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Nucleotide excision repair
Interstrand crosslink repair
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SUNY Stony Brook
Nucleotide Excision Repair (NER)
NER is the main pathway by which various bulky DNA lesions, including those induced by UV light, are repaired. In the process, the damage is excised from DNA as part of an oligonucleotide of about 30 bases in length in a process that requires over 20 proteins. Defects in NER are associated with the inherited disorder xeroderma pigmentosum (XP), which is characterized by extreme UV sensitivity and predisposition to skin cancer. NER has been reconstituted using purified proteins and human and hamster cell lines containing mutations in nearly every NER gene are available. The NER excision reaction is therefore ideally suited to study the enzymology of a multi protein system. NER involves the following steps: Damage recognition, DNA helix opening and open intermediate stabilization, dual incision around the lesion, removal of the damaged oligonucleotide and gap filling by replication proteins. We are addressing the following questions to elucidate the detailed molecular mechanisms of NER.
How is broad substrate recognition in NER achieved?
One set of projects is focused on the mechanism of damage recognition in NER, specifically how the NER machinery manages to recognize structurally diverse lesions in DNA in a vast sea of unmodified DNA. We have designed and synthesized photoreactive and fluorescent analogs of an acetyl aminofluorene adduct of dG, an efficient NER substrate. These analogs are efficiently repaired by NER and we are using currently using them in photocrosslinking and fluorescence spectroscopy studies to study how various NER proteins interact with damaged sites in DNA. These studies will be used to provide experimental evidence for current NER models hypothesizing that the initial damage recognition factos, XPC/HR23B recognizes distortion of the DNA helix, a second factor, which we believe is one of the two helicase subunits of TFIIH, verifies the presence of the damage and allows progression through the NER pathway.

How do protein-protein interactions control NER?
Another aim is the characterization of protein-protein interactions involved in NER. One essential interaction in NER is between the XPA and ERCC1 proteins. Together with Tom Ellenberger (Washington University, St. Louis), we have structurally characterized the interaction of a short peptide of XPA, which is sufficient to mediate the interaction with ERCC1. Structural characterization has revealed that this peptide undergoes a disorder to order transition upon binding ERCC1, assuming a tight turn conformation. This study provides the basis for the further biochemical and cell biological characterization of this interaction using mutational analysis of the two proteins as well as the generation of small molecules that block this interaction. We also carried out a biochemical study of this interaction by introducing mutations in ERCC1 and XPA. These mutations abolished the interaction between ERCC1 and XPA as well as NER activity in vitro and in vivo. The availability of such mutants in ERCC1 also makes it possible to investigate additional roles of the ERCC1-XPF in additional DNA repair pathways including interstrand crosslink repair and homologous recombination. We are also actively pursuing the characterization of additional protein-protein interactions in NER.
How are the catalytic steps in NER coordinated?
The two NER endonucleases ERCC1/XPF and XPG make incisions in the DNA 5’ and 3’ to the lesion. We are addressing the question of how the activity of these two proteins is regulated as part of a mutliprotein complex. In particular we are investigating how NER ensures that the two proteins do not randomly chop up genomic DNA and how the two incisions are coordinated that not ssDNA gap is formed in the process. We have recently found that a tight coordination of the dual incision and repair synthesis steps are likely responsible ensure a smooth progression through the NER pathway. Accordingly, XPF incises first 5’ to the lesion, repair synthesis is initiated and only then is the incision 3’ to the lesion made by XPG. We are now studying how various factors, including post-translational modifications, are involved in coordinating various steps in NER.