1)Comparative Genomics and Evolutionary Trajectories of Viral ATP Dependent DNA-Packaging Systems.? We performed a comparative genomics study of ATP-dependent DNA packaging systems of viruses. Several distinct ATPase motors and accessory proteins have been identified in DNA-packaging systems of viruses such as terminase-portal systems, the phi29-like packaging apparatus, and packaging systems of lipid inner-membrane-containing viruses. Sequence and structure analysis of these proteins suggest that there were two major independent innovations of ATP-dependent DNA packaging systems in the viral universe. The first of these utilizes a HerA/FtsK superfamily ATPase and is seen in prokaryotic viruses with inner lipid membranes, large eukaryotic nucleo-cytoplasmic DNA viruses (including poxviruses) and a group of eukaryotic mobile DNA transposons. We showed that ATPases of the phi29-like packaging system are also divergent versions of the HerA/FtsK superfamily that functions in viruses without an inner membrane. The second system, the terminase-portal system, is dominant in prokaryotic tailed viruses and typically functions with linear chromosomes. The large subunit of this system contains a distinct ATPase domain and a C-terminal nuclease domain of the RNAse H fold. We developed the classification of these ATPases within the P-loop NTPases, genomic demography and positioning of their genes in the viral chromosome. We showed that diverse portal proteins utilized by these systems share a common evolutionary origin and might have frequently displaced each other in evolution. Examination of conserved gene neighborhoods indicates repeated acquisition of Helix-turn-Helix domaincontaining terminase small subunits and a third accessory component, the MuF protein. Adenoviruses appear to have evolved a third packaging ATPase, unique to their lineage. Relationship between one major type of packaging ATPases and cellular chromosome pumps like FtsK suggests an ancient common origin for viral packaging and cellular chromosome partitioning systems.? ? 2)Comparative genomics of transcription factors and chromatin proteins in parasitic protists and other eukaryotes.? Comparative genomics of parasitic protists and their free-living relatives are profoundly impacting our understanding of the regulatory systems involved in transcription and chromatin dynamics. While some parts of these systems are highly conserved, other parts are rapidly evolving, thereby providing the molecular basis for the variety in the regulatory adaptations of eukaryotes. The gross number of specific transcription factors and chromatin proteins are positively correlated with proteome size in eukaryotes. However, the individual types of specific transcription factors show an enormous variety across different eukaryotic lineages. The dominant families of specific transcription factors even differ between sister lineages, and have been shaped by gene loss and lineage-specific expansions. Recognition of this principle has helped in identifying the hitherto unknown, major specific transcription factors of several parasites, such as apicomplexans, Entamoeba histolytica, Trichomonas vaginalis, Phytophthora and ciliates. Comparative analysis of predicted chromatin proteins from protists allows reconstruction of the early evolutionary history of histone and DNA modification, nucleosome assembly and chromatin-remodeling systems. Many key catalytic, peptide-binding and DNA-binding domains in these systems ultimately had bacterial precursors, but were put together into distinctive regulatory complexes that are unique to the eukaryotes. In the case of histone methylases, histone demethylases and SWI2/SNF2 ATPases, proliferation of paralogous families followed by acquisition of novel domain architectures, seem to have played a major role in producing a diverse set of enzymes that create and respond to an epigenetic code of modified histones. The diversification of histone acetylases and DNA methylases appears to have proceeded via repeated emergence of new versions, most probably via transfers from bacteria to different eukaryotic lineages, again resulting in lineage-specific diversity in epigenetic signals. Even though the key histone modifications are universal to eukaryotes, domain architectures of proteins binding post-translationally modified-histones vary considerably across eukaryotes. This indicates that the histone code might be """"""""interpreted"""""""" differently from model organisms in parasitic protists and their relatives. The complexity of domain architectures of chromatin proteins appears to have increased during eukaryotic evolution. Thus, Trichomonas, Giardia, Naegleria and kinetoplastids have relatively simple domain architectures, whereas apicomplexans and oomycetes have more complex architectures. RNA-dependent post-transcriptional silencing systems, which interact with chromatin-level regulatory systems, show considerable variability across parasitic protists, with complete loss in many apicomplexans and partial loss in Trichomonas vaginalis. This evolutionary synthesis offers a robust scaffold for future investigation of transcription and chromatin structure in parasitic protists.? ? 3)Anatomy of the E2 ligase fold: implications for enzymology and evolution of ubiquitin/Ub-like protein conjugation.? The configuration of the active site of E2 ligases, central enzymes in the ubiquitin/ubiquitin-like protein (Ub/Ubl) conjugation systems, has long puzzled researchers. Taking advantage of the wealth of newly available structures and sequences of E2s from diverse organisms, we performed a large-scale comparative analysis of these proteins. As a result we identified a previously under-appreciated diversity in the active site of these enzymes, in particular, the spatial location of the catalytic cysteine and a constellation of associated conserved residues that potentially contributes to catalysis. We observed structural innovations of differing magnitudes occurring in various families across the E2 fold that might correlate in part with differences in target interaction. A key finding was the independent emergence on multiple occasions of a polar residue, often a histidine, in the vicinity of the catalytic cysteine in different E2 families. We propose that these convergently emerging polar residues have a common function, such as in the stabilization of oxyanion holes during Ub/Ubl transfer and spatial localization of the Ub/Ubl tails in the active site. Thus, the E2 ligases represent a rare example in enzyme evolution of high structural diversity of the active site and position of the catalytic residue despite all characterized members catalyzing a similar reaction. Our studies also indicated certain evolutionarily conserved features in all active members of the E2 superfamily that stabilize the unusual flap-like structure in the fold. These features are likely to form a critical mechanical element of the fold required for catalysis. The results presented here could aid in new experiments to understand E2 catalysis. ? ? BEN: a novel domain in chromatin factors and DNA viral proteins.? We reported a previously uncharacterized alpha-helical module, the BEN domain, in diverse animal proteins such as BANP/SMAR1, NAC1 and the Drosophila mod(mdg4) isoform C, in the chordopoxvirus virosomal protein E5R and in several proteins of polydnaviruses. Contextual analysis suggests that the BEN domain mediates protein-DNA and protein-protein interactions during chromatin organization and transcription. The presence of BEN domains in a poxviral early virosomal protein and in polydnaviral proteins also suggests a possible role for them in organization of viral DNA during replication or transcription.
