2009 Scientific Report

More documents

Recommendations

Info

Van Andel Research Institute | Scientific Report Figure 2 Figure 2. Functional regions of Dbf4 determined by deletion analysis. Dbf4 contains three regions called motifs N, M, and C that are conserved among Dbf4 orthologs. A BRCT-like domain spans motif N. Motif C encodes a single C 2 H 2 type Zn-finger. Finally, two regions spanning motif M and C interact with Cdc7 and activate its kinase activity. In Figure 2, we summarize our analysis of Dbf4 functional regions. The N-terminal third of Dbf4 is dispensable for DNA replication. However, the N-terminus encodes several functions that are important for Dbf4 function. There are two putative classic nuclear localization sequences (NLSs) at 55-61 and 251-257. Although the first sequence is dispensable, deletion of both sequences is lethal. Functionality is restored to a dbf4 mutant lacking the first 265 residues by addition of a heterologous NLS from the SV40 large T-antigen, suggesting that there are no NLS sequences in the Dbf4 C-terminus. We also identified a BRCT-like domain from residues 115-219 that is apparently conserved in all Dbf4 orthologs. BRCT domains are often present in DNA damage-responsive proteins and they interact with phosphorylated residues. Although Dbf4 mutants deleting this region are viable, they exhibit a slow S-phase and defects in response to DNA-damaging agents such as hydroxyurea, bleomycin, and methylmethane sulfonate. Whether the BRCT-like region governs a DNA repair function for Dbf4 is uncertain, because addition of an SV40 NLS to the dbf4-ND221 deletion mutant reverses most of these DNA replication and damage phenotypes. This suggests that the damage sensitivity is a secondary consequence of lowered nuclear localization and, therefore, compromised initiation activity. Consistent with this explanation, we found that many initiation mutants also exhibit secondary DNA damage sensitivities. Interestingly, deletion of the BRCT-like domain causes defects in late-origin activation, but early origins are activated normally. This raises the intriguing possibility that the BRCT-like domain targets the kinase to late replication origins. We also constructed a series of C-terminal deletion Dbf4 mutants; such mutants that remove a conserved Zn-finger motif are viable. This indicates that C-terminal residues are not essential for Dbf4 activity. However, deletion of C-terminal residues results in a markedly slower S-phase progression, temperature sensitivity, and DNA damage sensitivity. This suggests that the C-terminus is required to activate full Cdc7 kinase activity or to target it to important replication substrates. Using recombinant Cdc7 and Dbf4 proteins, we found that Dbf4 mutants lacking the C-terminus have a profound defect in Cdc7 kinase activation. The Zn-finger motif also interacts with Cdc7 via a two-hybrid assay, and this interaction depends on conserved residues in the Zn-finger. Lastly, there is a second Cdc7 binding site that overlaps motif M. We found that either Cdc7 binding region could be deleted individually and still allow Cdc7 binding, but deletion of both domains does not allow Cdc7 binding. We would like to identify proteins that interact with the Dbf4 N-terminus and determine the functional consequences of those interactions. Clearly the N-terminal third of Dbf4 is not required for DNA replication, but these residues are conserved in mouse and human cells and so must confer some critical function. Using a two-hybrid approach, we found that the Dbf4 N-terminus interacts with Polo kinase, a key regulator of mitotic progression. Detailed analysis of this interaction suggests that Dbf4 influences chromosome segregation, which represents a totally new activity for Cdc7-Dbf4 kinase. 66
VARI | 2009 External Collaborators Catherine Fox, University of Wisconsin–Madison Carol Newlon, University of Medicine and Dentistry of New Jersey, Newark Philippe Pasero, CNRS, Montpellier, France Alain Verreault, University of Montreal, Quebec, Canada Wolfgang Zachariae, Max Plank Institute, Dresden, Germany Recent Publications Harkins, V., Carrie Gabrielse, L. Haste, and M. Weinreich. In press. Budding yeast Dbf4 sequences required for Cdc7 kinase activation and identification of a functional relationship between the Dbf4 and Rev1 BRCT domains. Genetics. Miller, Charles T., Carrie Gabrielse, Ying-Chou Chen, and Michael Weinreich. 2009. Cdc7p-Dbf4p regulates mitotic exit by inhibiting polo kinase. PLoS Genetics 5(5): e1000498. Bonte, Dorine, Charlotta Lindvall, Hongyu Liu, Karl Dykema, Kyle Furge, and Michael Weinreich. 2008. Cdc7-Dbf4 kinase overexpression in multiple cancers and tumor cell lines is correlated with p53 inactivation. Neoplasia 10(9): 920–931. Chang, FuJung, James F. Theis, Jeremy Miller, Conrad A. Nieduszynski, Carol S. Newlon, and Michael Weinreich. 2008. Analysis of chromosome III replicators reveals an unusual structure for the ARS318 silencer origin and a conserved WTW sequence within the origin recognition complex binding site. Molecular and Cellular Biology 28(16): 5071–5081. Fox, Catherine A., and Michael Weinreich. 2008. Beyond heterochromatin: SIR2 inhibits the initiation of DNA replication. Cell Cycle 7(21): 3330–3334. From left: Savreux, Gabrielse, Chang, Miller, Chen, Weinreich 67
Page 1 and 2:
Van Andel Research Institute Scient
Page 3 and 4:
VARI | 2009 Van Andel Research Inst
Page 5 and 6:
VARI | 2009 Director’s Introducti
Page 7 and 8:
VARI | 2009 Director’s Introducti
Page 9 and 10:
VARI | 2009 We continued to receive
Page 11 and 12:
Van Andel Research Institute ® The
Page 13 and 14:
VARI | 2009 Laboratory Reports 7
Page 15 and 16:
VARI | 2009 Research Interests The
Page 17 and 18:
VARI | 2009 Brian Cao, M.D. Laborat
Page 19 and 20:
VARI | 2009 • Generation of monoc
Page 21 and 22: VARI | 2009 Research Interests Mass
Page 23 and 24: VARI | 2009 Nicholas S. Duesbery, P
Page 25 and 26: VARI | 2009 Recent Publications Alf
Page 27 and 28: VARI | 2009 Research Interests The
Page 29 and 30: VARI | 2009 Kyle A. Furge, Ph.D. La
Page 31 and 32: VARI | 2009 Recent Publications Eis
Page 33 and 34: VARI | 2009 Research Interests All
Page 35 and 36: VARI | 2009 We are pursuing the hyp
Page 37 and 38: VARI | 2009 Research Interests The
Page 39 and 40: VARI | 2009 External Collaborators
Page 41 and 42: VARI | 2009 Cindy K. Miranti, Ph.D.
Page 43 and 44: VARI | 2009 Project 2: Integrin and
Page 45 and 46: VARI | 2009 James H. Resau, Ph.D. D
Page 47 and 48: VARI | 2009 Mouse models of Parkins
Page 49 and 50: VARI | 2009 Pamela J. Swiatek, Ph.D
Page 51 and 52: VARI | 2009 Cytogenetics Our lab al
Page 53 and 54: VARI | 2009 Bin T. Teh, M.D., Ph.D.
Page 55 and 56: VARI | 2009 Targeted therapeutic st
Page 57 and 58: VARI | 2009 Steven J. Triezenberg,
Page 59 and 60: VARI | 2009 We took several approac
Page 61 and 62: VARI | 2009 George F. Vande Woude,
Page 63 and 64: VARI | 2009 We had previously gener
Page 65 and 66: VARI | 2009 Craig P. Webb, Ph.D. Pr
Page 67 and 68: VARI | 2009 Predictive therapeutics
Page 69 and 70: VARI | 2009 Retina whole mount Imag
Page 71: VARI | 2009 Research Interests We s
Page 75 and 76: VARI | 2009 Research Interests Our
Page 77 and 78: VARI | 2009 Recent Publications Li,
Page 79 and 80: VARI | 2009 Research Interests My m
Page 81 and 82: VARI | 2009 External Collaborators
Page 83 and 84: VARI | 2009 Daniel Nathans Memorial
Page 85 and 86: VARI | 2009 Postdoctoral Fellowship
Page 87 and 88: VARI | 2009 Grand Rapids Area Pre-C
Page 89 and 90: VARI | 2009 Summer interns 2008: 1.
Page 91 and 92: VARI | 2009 Han-Mo Koo Memorial Sem
Page 93 and 94: VARI | 2009 October Teresa K. Woodr
Page 95 and 96: VARI | 2009 Van Andel Research Inst
Page 97 and 98: VARI | 2009 Office of the Director
Page 99 and 100: VARI | 2009 Information Technology
Page 101 and 102: VARI | 2009 Van Andel Research Inst
Page 104: 333 Bostwick Avenue, N.E., Grand Ra
show all

2009 Scientific Report

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?