Abstract

Polyubiquitin (polyUb) chains linked through lysine-48 (K48) of Ub represent the predominant targeting signal in the Ub-proteasome proteolytic pathway, whereas polyUb chains linked through K63 function in postreplicative DNA repair by a mechanism which does not appear to involve proteolysis. These observations suggest that the assembly of Ub into distinct types of polymers may be a mechanism to diversify the signaling functions of Ub. To test this hypothesis we will apply methods of biochemistry and yeast molecular genetics to characterize the assembly and recognition of K63-linked polyUb chains, and elucidate the signaling function of these polymers in DNA repair. Our discovery of yeast enzymes which assemble homopolymeric K63-linked polyUb chains in vitro, and function in DNA repair in vivo, provides the starting point for this work. These proteins are Ubcl3p (a Ub conjugating enzyme) and Mms2p (a Ub E2 variant or UEV protein). By characterizing the mechanism and function of the Mms2p/Ubc13p complex, we will explicate the process of signal generation; by identifying cellular proteins that are linked to K63-linked chains, we will gain insight into the functional consequences of signal recognition; finally, by using an in vitro-assembled chain as a tool, we will directly address the proteolytic signaling competence of K63-linked polyUb chains, and isolate chain-recognizing factors that may represent downstream elements in the proposed DNA repair signaling pathway. The results of these studies will provide new insights into the mechanistic role of Ub in DNA repair, and may ultimately lead to improved strategies for the treatment of cancer and other diseases. Moreover, if our results support the hypothesis that polyUb chains serve to diversify the signaling functions of Ub, it will help to explain how this small molecule is able to act as a distinct signal in multiple cellular processes. Finally, our results will provide the first biochemical insight into the functioning of UEV protein family, which in mammals includes the tumor suppressor Tsgl0l, and other proteins implicated in differention and cell transformation.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM060372-03
Application #
6498710
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Wolfe, Paul B
Project Start
2000-02-01
Project End
2004-01-31
Budget Start
2002-02-01
Budget End
2003-01-31
Support Year
3
Fiscal Year
2002
Total Cost
$277,648
Indirect Cost

  Institution

Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Public Health
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218

  Publications

Carlile, Candice M; Pickart, Cecile M; Matunis, Michael J et al. (2009) Synthesis of free and proliferating cell nuclear antigen-bound polyubiquitin chains by the RING E3 ubiquitin ligase Rad5. J Biol Chem 284:29326-34
Zhang, Daoning; Raasi, Shahri; Fushman, David (2008) Affinity makes the difference: nonselective interaction of the UBA domain of Ubiquilin-1 with monomeric ubiquitin and polyubiquitin chains. J Mol Biol 377:162-80
Eddins, Michael J; Carlile, Candice M; Gomez, Kamila M et al. (2006) Mms2-Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation. Nat Struct Mol Biol 13:915-20
Raasi, Shahri; Varadan, Ranjani; Fushman, David et al. (2005) Diverse polyubiquitin interaction properties of ubiquitin-associated domains. Nat Struct Mol Biol 12:708-14
Varadan, Ranjani; Assfalg, Michael; Raasi, Shahri et al. (2005) Structural determinants for selective recognition of a Lys48-linked polyubiquitin chain by a UBA domain. Mol Cell 18:687-98
Tsui, Colleen; Raguraj, Arani; Pickart, Cecile M (2005) Ubiquitin binding site of the ubiquitin E2 variant (UEV) protein Mms2 is required for DNA damage tolerance in the yeast RAD6 pathway. J Biol Chem 280:19829-35
Pickart, Cecile M (2004) Back to the future with ubiquitin. Cell 116:181-90
Pickart, Cecile M; Eddins, Michael J (2004) Ubiquitin: structures, functions, mechanisms. Biochim Biophys Acta 1695:55-72
Varadan, Ranjani; Assfalg, Michael; Haririnia, Aydin et al. (2004) Solution conformation of Lys63-linked di-ubiquitin chain provides clues to functional diversity of polyubiquitin signaling. J Biol Chem 279:7055-63
Wu, Pei-Ying; Hanlon, Mary; Eddins, Michael et al. (2003) A conserved catalytic residue in the ubiquitin-conjugating enzyme family. EMBO J 22:5241-50

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