Evolutionary loss of 8-oxo-G repair components among eukaryotes
© Jansson et al; licensee BioMed Central Ltd. 2010
Received: 14 June 2010
Accepted: 1 September 2010
Published: 1 September 2010
We have examined the phylogenetic pattern among eukaryotes of homologues of the E. coli 7,8-dihydro-8-oxoguanine (8-oxo-G) repair enzymes MutY, MutM, and MutT.
These DNA repair enzymes are present in all large phylogenetic groups, with MutM homologues being the most universally conserved. All chordates and echinoderms were found to possess all three 8-oxo-G repair components. Likewise, the red and green algae examined have all three repair enzymes, while all land-living plants have MutY and MutM homologues, but lack MutT. However, for some phyla, e.g. protostomes, a more patchy distribution was found. Nematodes provide a striking example, where Caenorhabditis is the only identified example of an organism group having none of the three repair enzymes, while the genome of another nematode, Trichinella spiralis, instead encodes all three. The most complex distribution exists in fungi, where many different patterns of retention or loss of the three repair components are found. In addition, we found sequence insertions near or within the catalytic sites of MutY, MutM, and MutT to be present in some subgroups of Ascomycetes.
The 8-oxo-G repair enzymes are ancient in origin, and loss of individual 8-oxo-G repair components at several distinct points in evolution appears to be the most likely explanation for the phylogenetic pattern among eukaryotes.
To maintain structural integrity of DNA, organisms have developed DNA repair mechanisms. These have evolved both in complexity and specificity to ensure genomic integrity against the constant threats from damaging agents of endogenous and exogenous origins. Damage to DNA bases resulting from alkylation, oxidation, deamination, and UV-induced crosslinking, is mainly repaired by the base excision repair (BER) pathway, which is highly conserved throughout evolution and ubiquitously present in bacteria, archaea, and eukaryotes . BER is the major pathway for repair of oxidative base damage, transcription-coupled repair (TCR) and mismatch repair (MMR) being important backup pathways. Moreover, several of the DNA glycosylases that initiate BER of oxidative damage have overlapping specificities and serve as alternative pathways for various DNA lesions . Oxidative damage in DNA, specifically the 8-oxo-G lesion, is removed or prevented by the 8-oxo-G-specific BER enzymes MutY, MutM, and MutT . MutT is an 8-oxo-dGTPase that prevents incorporation of 8-oxo-G into DNA . MutM excises 8-oxo-G paired with C , while MutY is an adenine-DNA glycosylase that excises A paired with 8-oxo-G . The MutY and MutM glycosylases are both members of the helix-hairpin-helix (HhH) superfamily. This gene subfamily is the most diverse of the DNA glycosylases, with differing substrate specificities . The MutT homologue belongs to the group of nudix hydrolases and is not classified as a DNA glycosylase, despite being a component of the 8-oxo-G repair system .
The existence of an 8-oxo-G repair system in all main organism groups; archaea, bacteria, fungi, animals, and plants, underscores the importance of this system to defend against deleterious 8-oxo-G mutagenesis. Despite the widespread conservation and importance of this repair system in maintaining genomic stability, limited phylogenetic data is available about the highly diverse and adaptable HhH gene family of DNA repair enzymes among eukaryotes. The expanding number of entire genome sequences from a wide range of eukaryotic groups therefore encouraged an analysis of the phylogenetic distribution of the 8-oxo-G repair genes. As a broad phylogenetic analysis of the HhH superfamily of BER DNA glycosylases among prokaryotes has already been presented , prokaryotes were omitted from this study. Here, we have identified 8-oxo-G repair genes from metazoans, fungi and plants, along with a sequence analysis of the identified proteins.
We find that their phylogenetic distribution among eukaryotes strongly argues for group-specific gene losses. Thus, we reveal several cases of unexpected gene distributions, despite the fact that our analysis includes organisms where DNA repair has been extensively characterised both biochemically and genetically: yeast, mammals, and higher plants.
The situation in fungi looks more diverse. Overall, all three 8-oxo-G repair homologues are found in basidiomycetous fungi (Fig. 3). However, among ascomycotes, the phylogenetic distribution pattern is clear but patchy. Of the three proteins, MutT is the least prevalent in Ascomycota, and is retained only in the "Candida" group. All ascomycetous fungi were found to harbour the MutM homolog, with the exception of Schizosaccharomyces, although in this clade the single species Sz. japonicus seems to have the MutM protein as well. The MutY homologue is absent not only from Saccharomyces cerevisiae and closely related species, but also from other organisms of Saccharomycotina. Interestingly, the MutY component is found in Sordariomycetes and Dothideomycetes (subgroups of Pezizomycotina), but not in Eurotiomycetes, indicating it has been lost several times during evolution of the ascomycotes. MutY was present in all Schizosaccharomyces species, which was expected as Sz. pombe has a well-characterised MutY homologue.
Overall, the MutM homologue emerges as the most prevalent repair 8-oxo-G component among eukaryotes.
Sequence divergence between orthologues
The "Saccharomyces", like the Eurotiomycetes subgroups, harbour only the MutM homolog. All subgroups of Ascomycota clearly harbour the MutM repair enzyme, except Schizosaccharomyces. Interestingly, in this clade the single species Sz. japonicus harbours a MutM homologue, with a sequence similar to other fungal homologues. By contrast, in Sz. pombe, MutT- and MutM-like proteins have not been identified either by sequence or experimental analysis . Also, the nematode C. elegans deviates from the expected distribution pattern of 8-oxo-G repair enzymes, in that it harbours none of the 8-oxo-G repair components. However, C. elegans does hold the Nth HhH superfamily homologue, which may function in an alternative 8-oxo-G repair pathway . It is noteworthy that Caenorhabditis thus only has one HhH superfamily member. By contrast, the nematode Trichinella spiralis harbours all three of the Mut proteins. This probably reflects the large evolutionary distance between nematode subgroups, with T. spiralis belonging to a basal lineage and evolutionary distant to C. elegans. Another noteworthy example is Drosophila melanogaster, which only has a MutM homologue, whereas most arthropods have two to three 8-oxo-G repair proteins. Therefore, the common picture of 8-oxo-G repair gene distribution, predicted from a typical "model organism", is not always the most representative view.
The widespread distribution of the MutM homologue in eukaryotic genomes, and the lack of either the MutY or the MutT homolog, or both, probably indicates 8-oxo-G in non-replicated DNA as the most abundant and important oxidative DNA damage to correct. The post-replicative adenine DNA glycosylase MutY mainly serves to excise adenines misincorporated opposite 8-oxo-G by replication, in cooperation with the MMR system . This likely provides redundancy in post-replicative mismatch repair by separate pathways. All three 8-oxo-G repair components, however, are highly specific for their substrates , and possibly may have evolved from more "promiscuous" BER repair enzymes with catalytic activity toward alternative substrates. The situation of combining "promiscuous" broad substrate enzymes with highly specific ones may provide an advantage in terms of specificity and redundancy within and between separate DNA repair pathways . The organism thereby holds the capacity to deal with a larger variety of DNA damage in a new complex chemical environment.
The identified sequence insertions in MutY, MutM, and MutT, respectively, among subgroups of fungi, do not obviously correlate with the presence or absence of the other 8-oxo-G repair homologues. It is also not possible to predict the functional importance of these sequence insertions for specific enzymatic activities. The sequence diversity among HhH glycosylases, and to some extent observed among subgroups of fungi in this study of the 8-oxo-G repair system, may reflect the need of specific and highly adaptable systems to process complex patterns of DNA damage, caused by different environmental factors. Disparities in catalytic mechanisms and in DNA repair pathways, by which an organism processes DNA damage, probably is part of the explanation . Even though there is an established functional redundancy between the MutY, MutM, and MutT proteins , and between separate repair pathways, in protecting against oxidative damage, more experimental data in substrate specificity and DNA repair pathway redundancy are clearly needed.
Eukaryotic organisms were included in the study if the entire genome sequence was available, with the aim of covering as wide a selection of organism groups as possible. Published sequences of MutY, MutM and MutT from Schizosaccharomyces pombe, Saccharomyces cerevisiae, human, and Arabidopsis thaliana were used in BLASTP searches (E-values < 1 × 10-20) to retrieve candidate homologues in the respective groups of fungi, metazoans, and plants, plus the slime mold Dictyostelium and the choanoflagellate Monosiga (for summary of data, see Additional file 1, 2, 3 and 4). Evaluation of candidates was based on the identification of domains in the NCBI Conserved Domains Database (CDD) that are specific for the different proteins: Endo3c (cd00056) and DNAglycosylase_C (cd03431) for MutY, OGG_N (pfam07934, or in some cases cl06806) and Endo3c for MutM, and MTH1 (cd03427) for MutT. The domains were identified as part of the NCBI web-based BLAST interface which includes an RPS-BLAST search vs. the position-specific scoring matrices in CDD. While E-values were different for the four domains, they were always lower than 1 × 10-05 (cd03431). Only specific hits to domains were considered and best hits to similar domains (for instance Nth) were used as evidence to reject a candidate. For MutY and MutM, both domains had to be identified to score as positive. Found sequences were also subjected to reciprocal BLASTP searches, ensuring that they indeed were most similar to proteins of the respective family. Most searches were conducted using the non-redundant protein database at NCBI of December 2009.
Although the domain search made identifying proteins with low similarity to the initial query sequences easier, reliable candidates from the performed searches were also used as queries to retrieve more sequences from closely related groups, especially when a thorough evaluation was needed because the identified candidate had a different length.
As most protein sequences are based on gene predictions, many sequences had truncated ends due to the problems of identifying the exon-intron structure and thus the true ends of the gene. In those cases, the candidate protein sequence was extended by matching full sequences from closely related organisms to the genome using TBLASTN and adding the found segments to the protein sequence. For some organisms, also EST data were used to validate a predicted sequence. Long gaps or insertions within sequences that are not conserved in related species, and thus are indications of erroneous gene predictions, were left uncorrected as long as they did not interfere with the identification of domains and could be aligned properly to the other sequences.
Organism-specific databases utilised in this study.
Univ. of Tokyo
Stagonospora (Phaeosphaeria) nodorum
Xenopus (Silurana) tropicalis
Multiple alignments were constructed using clustalw or t_coffee and visualised in JalView where also annotated features, such as the MutY "adenine recognition site", were included (Figs 1 and 2). The locations of found insertions were mapped to known 3D structures of the proteins from the Protein Data Bank, accessed via NCBI Structure database, using the program Cn3D. Structures used in the analysis were: MutY from Geobacillus stearothermophilus, PDB:1RRQ; MutM from Homo sapiens, PDB:2NOF; MutT from E. coli, PDB:3A6S.
This work was supported by the Swedish Research Council (2007-5460) and the University of Gothenburg Science Faculty research platform in Ecotoxicogenomics.
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