F-CphI represents a new homing endonuclease family using the Endo VII catalytic motif

Xiaoting Fang,Yongliang Jiang,Kim Li,Qinglu Zeng

doi:10.1186/s13100-018-0132-5

Abstract

BackgroundThere are six known families of homing endonucleases, LAGLIDADG, GIY-YIG, HNH, His-Cys box, PD-(D/E)-XK, and EDxHD, which are characterized by their conserved residues. Previously, we discovered a novel homing endonuclease F-CphI encoded by ORF177 of cyanophage S-PM2. F-CphI does not resemble any characterized homing endonucleases. Instead, the C-terminus of F-CphI aligns well with the N-terminal catalytic domain of a Holliday junction DNA resolvase, phage T4 endonuclease VII (Endo VII).ResultsA PSI-BLAST search resulted in a total of 313 Endo VII motif–containing sequences in sequenced genomes. Multiple sequence alignment showed that the catalytically important residues of T4 Endo VII were all well conserved in these proteins. Our site-directed mutagenesis studies further confirmed that the catalytically important residues of T4 Endo VII were also essential for F-CphI activity, and thus F-CphI might use a similar protein fold as Endo VII for DNA cleavage. A phylogenetic tree of the Endo VII motif–containing sequences showed that putative resolvases grouped into one clade while putative homing endonucleases and restriction endonucleases grouped into another clade.ConclusionsBased on the unique conserved residues, we proposed that F-CphI represents a new homing endonuclease family, which was named the DHHRN family. Our phylogenetic analysis could be used to predict the functions of many previously unknown proteins.

Highlights

There are six known families of homing endonucleases, LAGLIDADG, GIY-YIG, HNH, His-Cys box, PD-(D/E)-XK, and EDxHD, which are characterized by their conserved residues
In a process called intron-homing, a homing endonuclease cleaves an intronless allele near the intron insertion site (IIS) and repair of the DNA break using the intron-containing allele as template transfers the intron and the homing endonuclease gene into the intronless allele [1]
In order to further study the biochemical properties of this novel homing endonuclease, we cloned S-PM2 ORF177 into pBAD/Myc-HisB and induced His-tagged F-CphI expression in Escherichia coli cells

Summary

Introduction

There are six known families of homing endonucleases, LAGLIDADG, GIY-YIG, HNH, His-Cys box, PD-(D/E)-XK, and EDxHD, which are characterized by their conserved residues. Homing endonuclease genes are found as optional free-standing genes inserted between two conserved genes. Analogous to intron-homing, Homing endonucleases have been grouped into six families, LAGLIDADG, GIY-YIG, HNH, His-Cys box, PD-(D/E)-XK, and EDxHD, which were named for the conserved amino acid residues (reviewed by [4]). Crystal structures of the six homing endonuclease families have been determined [5,6,7,8,9,10] Based on their structural similarities, it was suggested that the HNH and His-Cys box families should be combined to a ββα-metal superfamily [11]. The catalytic motifs of PD-(D/E)-XK and EDxHD families were shown to be related [4]

Methods

Results

Discussion

Conclusion