Bacteria and archaea have developed CRISPR-Cas surveillance complexes to protect themselves against invading phages and plasmids, with the pathway defined by spacer acquisition, crRNA maturation and target interference steps. Our laboratory has focused its efforts on structural characterization of the interference step by studying single-subunit Cas and multi-subunit Cascade complexes with guide RNAs and target dsDNAs and/or RNAs. We are also investigating how evolved anti-CRISPR proteins suppress the CRISPR-Cas pathway, the role of accessory CRISPR nucleases and CRISPR-mediated DNA transposition. Host defense against infection by viral and bacterial pathogens is critically dependent on the initiation and maintenance of the finely tuned primary innate immune response. Our efforts are primarily focused on the metazoan second messenger cyclic GAMP produced by DNA-activated cGAS synthase, together with elucidation of the principles underlying activation of STING by cGAMP. We continue to make progress in identifying and characterizing anti-viral small molecules targeted to cGAS and STING.

Structure Maintenance of Chromosome (SMC) complexes are central to chromosome segregation, compaction and DNA repair, thereby impacting on gene expression and regulation. SMC family members control chromosome topology during the cell cycle with ATP-regulated Smc5/6 involved in rescue of stalled replication forks, while ATP- and nuclease-regulated MRE11-Rad50 (MR) is involved in preservation of genomic integrity and suppression of tumorigenesis. Our group has focused on the structural biology of DNA-bound Smc5/6 and MR complexes towards a molecular understanding the mechanisms underlying preservation of genomic integrity and suppression of tumorigenesis. Our group has also focused its efforts on a structure-based mechanistic understanding of homologous recombination regulation during meiosis initiated by Spo11-induced DNA DSBs and the role of accessory factors in modulating the process.

The packaging of DNA within chromosomes, the orderly replication and distribution of chromosomes, the maintenance of genomic integrity, and the regulated expression of genes depend upon nucleosomal histone proteins. Our long-term goals are directed toward gaining structural and mechanistic insights into the functional readout of histone covalent modification(s), either at the single or combinatorial readout level by writers, readers and erasers of such marks, in a context-dependent manner, both at the histone tail and nucleosome level. Methylation of cytosine in the CpG context has pronounced effects on gene expression with DNA methylation patterns established during embryonic development by de novo DNA methyltransferases (DNMTases) and then faithfully replicated by maintenance DNMTases during subsequent somatic cell division. Current efforts are focused on structure-function studies of writers, readers and erasers of DNA methylation marks, as well as elucidation of the impact of crosstalk between DNA and histone methylation marks on gene regulation. We have also extended these studies to RNA-dependent DNA methylation in plants.

RNA interference is a conserved biological response to double-stranded RNA that regulates gene expression. The response is mediated by small interfering RNAs (siRNAs), which guide the sequence-specific degradation of cognate messenger RNAs (mRNAs). Our group has structurally characterized events associated with processing of dsRNAs into siRNAs by the endonuclease activity of Dicer and guide-strand-mediated cleavage of target RNAs by Argonaute (Ago). Germline-specific piRNAs and Piwi proteins play a critical role in genome defense against transposable elements, thereby protecting the genome against transposon-induced defects in gametogenesis and fertility. Current efforts are addressed towards understanding the role of proteins and associated RNAs in mechanisms underlying piRNA biogenesis and transposon suppression.

The biology of histone proteins encompasses their synthesis in the cytosol, nuclear import and incorporation into nucleosomes, as well as subsequent eviction from chromatin, redeposition, storage or degradation. Histone chaperones represent a structurally and functionally diverse family of histone-binding proteins that prevent promiscuous interactions of histones before their assembly into chromatin. Our efforts to date have focused on histone chaperones Daxx, Mcm2 and DNAJC9 and their complexes with histone peptides to address the specificity underlying histone chaperone binding to histones and their variants. Lipid transfer proteins are important in membrane vesicle biogenesis and trafficking, signal transduction and immunological presentation processes. We have an ongoing structural biology program towards defining a framework for understanding how mammalian glycolipid transfer proteins (GLTPs) acquire and release glycosphingolipids (GSLs) during lipid intermembrane transfer and presentation processes. This research has been extended to transfer of phospholipid ceramide-1-phosphate by its transfer protein CPTP.

The role of RNA in information transfer and catalysis highlights its dual functionalities, thereby impacting on RNA folding, recognition and catalysis. Our crystallographic structural efforts have focused on natural riboswitches that target nucleotides, anions, amino acids and cofactor ligands to elucidate principles of ligand pocket architecture, the network of intermolecular contacts, the role of hydrated divalent cations and shape complementarity to the exquisite specificity of molecular recognition. Small self-cleaving nucleolytic ribozymes contain catalytic domains that use in-line alignment and acid-base chemistry to accelerate site-specific cleavage/ligation of phosphodiester backbones with implications for viral genome replication, pre-mRNA processing and alternative splicing. We have contributed to the field through structural and biochemical studies of twister, twister-sister, pistol and hatchet nucleolytic ribozymes and are currently focused on trapping the cleavage-competent vanadate transition states of these ribozymes.

Our group has been interested in a mechanistic understanding of RNA-mediated protein function, that include exonucleases and endonucleases, as well as proteins involved in RNA decapping, export and toxin-antitoxin regulation. We have structurally investigated the impact of misregulation of protein-RNA complexes on diseases ranging from leukemia, neurodegenerative, autoimmune, myelination and muscular dystrophy. We have ongoing projects identifying and characterizing inhibitors targeting protein scaffolds mediating SARS-CoV-2 viral infection, and those involved in leukemic transformation. Structure-function studies have also been undertaken on protein-DNA complexes towards a mechanistic understanding of mycobacterial helicases and motor-nucleases, as well as complexes involved in DSB repair and in transcriptional repression.