Our lab works on a variety of different biological systems including, DNA replication and repair, nuclear receptors, and heparan and chondroitin sulfate biosynthesis. Listed below are the main focuses from our research in this past year: 1) SMCHD1 (structural maintenance of chromosome flexible hinge domain containing 1) is a 2005 amino acid protein involved in gene silencing with reported roles in X inactivation, genomic imprinting, and non-homologous end joining (NHEJ). Missense mutations in SMCHD1 are associated with isolated Arhinia, Bosma Arhinia Microphthalmia Syndrome (BAMS) and Facioscapulohumeral Muscular Dystrophy Type 2 (FSHD2). Based on previous work it had been shown that the N-terminal 703 amino acids contain a GHKL-ATPase domain based on low sequence identity and a weak ATPase activity. Unlike other GHKL-ATPase enzymes, these constructs did not appear to dimerize upon exposure to ATP. Our lab has been able to express a construct of the N-terminal domain in Ecoli. Using differential scanning fluorimetry and gel filtration chromatography our lab has been able to demonstrate that like the other GHKL-ATPase enzymes, the N-terminal domain of SMCHD1 does indeed form a dimer in solution. Recently we have been able to solve the first crystal structure of this enzyme. Preliminary structural evidence reveals a similar GHKL-ATPase domain as found in MutL, MORC proteins, and heat shock proteins, that forms a domain swap of an N-terminal strand that extends the beta-sheet of the other monomer in the dimer. Unique to SMCHD1 is a ubiquitin like domain on the N-terminus thats function is unknown. We are currently in the process of refining and analyzing the initial structure to better understand how the proteins function. 2) Classical non-homologous end-joining (NHEJ) is the pathway through which DNA double strand breaks, as a result of immunoglobulin gene maturation or genotoxic DNA damage, are repaired. Human X family polymerase Pol Mu and Pol Lambda are involved in faithful repair of these breaks by incorporating correctly paired nucleotides opposite the complementary strand of the other side of the DNA break, while DNA ligase IV (LigIV) utilizes ATP to catalyzes the ligation step of the 3-hydroxyl and the 5-phosphate of the break to generate a new phosphodiester bond. In support of research carried out in the Kunkel and Wilson laboratories at NIEHS, our lab has helped determine crystal structures of polymerase Mu and Lambda with damaged and mispaired bases to better understand functional differences between these related enzymes and how these enzymes repair these breaks. In addition, our lab determined the first two crystal structures of the catalytic domain of human LigIV in complex with DNA at two different steps of the catalytic cycle including a lysyl-AMP intermediate in an open conformation and the DNA-adenylate in a closed conformation just prior to bond formation. These structures were the foundation for structural guided mutagenesis work probing residues involved in catalysis to better understand the enzymes function. The polymerase and ligase work combined provide a deeper understanding at the mechanistic level for how double strand DNA breaks are repaired.
