DNA repair pathways have evolved to counteract the constant threat of endogenous and exogenous DNA damaging agents. Defects in DNA repair pathways are associated with cancer-prone disorders such as xeroderma pigmentosum or Fanconi anemia. Conversely, DNA repair enzymes also counteract the effects of antitumor agents. Research in our laboratory aims to develop a deep fundamental understanding of the molecular mechanisms of DNA repair pathways and leverage this knowledge to improve cancer therapy. We are using a combination of organic and analytical chemistry, biochemistry and molecular and cell biology we are pursuing the four main project areas discussed below. An critical component of our research program are collaboration with other researchers in Korea, Asia, Europe and the US in many areas including organic chemistry, structural biology, single molecule biophysics and oncology.
Molecular Mechanisms of Nucleotide Excision Repair (NER)
NER is the main pathway for the repair of bulky DNA lesions formed by UV light, environmental and cancer chemotherapeutic agents. NER is highly dynamic and operates by the sequential assembly of proteins at damaged sites, culminating with the excision of the damage as part of an oligonucleotide. Our group has made numerous contributions to the to the understanding of the mechanism of damage recognition and dual incision in NER using chemical, biochemical and cell biological approaches. Our long-term goal is to elucidate the mechanistic basis of key features of the NER pathway: i) damage recognition by XPC-RAD23B and verification by TFIIH, ii) protein-protein and protein-DNA interactions that drive the NER pathway, iii) assembly of the pre-incision complex and coordinated dual incision. Recent advances in protein biochemistry, single molecule biophysics, mass spectrometry and structural biology provide new opportunities to achieve such a comprehensive mechanistic understanding. Recently, we have reconstituted the NER pathway with purified proteins, characterized crucial interactions between XPA and RPA that are crucial for NER, characterized novel patient mutations in NER genes, and used single molecule DNA curtain assays to study how XPC-RAD23B finds lesions in DNA.
Chemical Approaches for Studying DNA Repair Pathways
Studies of DNA repair pathways have greatly benefited from the generation and synthesis of DNA probes, in the form of defined site-specific substrates or specific probes for biochemical and cell biological studies. Our laboratory has been developing new methodology to synthesize chemically defined DNA interstrand crosslinks (ICLs), lesion formed by antitumor agents such as cisplatin or nitrogen mustards. We and many laboratories around the world have used these ICLs to delineate the cellular pathways of ICL repair. We are continuing to develop new synthetic probes for the study of ICL repair and other human DNA repair pathways. We are using these probes extensively to investigate various aspects of DNA repair. One recent focus has been the study of how DNA polymerases interact with structurally diverse ICLs. These studies shed new light on the role of DNA polymerases in ICL repair and insights may be used to design more effective therapeutics based on how their DNA adducts interact with DNA polymerases.
Mechanistic Basis of Resistance and Diagnostic Tools
for Platinum Anticancer Therapy
Cisplatin, carboplatin and oxaliplatin are among the most successful and widely used antitumor agents, yet it is still not known which of the different DNA adducts formed are the most clinically relevant and how various DNA repair pathways contribute to the resistance to therapy. We have developed a mass-spectrometry based approach to detect the levels of various platinum-DNA adducts. We are use this method together with cell biology and genetics to correlate adduct levels with resistance to therapy in model- and tumor cell lines. These studies will yield an understanding of how cisplatin, carboplatin and oxaliplatin lesions are processed in cells. We will aim to develop our MS approach it into a diagnostic and predictive tool for cisplatin therapy outcomes in the clinic. We expect that we will a similar approach for additional antitumor agents in the future.
Hijacking Transcription-Coupled Nucleotide Excision Repair
for Cancer Therapy
The NER pathway has significant impact on the responsiveness of tumors to DNA damaging drugs, such as cisplatin. High levels of the NER gene ERCC1 are a reliable predictor for resistance to cisplatin treatment. By contrast, precision oncology strategies to specifically target tumors based on the NER status are not available. Using the known, but relatively rarely used, drug trabectedin as a starting point, we are studying natural product and their derivatives that can induce breaks in genomic DNA in NER-proficient cells. We are exploring a specific mechanistic hypothesis for how trabectedin induces DNA breaks in an NER-dependent fashion and will aim to generate new tool compounds with similar properties. We will explore how this unique property can be applied to treat tumor cells with elevated levels of DNA repair that are resistant to treatment with drugs, such as cisplatin.