Structural biology of genome maintenance

DNA damage arising from exposure to environmental toxins and cellular metabolites thwarts DNA replication and leads to genome instability, cell death, and diseases including cancer. Our laboratory uses the tools of structural biology and biochemistry to investigate molecular mechanisms of proteins involved in repairing DNA damage and maintaining replication fork progression. We primarily use X-ray crystallography, together with NMR and EM, as a starting point to understand how these proteins recognize and manipulate DNA structure to carry out their particular functions. Current work focuses on base excision repair of DNA alkylation damage by DNA glycosylases, repair of stalled replication forks by structure-specific DNA translocases, and priming of DNA synthesis during eukaryotic replication. The long-term goals are to understand the fundamental processes underlying genome maintenance and to develop new therapeutic strategies that target genetic diseases.

Repair of DNA alkylation damage

The bases of DNA are alkylated by cellular metabolites, environmental and dietary toxins, as well as by chemotherapeutic agents designed to kill tumor cells. DNA glycosylases protect the cell by excising the most commonly formed alkylated bases from the DNA backbone. We are investigating the structural basis for how these enzymes discriminate damaged from undamaged bases.

Past work focused on bacterial enzymes from the helix-hairpin-helix superfamily because these enzymes have dramatically different specificities for 3-methyladenine (PDF) and 1,N6-ethenoadenine (PDF) despite their similar architectures. Our work on Schizosaccharomyces pombe Mag2 provided clues into how these enzymes sense altered base pairs within the DNA helix (PDF).

More recently, we have focused on the recently discovered AlkC/AlkD family of alkylpurine DNA glycosylases because of their unique specificity toward positively charged DNA bases. Our crystal structures of Bacillus cereus AlkD revealed a three-dimensional architecture never before observed in a DNA binding protein or enzyme (PDF), as well as a novel mode of enzymatic capture of DNA damage (PDF). We have recently found through a series of time-resolved crystal structures that AlkD does not operate by a base flipping mechanism common to all other glycosylases (PDF), a feature that allows this unique BER enzyme to excise bulky DNA adducts (PDF). Our work on AlkC provided a molecular basis for how a non-base-flipping glycosylase selects for discrete methylated bases, and described a potential alternative mechanism for removal of 1mA and 3mC in bacteria (PDF).

Funded by NSF grants MCB-1517695 and MCB-1928918

Repair of stalled replication forks

Eukaryotic DNA replication is carried out by large multiprotein machines that coordinate DNA unwinding and synthesis at the replication fork. DNA damage, nucleotide depletion, and repetitive DNA sequences can stall progression of the replisome and lead to genome instability. We are interested in the molecular mechanisms of how the replisome maintains DNA synthesis upon encountering DNA damage.

Previous work in the lab focused on Mcm10, one of the first proteins loaded onto chromatin during S-phase and required for replisome assembly. We identified the domain architecture of vertebrate Mcm10 (PDF) and determined the structural basis for DNA binding (PDF, PDF) and oligomerization (PDF).

Current work is focused on interesting, recently discovered enzymes that promote repair or bypass of DNA damage at stalled replication forks. The DNA motor proteins SMARCAL1 and HLTF catalyze regression of stalled forks into four-stranded junctions that promote replication restart through recombination. In collaboration with David Cortez, we characterized the domain architectore and fork recognition properties of SMARCAL1 (PDF, PDF), and with Karlene Cimprich established how the potential tumor suppressor Helicase Like Transcription Factor (HLTF) recognizes stalled forks via a specific interaction with the 3'-end of the nascent leading strand (PDF, PDF).

We are also studying the mechanisms by which DNA glycosylases — normally associated with base excision repair of small lesions — unhook interstrand crosslinks that tether opposing DNA stands and thereby inhibit replication. We have determined the structure of a novel bacterial glycosylase, which we have named AlkZ, and that unhooks ICLs derived from the secondary metabolite azinomycin B (PDF).

Finally, we have recently established how the SRAP (SOS-response associated peptidase) protein HMCES/YedK covalently attaches itself to abasic sites at DNA fork structures as a means of supressing mutageneic translesion bypass (PDF).

Funded by NIH grants R01GM117299 and R01GM131071