Our long-term goal is to help decipher how cells process DNA lesions in the maintenance of genomic integrity. The DNA damage response (DDR) involves protein complexes that sense DNA lesions and coordinate cell cycle progression along with DNA repair, apoptosis and DNA damage tolerance pathways. Reversible post-translational modifications (PTMs) of histone and non-histone proteins are essential for the DDR machinery to assemble at DNA damage sites but the mechanisms are poorly understood. Our proposed research focuses on probing, from a structural perspective, how PTMs participate in the control of time- and space-dependent protein associations in the DDR. Specifically, we will investigate the roles of lysine methylation and ubiquitylation in the repair of DNA double-strand breaks (DSBs) and in DNA damage tolerance by translesion DNA synthesis (TLS). 53BP1 is an important mediator of DNA repair by non-homologous end joining. In response to DSBs, 53BP1 associates with histone H4 dimethylated at lysine 20 (H4K20me2), which, in the absence of damage, is thought to be complexed to the lysine demethylase JMJD2A. Upon DSB induction, several ubiquitin ligases such as RNF8 are recruited to damage sites where they ubiquitylate histones and JMJD2A. The ubiquitylation of JMJD2A triggers its degradation, facilitating 53BP1 interaction with H4K20me2 and relocalization to DSBs.
Under Aim 1, we will probe the recruitment mechanisms of 53BP1, JMJD2A and structurally related protein PHF20. We will characterize interactions of these proteins with the nucleosome core particle.
Under Aim 2, we will investigate how mono-ubiquitylation of the DNA clamp PCNA in response to DNA damage activates damage tolerance by TLS. We will probe how the TLS DNA polymerase Rev1 and TLS are activated by PCNA ubiquitylation. Our hypothesis is that ubiquitylation alters PCNA?Rev1 interaction and functions as a double-switch to turn on TLS. Our studies will be the basis for designing in vivo assays that can elevate our knowledge of the DDR. Alteration of H4K20 methylation is a hallmark of human tumor cells and mutations in several DDR proteins are linked to cancer predispositions in humans. Because loss of 53BP1 reverses cancer phenotypes of BRCA1 mutant cells, inhibition of 53BP1 recruitment to DSB sites could have potential for cancer therapy. Rev1 and TLS contribute to the acquired therapeutic resistance of cancer cells.

Public Health Relevance

The proposed studies will contribute to the elucidation of fundamental cellular processes involved in the detection and repair of DNA double-strand breaks, one of the most harmful DNA lesions, and to our understanding of how DNA translesion synthesis is activated, a major cause of tumor drug resistance. The work has relevance to public health because by understanding the mechanisms of these important processes, we may be able to find ways to prevent and treat human malignancies, particularly cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA132878-06A1
Application #
8627747
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Pelroy, Richard
Project Start
2013-12-26
Project End
2018-11-30
Budget Start
2013-12-26
Budget End
2014-11-30
Support Year
6
Fiscal Year
2014
Total Cost
$357,750
Indirect Cost
$132,750
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
Klein, Christopher J; Wu, Yanhong; Vogel, Peter et al. (2014) Ubiquitin ligase defect by DCAF8 mutation causes HMSN2 with giant axons. Neurology 82:873-8
Tang, Jiangbo; Cho, Nam Woo; Cui, Gaofeng et al. (2013) Acetylation limits 53BP1 association with damaged chromatin to promote homologous recombination. Nat Struct Mol Biol 20:317-25
Cui, Gaofeng; Botuyan, Maria Victoria; Mer, Georges (2013) (1)H, (15)N and (13)C resonance assignments for the three LOTUS RNA binding domains of Tudor domain-containing protein TDRD7. Biomol NMR Assign 7:79-83
Mallette, Frederick A; Mattiroli, Francesca; Cui, Gaofeng et al. (2012) RNF8- and RNF168-dependent degradation of KDM4A/JMJD2A triggers 53BP1 recruitment to DNA damage sites. EMBO J 31:1865-78
Singh, Namit; Wiltshire, Timothy D; Thompson, James R et al. (2012) Molecular basis for the association of microcephalin (MCPH1) protein with the cell division cycle protein 27 (Cdc27) subunit of the anaphase-promoting complex. J Biol Chem 287:2854-62
Su, Dan; Hu, Qi; Li, Qing et al. (2012) Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106. Nature 483:104-7
Fazly, Ahmed; Li, Qing; Hu, Qi et al. (2012) Histone chaperone Rtt106 promotes nucleosome formation using (H3-H4)2 tetramers. J Biol Chem 287:10753-60
Bian, Chuanbing; Xu, Chao; Ruan, Jianbin et al. (2011) Sgf29 binds histone H3K4me2/3 and is required for SAGA complex recruitment and histone H3 acetylation. EMBO J 30:2829-42
Su, Dan; Hu, Qi; Zhou, Hui et al. (2011) Structure and histone binding properties of the Vps75-Rtt109 chaperone-lysine acetyltransferase complex. J Biol Chem 286:15625-9
Klein, Christopher J; Botuyan, Maria-Victoria; Wu, Yanhong et al. (2011) Mutations in DNMT1 cause hereditary sensory neuropathy with dementia and hearing loss. Nat Genet 43:595-600

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