Research

Structural biology of genome maintenance

DNA damage generated by environmental toxins and cellular metabolites interferes with 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. Atomic structures determined by electron microscopy and X-ray crystallography provide detailed snapshots of these proteins as they recognize and remodel DNA to carry out a particular function. Current work focuses on repair of stalled replication forks by structure-specific DNA translocases, priming of DNA synthesis during eukaryotic replication, and base excision repair of DNA alkylation damage and interstrand DNA crosslinks by DNA glycosylases. 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 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 and other impediments can stall progression of the replisome and lead to genome instability. We are interested in how the replisome maintains DNA synthesis upon encountering DNA damage, and are studying the molecular mechanisms of enzymes that promote repair or tolerance of DNA damage at stalled replication forks.

The DNA motor proteins SMARCAL1, HLTF, and ZRANB3, and FBH1 catalyze fork reversal, which involves reannealing parental DNA strands and unwinding daughter strands to generate branched structures that facilitate replication restart. We characterized the structural basis for recognition of stalled forks by SMARCAL1, HLTF, and FBH1, and are interested in understanding how these enzymes operate in the context of the replisome and the RAD51 recombinase.

In collaboration with David Cortez, we established the molecular basis for how HMCES/YedK proteins form covalent DNA-protein crosslinks (DPCs) to abasic sites in single-stranded DNA located at DNA junctions, such as those found at stalled replication forks, as a means of supressing strand breaks and mutagenic translesion bypass synthesis. We also characterized how these enzymes catalyze the reverse reaction to remove the DPC in cells.

Synthesis of DNA during replication is intiated by DNA polymerase α–primase, which generates an RNA-DNA primer that is extended by replicative polymerases. We determined a series of cryo-EM structures of pol α–primase with primed templates representing various stages of DNA synthesis. The structures reveal a rermarkable range of motion that allows for primer synthesis across its two active sites. The structures also provide a structural basis for the defined length of both RNA and DNA segments of the primer, an important factor for replication fidelity and genome stability.

Funded by NIH grants R35GM136401 and P01CA092584

Repair of DNA alkylation damage

The bases of DNA are susceptible to alkylation from cellular metabolites, environmental and dietary toxins, and chemotherapeutic agents. DNA glycosylases protect cells by excising chemically modified bases from DNA, initiating the base excision repair (BER) pathway. We are investigating the structural basis for how several unique DNA glycosylases excise highly functionalized alkyl-DNA lesions to act as self-resistance mecahnisms to antibiotic producing bacteria, or as a means of repairing highly cytotoxic interstrand DNA crosslinks (ICLs) that covalentely tether the opposing DNA stands.

We discovered through a series of time-resolved crystal structures that the bacterial AlkD glycosylase does not operate by a base flipping mechanism common to all other glycosylases, which enables excision of bulky DNA adducts of the duocarmycin/yatakemycin family of antibiotics. Our work on the related AlkC enzyme 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.

We are working to understand how the mammalian NEIL3 excises ICLs dervied from abasic sites and is recruited to stalled replication forks. We are also characerizing a new family of bacterial glycosylases that unhook ICLs and excise bulky adducts generated from endogenous secondary metabolites. We discovered that a related and previously unknown glycosylase in E. coli (YcaQ) initiates an ICL repair pathway distinct from nucleotide excision repair in bacteria. More recently, we found that the YcaQ ortholog in the human pathogen Acinetobacter baumannii, which we named AlkX, is important for bacterial colonization of human tissues.

Funded by NSF (MCB-2341288) and NIH (R35GM136401)