf1p). We found that all three proteins exhibit relatively weak binding to intact dsDNA. In fact, ScAbf2p and YlMhb1p bind quantitatively to this substrate only at very high protein to DNA ratios and CpGcf1p shows only negligible binding to dsDNA. In contrast, the proteins exhibit much higher preference for recombination intermediates such as Holliday junctions (HJ) and replication forks (RF). Therefore, we hypothesize that the roles of the yeast mtHMG proteins in maintenance and compaction of mtDNA invivo are in large part mediated by their binding to recombination/replication intermediates. We also speculate that the distinct biochemical properties of CpGcf1p may represent one of the prerequisites for frequent evolutionary tinkering with the form of the mitochondrial genome in the CTG-clade of hemiascomycetous yeast species.

Highlights

The compaction of DNA into chromosomes enables its accommodation into the confines of the cell, and provides a means for spatial regulation of gene expression and protection from DNA damage
Yeast mobility group (HMG)-box containing (mtHMG) proteins differ in their affinity to dsDNA To initiate the biochemical characterization of mtHMG proteins, we expressed recombinant genes encoding the corresponding protein without the N-terminal mitochondrial targeting sequence in fusion with GST using the pGEX-6P-2 vector
The nature of this complex is unclear, it is evident that the affinities of the mtHMG proteins to dsDNA as well as the lengths of their corresponding DNA-binding sites differ, and that the CpGcf1p exhibits the lowest affinity towards intact dsDNA

Summary

Introduction

The compaction of DNA into chromosomes enables its accommodation into the confines of the cell, and provides a means for spatial regulation of gene expression and protection from DNA damage. It is not surprising that the compaction of DNA into nucleosomes is mediated by a complex of highly conserved proteins called histones [1]. The necessity for DNA compaction is not limited to the eukaryotic nucleus, as it extends to bacterial cells as well as DNA-containing organelles, namely chloroplasts and mitochondria. In the latter, the DNA is compacted into nucleoprotein structures called mitochondrial nucleoids (mt-nucleoids) [6,7,8,9,10,11].

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Results

Conclusion