DNA interstrand crosslinks (ICLs) can be induced endogenously (e.g. lipid peroxidation) and by environmental agents (e.g. cigarette smoking, automobile exhausts, and pollution). The ICL damage tethers both strands of DNA duplex and blocks essential DNA metabolic functions such as replication. It remains poorly understood how the ICL damage is repaired in humans. ICLs are also the primary toxic lesions induced by many bi-functional chemotherapeutic drugs to kill cancerous cells. Cells develop resistance to such agents through up-regulating their repair capacity of ICLs, thereby compromising the therapeutic efficacy. Fanconi anemia (FA, FANC) is a hereditary disorder characterized by bone marrow failure, developmental defects, predisposition to cancers, hypersensitivity to crosslinking agents, indicating involvement of FA proteins in repair and tolerance of ICLs. At least 13 FANC genes have been identified thus far. Eight (FANC-A, B, C, E, F, G, L, and M) of the thirteen FANC gene products are found in a protein complex, termed as the FA core complex. Based on the preliminary data and previous observations, we hypothesize that FANCM participates in the incision step of ICL repair, and the FA core complex is involved in the regulation of incision endonuclease activities for precise and efficient incision of ICLs. The overall goal of this proposal is to delineate the mechanism of the dual incisions on both sides of ICL damage (unhooking) and to determine how the FA core complex helps maintaining stability of replication forks and contributes to the ICL unhooking when the DNA replication fork encounters an ICL. We will characterize the enzymatic properties of FANCM, identify the endonucleases that carry out the ICL incision, and test whether they collaborate with each other for successful ICL unhooking. By employing RNA interference and cDNA complementation analyses, we will verify the in vitro discoveries in human fibroblast cells through monitoring the ICL incision-induced production of DNA double strand breaks. We will test how components of the FA core complex are involved in maintaining stability of replication forks and in regulating activities of the ICL incision endonucleases. We will purify all components of the FA core complex, evaluate their DNA damage recognition activity, profile their physical and functional interactions with the incision endonucleases, and delineate the regulatory mechanism of damage incision in a biochemically defined in vitro system. We will also determine whether the FA core complex recruits and regulates the endonucleases in human cells through RNA interference and confocal microscopy. Understanding the mechanism of the ICL recognition and incision will not only contribute to the overall clarification of the ICL repair process, but also provide a novel basis for interventional strategies. For example, developing inhibitors of the ICL repair would lead to future translational research for chemosensitizers to overcome the clinically observed drug resistance in cancer patients.

Public Health Relevance

The characterization of DNA interstrand cross-link repair is directly related to human health because DNA cross-linking agents are widely used in cancer chemotherapy and deficient interstrand cross-link repair underlies the cancer-prone genetic syndromes such as Fanconi anemia and hereditary breast and ovarian cancers. Our proposal research will provide meaningful insights to the design of effective strategies for potentiating the cancer-specific toxicity of chemotherapeutic drugs and improving therapy for the cancer-prone syndromes.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL105631-03
Application #
8386961
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Qasba, Pankaj
Project Start
2010-12-01
Project End
2015-11-30
Budget Start
2012-12-01
Budget End
2013-11-30
Support Year
3
Fiscal Year
2013
Total Cost
$361,984
Indirect Cost
$123,984
Name
University of Miami School of Medicine
Department
Biochemistry
Type
Schools of Medicine
DUNS #
052780918
City
Coral Gables
State
FL
Country
United States
Zip Code
33146
Palovcak, Anna; Liu, Wenjun; Yuan, Fenghua et al. (2017) Maintenance of genome stability by Fanconi anemia proteins. Cell Biosci 7:8
Mittal, Rahul; Grati, M'hamed; Sedlacek, Miloslav et al. (2016) Characterization of ATPase Activity of P2RX2 Cation Channel. Front Physiol 7:186
Radhakrishnan, Rangasudhagar; Li, Yixuan; Xiang, Shengyan et al. (2015) Histone deacetylase 10 regulates DNA mismatch repair and may involve the deacetylation of MutS homolog 2. J Biol Chem 290:22795-804
Yuan, Fenghua; Dutta, Tanmay; Wang, Ling et al. (2015) Human DNA Exonuclease TREX1 Is Also an Exoribonuclease That Acts on Single-stranded RNA. J Biol Chem 290:13344-53
Panneerselvam, Jayabal; Pickering, Anna; Han, Bing et al. (2014) Basal level of FANCD2 monoubiquitination is required for the maintenance of a sufficient number of licensed-replication origins to fire at a normal rate. Oncotarget 5:1326-37
Gwon, Gwang Hyeon; Kim, Youngran; Liu, Yaqi et al. (2014) Crystal structure of a Fanconi anemia-associated nuclease homolog bound to 5' flap DNA: basis of interstrand cross-link repair by FAN1. Genes Dev 28:2276-90
Xu, Meng; Lai, Yanhao; Torner, Justin et al. (2014) Base excision repair of oxidative DNA damage coupled with removal of a CAG repeat hairpin attenuates trinucleotide repeat expansion. Nucleic Acids Res 42:3675-91
Benitez, Anaid; Yuan, Fenghua; Nakajima, Satoshi et al. (2014) Damage-dependent regulation of MUS81-EME1 by Fanconi anemia complementation group A protein. Nucleic Acids Res 42:1671-83
Qian, Liangyue; Yuan, Fenghua; Rodriguez-Tello, Paola et al. (2013) Human Fanconi anemia complementation group a protein stimulates the 5' flap endonuclease activity of FEN1. PLoS One 8:e82666
Yan, Denise; Zhu, Yan; Walsh, Tom et al. (2013) Mutation of the ATP-gated P2X(2) receptor leads to progressive hearing loss and increased susceptibility to noise. Proc Natl Acad Sci U S A 110:2228-33

Showing the most recent 10 out of 14 publications