Screening of glycosylase activity on oxidative derivatives of methylcytosine: Pedobacter heparinus SMUG2 as a formylcytosine- and carboxylcytosine-DNA glycosylase.

5-Methylcytosine (mC) is an epigenetic mark that impacts transcription, development, diseases including cancer and aging. The demethylation process involves Tet-mediated stepwise oxidation of mC to hmC, fC, or caC, excision of fC or caC by thymine-DNA glycosylase (TDG), and subsequent base excision repair. Thymine-DNA glycosylase (TDG) belongs to uracil-DNA glycosylase (UDG) superfamily, which is a group of enzymes that are initially found to be responsible for excising the deaminated bases from DNA and generating apurinic/apyrimidinic (AP) sites. mC oxidative derivatives may also be generated from Fenton chemistry and γ-irradiation. In screening DNA glycosylase activity in UDG superfamily, we identified new activity on fC- and caC-containing DNA in family 2 MUG/TDG and family 6 HDG enzymes. Surprisingly, we found a glycosylase SMUG2 from bacterium Pedobacter heparinus (Phe), a subfamily of family 3 SMUG1 DNA glycosylase, displayed catalytic activity towards not only DNA containing uracil, but also fC and caC. Given the sequence and structural differences between the family 3 and other family enzymes, we investigated the catalytic mechanism using mutational, enzyme kinetics and molecular modeling approaches. Mutational analysis and kinetics measurements identified I62, N63 and F76 of motif 1, and H205 of motif 2 in Phe SMUG2 as important catalytic residues, of which H205 of motif 2 played a critical role in catalyzing the removal of fC and caC. A catalytic model underlying the roles of these residues was proposed. The structural and catalytic differences between Phe SMUG2 and human TDG were compared by molecular modeling and molecular dynamics simulations. This study expands our understanding of DNA glycosylase capacity in UDG superfamily and provides insights into the molecular mechanism of fC and caC excision in Phe SMUG2.

An unconventional family 1 uracil DNA glycosylase in Nitratifractor salsuginis.

The uracil DNA glycosylase superfamily consists of at least six families with a diverse specificity toward DNA base damage. Family 1 uracil N-glycosylase (UNG) exhibits exclusive specificity on uracil-containing DNA. Here, we report a family 1 UNG homolog from Nitratifractor salsuginis with distinct biochemical features that differentiate it from conventional family 1 UNGs. Globally, the crystal structure of N. salsuginisUNG shows a few additional secondary structural elements. Biochemical and enzyme kinetic analysis, coupled with structural determination, molecular modeling, and molecular dynamics simulations, shows that N. salsuginisUNG contains a salt bridge network that plays an important role in DNA backbone interactions. Disruption of the amino acid residues involved in the salt bridges greatly impedes the enzymatic activity. A tyrosine residue in motif 1 (GQDPY) is one of the distinct sequence features setting family 1 UNG apart from other families. The crystal structure of Y81G mutant indicates that several subtle changes may account for its inactivity. Unlike the conventional family 1 UNG enzymes, N. salsuginisUNG is not inhibited by Ugi, a potent inhibitor specific for family 1 UNG. This study underscores the diversity of paths that a uracil DNA glycosylase may take to acquire its unique structural and biochemical properties during evolution. DATABASE: Structure data are available in the PDB under accession numbers 5X3G and 5X3H.

Substantial genome synteny preservation among woody angiosperm species: comparative genomics of Chinese chestnut (Castanea mollissima) and plant reference genomes.

BACKGROUND: Chinese chestnut (Castanea mollissima) has emerged as a model species for the Fagaceae family with extensive genomic resources including a physical map, a dense genetic map and quantitative trait loci (QTLs) for chestnut blight resistance. These resources enable comparative genomics analyses relative to model plants. We assessed the degree of conservation between the chestnut genome and other well annotated and assembled plant genomic sequences, focusing on the QTL regions of most interest to the chestnut breeding community. RESULTS: The integrated physical and genetic map of Chinese chestnut has been improved to now include 858 shared sequence-based markers. The utility of the integrated map has also been improved through the addition of 42,970 BAC (bacterial artificial chromosome) end sequences spanning over 26 million bases of the estimated 800 Mb chestnut genome. Synteny between chestnut and ten model plant species was conducted on a macro-syntenic scale using sequences from both individual probes and BAC end sequences across the chestnut physical map. Blocks of synteny with chestnut were found in all ten reference species, with the percent of the chestnut physical map that could be aligned ranging from 10 to 39 %. The integrated genetic and physical map was utilized to identify BACs that spanned the three previously identified QTL regions conferring blight resistance. The clones were pooled and sequenced, yielding 396 sequence scaffolds covering 13.9 Mbp. Comparative genomic analysis on a microsytenic scale, using the QTL-associated genomic sequence, identified synteny from chestnut to other plant genomes ranging from 5.4 to 12.9 % of the genome sequences aligning. CONCLUSIONS: On both the macro- and micro-synteny levels, the peach, grape and poplar genomes were found to be the most structurally conserved with chestnut. Interestingly, these results did not strictly follow the expectation that decreased phylogenetic distance would correspond to increased levels of genome preservation, but rather suggest the additional influence of life-history traits on preservation of synteny. The regions of synteny that were detected provide an important tool for defining and cataloging genes in the QTL regions for advancing chestnut blight resistance research.

Identical genomic organization of two hemichordate hox clusters.

Genomic comparisons of chordates, hemichordates, and echinoderms can inform hypotheses for the evolution of these strikingly different phyla from the last common deuterostome ancestor. Because hox genes play pivotal developmental roles in bilaterian animals, we analyzed the Hox complexes of two hemichordate genomes. We find that Saccoglossus kowalevskii and Ptychodera flava both possess 12-gene clusters, with mir10 between hox4 and hox5, in 550 kb and 452 kb intervals, respectively. Genes hox1-hox9/10 of the clusters are in the same genomic order and transcriptional orientation as their orthologs in chordates, with hox1 at the 3' end of the cluster. At the 5' end, each cluster contains three posterior genes specific to Ambulacraria (the hemichordate-echinoderm clade), two forming an inverted terminal pair. In contrast, the echinoderm Strongylocentrotus purpuratus contains a 588 kb cluster of 11 orthologs of the hemichordate genes, ordered differently, plausibly reflecting rearrangements of an ancestral hemichordate-like ambulacrarian cluster. Hox clusters of vertebrates and the basal chordate amphioxus have similar organization to the hemichordate cluster, but with different posterior genes. These results provide genomic evidence for a well-ordered complex in the deuterostome ancestor for the hox1-hox9/10 region, with the number and kind of posterior genes still to be elucidated.

Genomic tools development for Aquilegia: construction of a BAC-based physical map.

BACKGROUND: The genus Aquilegia, consisting of approximately 70 taxa, is a member of the basal eudicot lineage, Ranuculales, which is evolutionarily intermediate between monocots and core eudicots, and represents a relatively unstudied clade in the angiosperm phylogenetic tree that bridges the gap between these two major plant groups. Aquilegia species are closely related and their distribution covers highly diverse habitats. These provide rich resources to better understand the genetic basis of adaptation to different pollinators and habitats that in turn leads to rapid speciation. To gain insights into the genome structure and facilitate gene identification, comparative genomics and whole-genome shotgun sequencing assembly, BAC-based genomics resources are of crucial importance. RESULTS: BAC-based genomic resources, including two BAC libraries, a physical map with anchored markers and BAC end sequences, were established from A. formosa. The physical map was composed of a total of 50,155 BAC clones in 832 contigs and 3939 singletons, covering 21X genome equivalents. These contigs spanned a physical length of 689.8 Mb (~2.3X of the genome) suggesting the complex heterozygosity of the genome. A set of 197 markers was developed from ESTs induced by drought-stress, or involved in anthocyanin biosynthesis or floral development, and was integrated into the physical map. Among these were 87 genetically mapped markers that anchored 54 contigs, spanning 76.4 Mb (25.5%) across the genome. Analysis of a selection of 12,086 BAC end sequences (BESs) from the minimal tiling path (MTP) allowed a preview of the Aquilegia genome organization, including identification of transposable elements, simple sequence repeats and gene content. Common repetitive elements previously reported in both monocots and core eudicots were identified in Aquilegia suggesting the value of this genome in connecting the two major plant clades. Comparison with sequenced plant genomes indicated a higher similarity to grapevine (Vitis vinifera) than to rice and Arabidopsis in the transcriptomes. CONCLUSIONS: The A. formosa BAC-based genomic resources provide valuable tools to study Aquilegia genome. Further integration of other existing genomics resources, such as ESTs, into the physical map should enable better understanding of the molecular mechanisms underlying adaptive radiation and elaboration of floral morphology.

New resources for marine genomics: bacterial artificial chromosome libraries for the Eastern and Pacific oysters (Crassostrea virginica and C. gigas).

Large-insert genomic bacterial artificial chromosome (BAC) libraries of two culturally and economically important oyster species, Crassostrea virginica and C. gigas, have been developed as part of an international effort to develop tools and reagents that will advance our ability to conduct genetic and genomic research. A total of 73,728 C. gigas clones with an average insert size of 152 kb were picked and arrayed representing an 11.8-fold genome coverage. A total of 55,296 clones with an average insert size of 150 kb were picked and arrayed for C. virginica, also representing an 11.8-fold genome coverage. The C. gigas and C. virginica libraries were screened with probes derived from selected oyster genes using high-density BAC colony filter arrays. The probes identified 4 to 25 clones per gene for C. virginica and 5 to 50 clones per gene for C. gigas. We conducted a preliminary analysis of genetic polymorphism represented in the C. gigas library. The results suggest that the degree of divergence among similar sequences is highly variable and concentrated in intronic regions. Evidence supporting allelic polymorphism is reported for two genes and allelic and/or locus specific polymorphism for several others. Classical inheritance studies are needed to confirm the nature of these polymorphisms. The oyster BAC libraries are publicly available to the research community on a cost-recovery basis at (www.genome.clemson.edu).