Ellenberger Lab Home PageWe are studying the structure and function of proteins that assemble at a DNA replication fork to catalyze the rapid and accurate replication of genomic DNA. We are also studying DNA repair proteins that locate and excise damaged bases from DNA, as well as a site-specific DNA recombinase that cleaves, exchanges, and religates strands from 2 DNA molecules with limited homology. In each case we are determining high-resolution crystal structures of the proteins complexed to their DNA targets, and probing the mechanisms of substrate recognition and enzymatic catalysis with genetic and biochemical experiments.
DNA polymerases replicate genomic DNA in concert with accessory factors that unwind the double helix and prime the synthesis of Okazaki fragments on the lagging strand of the replication fork. We recently determined the crystal structure of bacteriophage T7 DNA polymerase complexed to a DNA primer-template, a nucleotide substrate, and its processivity factor E. coli thioredoxin. The crystal structure of the catalytic complex shows a conformational change in the polymerase that engages conserved residues and two metals with the DNA and nucleotide substrates, promoting the incorporation of the correct nucleotide specified by the DNA template. The T7 DNA polymerase physically interacts with a hexameric DNA helicase that unwinds the DNA duplex ahead of the replication complex. Our recent crystal structure of the T7 DNA helicase shows a close resemblance to RecA, a protein that promotes the exchange of homologous DNA strands. We are now examining how the helicase functions as a motor that couples the energy of nucleotide hydrolysis to the unwinding of duplex DNA, and how the helicase contacts the DNA polymerase to couple DNA synthesis on the leading and lagging strands of the replication fork.
DNA base excision glycosylases recognize modified bases in duplex DNA and cleave the N-glycosylic bond to release the damaged base from the DNA backbone during the first step of base excision repair. These enzymes flip modified nucleotides out of the DNA double helix and into the active site of the repair enzyme. The crystal structure of human 3-methyladenine DNA glycosylase complexed to alkylation-damaged DNA reveals how substrate bases are captured in the enzyme active site and it suggests the mechanism of glycosylic bond cleavage. A crystal structure of the functionally analogous E. coli enzyme, 3-methyladenine DNA glycosylase II, reveals a protein fold that is unrelated to that of the human enzyme, highlighting 2 different solutions to the problem of locating the same modified bases in DNA. The active sites of the human and E. coli enzymes are rich in aromatic amino acids that can stabilize electron-deficient alkylated bases in a "flipped-out" conformation by _-electron stacking interactions. We are presently studying how these enzymes find substrate bases in DNA, and the energetics of base flipping.
The bacteriophage lambda integrase protein (Int) is archetypic of site-specific DNA recombinases that cleave and rejoin two DNAs having limited sequence homology. The crystal structure of lambda Int’s catalytic domain shows that the tyrosine nucleophile initiating DNA cleavage is located on a flexible loop more than 20 Å from Int’s catalytic center. We are examining how the catalytic loopin Int’s active site is positioned for DNA cleavage by trapping the Int protein in a covalent phosphotyrosine linkage with a DNA substrate. Lambda Int’s catalytic activity is regulated by DNA bending factors and interactions with DNA sites distant from the site of strand exchange. Structures of these multiprotein complexes with bent DNA are being pursued.
Selected Publications:
Lau, A. Y., Schärer, O.D., Samson, L., Verdine, G.L., & T.
Ellenberger. (1998). Crystal Structure of a Human Alkylbase-DNA
Repair Enzyme Complexed to DNA: Mechanisms for Nucleotide Flipping and
Base Excision. Cell 95, 249-258.
Doublié, S., Tabor, S., Long, A.M., Richardson, C.C., & T. Ellenberger. (1998). Crystal Structure of a Bacteriophage T7 DNA Replication Complex. Nature 391, 251-258.
Kwon, H.J., R. Tirumalai, A. Landy, & T.E. Ellenberger. (1997).
Flexibility in DNA Recombination: Structure of the__ Integrase Catalytic
Core. Science 276, 126.