Structural Biology of Lysine Methylation in DNA Damage and Checkpoint Signaling

Summary

Principal Investigator: Georges Mer
Affiliation: Mayo Clinic
Country: USA
Abstract: DESCRIPTION (provided by applicant): Our long-term objective is to help decipher the mechanism of DNA double-strand break (DSB) repair, a process indispensable in maintaining genomic stability in all organisms, and an important barrier to cancer in higher eukaryotes. Reversible site-specific post-translational modifications of histone and non-histone proteins are essential for the DNA repair machinery to assemble at sites of DNA DSBs and to the concomitant checkpoint activation leading to cell cycle arrest or apoptosis, but how these modifications contribute to the DNA damage response is poorly understood. Our proposed research focuses on understanding the role of lysine methylation in DNA DSB and cell cycle checkpoint signaling. We will probe several molecular interactions driven by the methylation of histone H4 at lysine 20 (H4-K20) and p53 at lysine 370 (p53-K370), and their possible synergistic coupling to other post-translational modifications (e.g. phosphorylated histone H2AX). In particular, we will study the interactions of methylated histone H4-K20 and methylated p53-K370 with human proteins 53BP1, JMJD2A and PHF20. 53BP1 is a key transducer of the cell response to DNA DSBs that participates in the assembly of DNA repair proteins and in checkpoint activation regulated by p53. JMJD2A is a histone demethylase whose function is linked to p53-dependent DNA damage-induced apoptosis. PHF20 is a component of the MLL1/MOF histone acetyltransferase complex that acetylates histone H4 in a p53 dependent manner in connection to DNA damage. For each study, we will apply a combination of biophysical experiments centered on nuclear magnetic resonance (NMR) spectroscopy and crystallographic three-dimensional (3D) structure determination of selected domains in complex with their associated targets. In Aim1, we will test the hypothesis that 53BP1 simultaneously recognizes methylated H4-K20 and phosphorylated histone H2AX using tandem tudor and tandem BRCT domains, respectively. This dual binding mode would explain how 53BP1 is recruited to DNA damage sites. In Aims 2 and 3, we will test the hypotheses that methylation of p53 at lysine 370 triggers a tight interaction of p53 with 53BP1 and JMJD2A, preventing p53 from binding DNA. This would provide a mechanism for the known inhibition of p53 transcriptional activity by methylation of lysine 370. In Aim 4, we have identified a new motif in PHF20 that we call disulfide cross-linked tudor dimer. This tudor domain is found in tandem with an MBT domain. We will characterize the tudor, MBT and tandem MBT-tudor domains of PHF20 in complex with methylated H4-K20 and p53-K370 peptides. Our structural and interaction studies at the atomic level will be the basis for informed design of in vivo experiments that will elevate our knowledge of DNA DSB signaling and repair. Alteration of histone H4 methylation at Lys20 is a hallmark of human tumor cells and mutation of p53 occurs in about 50% of human cancers. In the long term, our studies involving methylated histone H4 and p53 may help control the DNA damage response therapeutically for the prevention and treatment of cancer. PUBLIC HEALTH RELEVANCE The proposed studies will contribute to the elucidation of fundamental cellular processes involved in detecting and repairing DNA double-strand breaks, one of the most harmful types of DNA damage. 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.
Funding Period: ----------------2008 - ---------------2013-
more information: NIH RePORT

Top Publications

  1. pmc Distinct binding modes specify the recognition of methylated histones H3K4 and H4K20 by JMJD2A-tudor
    Joseph Lee
    Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA
    Nat Struct Mol Biol 15:109-11. 2008
  2. pmc Dual recognition of phosphoserine and phosphotyrosine in histone variant H2A.X by DNA damage response protein MCPH1
    Namit Singh
    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA
    Proc Natl Acad Sci U S A 109:14381-6. 2012
  3. pmc PHF20 is an effector protein of p53 double lysine methylation that stabilizes and activates p53
    Gaofeng Cui
    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, USA
    Nat Struct Mol Biol 19:916-24. 2012
  4. pmc Acetylation limits 53BP1 association with damaged chromatin to promote homologous recombination
    Jiangbo Tang
    Department of Cancer Biology, Abramson Family Cancer Research Institute, Basser Research Center for BRCA, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
    Nat Struct Mol Biol 20:317-25. 2013
  5. pmc Heme-based sensing by the mammalian circadian protein CLOCK
    Gudrun S Lukat-Rodgers
    Department of Chemistry and Biochemistry, North Dakota State University, Fargo, North Dakota 58102, USA
    Inorg Chem 49:6349-65. 2010
  6. pmc Celastrol inhibits Hsp90 chaperoning of steroid receptors by inducing fibrillization of the Co-chaperone p23
    Ahmed Chadli
    Center for Molecular Chaperone Radiobiology and Cancer Virology, Medical College of Georgia, Augusta, Georgia 30912, USA
    J Biol Chem 285:4224-31. 2010
  7. pmc Structure of a second BRCT domain identified in the nijmegen breakage syndrome protein Nbs1 and its function in an MDC1-dependent localization of Nbs1 to DNA damage sites
    Chao Xu
    Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA
    J Mol Biol 381:361-72. 2008
  8. pmc Kinetic analysis of interaction of BRCA1 tandem breast cancer c-terminal domains with phosphorylated peptides reveals two binding conformations
    Yves Nominé
    Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 200 First Street Southwest, Rochester, Minnesota 55905, USA
    Biochemistry 47:9866-79. 2008
  9. pmc Structural basis for the recognition of methylated histone H3K36 by the Eaf3 subunit of histone deacetylase complex Rpd3S
    Chao Xu
    Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA
    Structure 16:1740-50. 2008
  10. pmc Preparation of recombinant peptides with site- and degree-specific lysine (13)C-methylation
    Gaofeng Cui
    Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA
    Biochemistry 48:3798-800. 2009

Scientific Experts

  • Christopher J Klein
  • Georges Mer
  • Maria Victoria Botuyan
  • Gaofeng Cui
  • James R Thompson
  • Chao Xu
  • Namit Singh
  • Dan Su
  • Ahmed Fazly
  • Zhiguo Zhang
  • Qi Hu
  • Robert C Benirschke
  • Joseph Lee
  • Yves Nominé
  • Jiangbo Tang
  • Frédérick A Mallette
  • Qing Li
  • Timothy D Wiltshire
  • Fergus J Couch
  • Chuanbing Bian
  • Nenad Juranic
  • Gudrun S Lukat-Rodgers
  • Slobodan Macura
  • Ahmed Chadli
  • Emeric Wasielewski
  • Niraj M Shanbhag
  • Nam Woo Cho
  • Roger A Greenberg
  • Erica M Manion
  • Sungman Park
  • Stephane Richard
  • Donghwa Kim
  • Annie Heroux
  • Leah C Young
  • Bruce Horazdovsky
  • Francesca Mattiroli
  • Michael B Yaffe
  • Michael J Hendzel
  • Titia K Sixma
  • Michael G Rosenfeld
  • Mark T Bedford
  • Duaa H Mohammad
  • Jin Q Cheng
  • Harihar Basnet
  • Aimee I Badeaux
  • Satoshi Kaneko
  • Brian A Davies
  • Fei Yan
  • Zengqiang Yuan
  • Kenneth K Lee
  • Jinrong Min
  • Tara L Burke
  • Jerry L Workman
  • Jin Qiu Zhou
  • Hui Zhou
  • Ariel Paulson
  • Bo O Zhou
  • Patrick A Grant
  • Brian E Fleharty
  • Jianbin Ruan
  • Minhao Wu
  • Wolfram Tempel
  • Jing Li
  • Rui Ming Xu
  • Abdellah Allali-Hassani
  • Jianye Zang
  • Dalia Barsyte
  • Qin Wang
  • Han Fang Tuan
  • Keiichi I Nakayama
  • Abdul Fauq
  • Cristina Correia
  • William P Sullivan
  • Marina Ramirez-Alvarado
  • Kenton R Rodgers
  • Sara J Felts
  • Shigetsugu Hatakeyama
  • Zeljko Bajzer
  • Whyte G Owen
  • Liming Wu
  • Junjie Chen
  • Ariel J Caride

Detail Information

Publications20

  1. pmc Distinct binding modes specify the recognition of methylated histones H3K4 and H4K20 by JMJD2A-tudor
    Joseph Lee
    Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA
    Nat Struct Mol Biol 15:109-11. 2008
    ....
  2. pmc Dual recognition of phosphoserine and phosphotyrosine in histone variant H2A.X by DNA damage response protein MCPH1
    Namit Singh
    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA
    Proc Natl Acad Sci U S A 109:14381-6. 2012
    ..X phosphorylation marks. We show that MCPH1 recruitment to sites of DNA damage is linked to both states of H2A.X...
  3. pmc PHF20 is an effector protein of p53 double lysine methylation that stabilizes and activates p53
    Gaofeng Cui
    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, USA
    Nat Struct Mol Biol 19:916-24. 2012
    ..Overall our work establishes that PHF20 functions as an effector of p53 methylation that stabilizes and activates p53...
  4. pmc Acetylation limits 53BP1 association with damaged chromatin to promote homologous recombination
    Jiangbo Tang
    Department of Cancer Biology, Abramson Family Cancer Research Institute, Basser Research Center for BRCA, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
    Nat Struct Mol Biol 20:317-25. 2013
    ..These findings demonstrate that acetylation in cis to H4K20me2 regulates relative BRCA1 and 53BP1 DSB chromatin occupancy to direct DNA repair mechanism...
  5. pmc Heme-based sensing by the mammalian circadian protein CLOCK
    Gudrun S Lukat-Rodgers
    Department of Chemistry and Biochemistry, North Dakota State University, Fargo, North Dakota 58102, USA
    Inorg Chem 49:6349-65. 2010
    ..Comparison of the spectroscopic properties and CO-binding kinetics of CLOCK PAS-A with other CO sensor proteins reveals that CLOCK PAS-A exhibits chemical properties consistent with a heme-based gas sensor protein...
  6. pmc Celastrol inhibits Hsp90 chaperoning of steroid receptors by inducing fibrillization of the Co-chaperone p23
    Ahmed Chadli
    Center for Molecular Chaperone Radiobiology and Cancer Virology, Medical College of Georgia, Augusta, Georgia 30912, USA
    J Biol Chem 285:4224-31. 2010
    ..This study reveals a unique inhibition mechanism of p23 by a small molecule that could be exploited in the dissection of protein fibrillization processes as well as in the therapeutics of steroid receptor-dependent diseases...
  7. pmc Structure of a second BRCT domain identified in the nijmegen breakage syndrome protein Nbs1 and its function in an MDC1-dependent localization of Nbs1 to DNA damage sites
    Chao Xu
    Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA
    J Mol Biol 381:361-72. 2008
    ....
  8. pmc Kinetic analysis of interaction of BRCA1 tandem breast cancer c-terminal domains with phosphorylated peptides reveals two binding conformations
    Yves Nominé
    Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 200 First Street Southwest, Rochester, Minnesota 55905, USA
    Biochemistry 47:9866-79. 2008
    ..The dynamic nature of BRCA1-BRCT may facilitate the binding of BRCA1 to different phosphorylated protein targets...
  9. pmc Structural basis for the recognition of methylated histone H3K36 by the Eaf3 subunit of histone deacetylase complex Rpd3S
    Chao Xu
    Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA
    Structure 16:1740-50. 2008
    ..This work also provides a general method for structure determination of low-affinity protein complexes implicated in methyllysine recognition...
  10. pmc Preparation of recombinant peptides with site- and degree-specific lysine (13)C-methylation
    Gaofeng Cui
    Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA
    Biochemistry 48:3798-800. 2009
    ..The peptides were used to probe the methylation state-dependent interactions of mono, di, and trimethylated p53 with three different proteins...
  11. pmc (1)H, (15)N and (13)C resonance assignments for the three LOTUS RNA binding domains of Tudor domain-containing protein TDRD7
    Gaofeng Cui
    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA
    Biomol NMR Assign 7:79-83. 2013
    ..These assignments will allow three-dimensional structure determination of the LOTUS domains and mapping of their interaction with RNA, steps toward deciphering the function of TDRD7...
  12. pmc RNF8- and RNF168-dependent degradation of KDM4A/JMJD2A triggers 53BP1 recruitment to DNA damage sites
    Frédérick A Mallette
    Terry Fox Molecular Oncology Group and the Bloomfield Center for Research on Aging, Sir Mortimer B Davis Jewish General Hospital, Lady Davis Institute for Medical Research, Montreal, Quebec, Canada
    EMBO J 31:1865-78. 2012
    ..We propose that the RNF8-dependent degradation of JMJD2A regulates DNA repair by controlling the recruitment of 53BP1 at DNA damage sites...
  13. pmc Histone chaperone Rtt106 promotes nucleosome formation using (H3-H4)2 tetramers
    Ahmed Fazly
    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA
    J Biol Chem 287:10753-60. 2012
    ..These results indicate that Rtt106 deposits H3-H4 heterotetramers onto DNA and provide the first description of a H3-H4 chaperone binding to (H3-H4)(2) heterotetramers in vivo...
  14. pmc Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106
    Dan Su
    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA
    Nature 483:104-7. 2012
    ..We show that the Rtt106-(H3-H4)(2) interaction is important for gene silencing and the DNA damage response...
  15. pmc Molecular basis for the association of microcephalin (MCPH1) protein with the cell division cycle protein 27 (Cdc27) subunit of the anaphase-promoting complex
    Namit Singh
    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA
    J Biol Chem 287:2854-62. 2012
    ..Collectively, our data define the biochemical, structural, and cellular determinants of the novel interaction between MCPH1 and Cdc27 and suggest that this interaction may occur within the larger context of MCPH1-APC/C...
  16. pmc Sgf29 binds histone H3K4me2/3 and is required for SAGA complex recruitment and histone H3 acetylation
    Chuanbing Bian
    School of Life Sciences, University of Science and Technology of China, Anhui, People s Republic of China
    EMBO J 30:2829-42. 2011
    ..Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation...
  17. pmc Structure and histone binding properties of the Vps75-Rtt109 chaperone-lysine acetyltransferase complex
    Dan Su
    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA
    J Biol Chem 286:15625-9. 2011
    ..These findings clarify the basis for the enhanced acetylation of histone H3 tail residues by Vps75-Rtt109...
  18. pmc Structural basis of ubiquitin recognition by translesion synthesis DNA polymerase ι
    Gaofeng Cui
    Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, United States
    Biochemistry 49:10198-207. 2010
    ..Instead, binding of UBM2 is centered on the recognition of Leu8 in ubiquitin, which is essential for the interaction...
  19. pmc Molecular basis for the association of human E4B U box ubiquitin ligase with E2-conjugating enzymes UbcH5c and Ubc4
    Robert C Benirschke
    Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA
    Structure 18:955-65. 2010
    ....
  20. pmc Mutations in DNMT1 cause hereditary sensory neuropathy with dementia and hearing loss
    Christopher J Klein
    Mayo Clinic, Department of Neurology, Division of Peripheral Nerve Diseases, Rochester, Minnesota, USA
    Nat Genet 43:595-600. 2011
    ..Our study shows that DNMT1 mutations cause the aberrant methylation implicated in complex pathogenesis. The discovered DNMT1 mutations provide a new framework for the study of neurodegenerative diseases...