Cisplatin is widely used anti-cancer chemotherapeutic drug that induces DNA damage by forming Cisplatin-DNA abducts in cells. In vivo and in citron studies strongly suggest that most of the Cisplatin-DNA abducts are repaired through nucleotide excision repair (NER) pathway. Due to extensive efforts, we now have significant knowledge about the mechanism of NER and the proteins involved. Recognition of DNA damage is a critical step in the early stage of repair. Xeroderma pigmentosum group A complementing protein (XPA), replication protein A (RPA), XPC-hHR23B, and XPE can independently bind damaged DNA. However, it is still in debate how the damaged recognition proteins function at the damaged DNA site. In this proposal, we will use biochemical and molecular approaches to address the following specific questions: 1) how multiple damage recognition factors function at the damaged DNA site? 2) do zinc-finger proteins (RPA and XPA) cause structural distortion at the damaged site? If so, is it necessary for dual incisions? 3) how do the damaged recognition factors affect the efficiency and accuracy of 3' and 5'- incisions? In the first aim, binding kinetics of individual damage recognition proteins to damaged DNA, interactions between damaged recognition factors, and assembly of a preincision complex will be analyzed using purified repair proteins (RPA,XPA,XPC-hHR23B, and TFIIH) and Cisplatin-induced intra strand crossed-linked DNA. In the second aim, we will analyze the molecular basis for structural dissertation of Cisplatin-damaged DNA. Conformational charges of damage recognition proteins following their interment with amazed DNA and the role of the zinc-finger motif of RPA and XPA in this event will be analyzed. We will use a foot printing assay to analyze the structural distortion of damaged DNA induced by damage recognition factors. In addition, fluorescence resonance energy transfer (FRET) method will be utilized to simultaneously monitor both the conformational change of damage recognition proteins and distortion of the damaged DNA. In the third aim, we will attempt to functionally define the role of damage recognition factors in 3' and 5' incision activity by XPG and ERCCI-XPF will be examined. Both the accuracy and efficiency of the 3' and 5' incisions will be analyzed in advance with the kinetics of incision activity. Various mutants of damage recognition factors (RPA and XPC) will be used to examine any unique role these proteins possess in 3' and 5' incising.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA092111-05
Application #
6906532
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Pelroy, Richard
Project Start
2001-06-01
Project End
2006-05-31
Budget Start
2005-06-01
Budget End
2006-05-31
Support Year
5
Fiscal Year
2005
Total Cost
$234,675
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Biochemistry
Type
Schools of Medicine
DUNS #
603007902
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
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Park, Su-Jung; Lee, Young-Ju; Beck, Brian D et al. (2006) A positive involvement of RecQL4 in UV-induced S-phase arrest. DNA Cell Biol 25:696-703

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