| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Papers In Press, published online ahead of print February 15, 2002
J. Biol. Chem, 10.1074/jbc.M107710200
Submitted on August 10, 2001
Revised on February 13, 2002
Accepted on February 15, 2002
Department of Molecular Biology and Genetics, Johns Hopkins University, Baltimore, MD 21205
Corresponding Author: tkelly{at}jhmi.edu
The origin recognition complex (ORC) plays a central role in the initiation of DNA replication in eukaryotic cells. It interacts with origins of DNA replication in chromosomal DNA and recruits additional replication proteins to form functional initiation complexes. These processes have not been well characterized at the biochemical level except in the case of Saccharomyces cerevisiae ORC. We report here the expression, purification, and initial characterization of Schizosaccharomyces pombe ORC (SpORC) containing six recombinant subunits. Purified SpORC binds efficiently to the ars1 origin of DNA replication via the essential N-terminal domain of the SpOrc4 subunit which contains nine AT-hook motifs. Competition-binding experiments demonstrated that SpORC binds preferentially to DNA molecules rich in AT-tracts, but does not otherwise exhibit a high degree of sequence specificity. The complex is capable of binding to multiple sites within the ars1 origin of DNA replication with similar affinities, indicating that the sequence requirements for origin recognition in S. pombe are significantly less stringent than in S. cerevisiae. We have also demonstrated that SpORC interacts directly with Cdc18p, an essential fission yeast initiation protein, and recruits it to the ars1 origin in vitro. Recruitment of Cdc18p to chromosomal origins is a likely early step in the initiation of DNA replication in vivo. These data indicate that the purified recombinant SpORC retains at least two of its primary biological functions and that it will be useful for the eventual reconstitution of the initiation reaction with purified proteins.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Marilley, P. Milani, J. Thimonier, J. Rocca-Serra, and G. Baldacci Atomic force microscopy of DNA in solution and DNA modelling show that structural properties specify the eukaryotic replication initiation site Nucleic Acids Res., November 29, 2007; 35(20): 6832 - 6845. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Siddiqui and B. Stillman ATP-dependent Assembly of the Human Origin Recognition Complex J. Biol. Chem., November 2, 2007; 282(44): 32370 - 32383. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Wang, S. E. Lindner, E. R. Leight, and B. Sugden Essential Elements of a Licensed, Mammalian Plasmid Origin of DNA Synthesis Mol. Cell. Biol., February 1, 2006; 26(3): 1124 - 1134. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Kusic, S. Kojic, A. Divac, and D. Stefanovic Noncanonical DNA Elements in the Lamin B2 Origin of DNA Replication J. Biol. Chem., March 18, 2005; 280(11): 9848 - 9854. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Dai, R.-Y. Chuang, and T. J. Kelly DNA replication origins in the Schizosaccharomyces pombe genome PNAS, January 11, 2005; 102(2): 337 - 342. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Giordano-Coltart, C. Y. Ying, J. Gautier, and J. Hurwitz Studies of the properties of human origin recognition complex and its Walker A motif mutants PNAS, January 4, 2005; 102(1): 69 - 74. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Gaczynska, P. A. Osmulski, Y. Jiang, J.-K. Lee, V. Bermudez, and J. Hurwitz Atomic force microscopic analysis of the binding of the Schizosaccharomyces pombe origin recognition complex and the spOrc4 protein with origin DNA PNAS, December 28, 2004; 101(52): 17952 - 17957. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. K. Chang, J. J. Donato, C. S. Chan, and B. K. Tye Mcm1 Promotes Replication Initiation by Binding Specific Elements at Replication Origins Mol. Cell. Biol., July 15, 2004; 24(14): 6514 - 6524. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Stefanovic, S. Stanojcic, A. Vindigni, A. Ochem, and A. Falaschi In Vitro Protein-DNA Interactions at the Human Lamin B2 Replication Origin J. Biol. Chem., October 31, 2003; 278(44): 42737 - 42743. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. J. Harvey and J. Newport CpG Methylation of DNA Restricts Prereplication Complex Assembly in Xenopus Egg Extracts Mol. Cell. Biol., October 1, 2003; 23(19): 6769 - 6779. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Vashee, C. Cvetic, W. Lu, P. Simancek, T. J. Kelly, and J. C. Walter Sequence-independent DNA binding and replication initiation by the human origin recognition complex Genes & Dev., August 1, 2003; 17(15): 1894 - 1908. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Ivessa and V. A. Zakian To fire or not to fire: origin activation in Saccharomyces cerevisiae ribosomal DNA Genes & Dev., October 1, 2002; 16(19): 2459 - 2464. [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
