Reconstitution and molecular analysis of the hRad9-hHus1-hRad1 (9-1-1) DNA damage responsive checkpoint complex

The Journal of Biological Chemistry
M A BurtelowL M Karnitz

Abstract

DNA damage activates cell cycle checkpoint signaling pathways that coordinate cell cycle arrest and DNA repair. Three of the proteins involved in checkpoint signaling, Rad1, Hus1, and Rad9, have been shown to interact by immunoprecipitation and yeast two-hybrid studies. However, it is not known how these proteins interact and assemble into a complex. In the present study we demonstrated that in human cells all the hRad9 and hHus1 and approximately one-half of the cellular pool of hRad1 interacted as a stable, biochemically discrete complex, with an apparent molecular mass of 160 kDa. This complex was reconstituted by co-expression of all three recombinant proteins in a heterologous system, and the reconstituted complex exhibited identical chromatographic behavior as the endogenous complex. Interaction studies using differentially tagged proteins demonstrated that the proteins did not self-multimerize. Rather, each protein had a binding site for the other two partners, with the N terminus of hRad9 interacting with hRad1, the N terminus of hRad1 interacting with hHus1, and the N terminus of hHus1 interacting with the C terminus of hRad9's predicted PCNA-like region. Collectively, these analyses suggest a model of how these three ...Continue Reading

References

Sep 1, 1995·Molecular and Cellular Biology·G CullmannB Stillman
Nov 26, 1996·Proceedings of the National Academy of Sciences of the United States of America·H B LiebermanS Davey
Feb 7, 1997·Cell·A G PaulovichL H Hartwell
Apr 4, 1998·Molecular Biology of the Cell·M DahlenP Sunnerhagen
Jul 11, 1998·The Journal of Biological Chemistry·A E ParkerW H Luyten
Jul 11, 1998·The Journal of Biological Chemistry·A E ParkerW H Luyten
Oct 2, 1998·The EMBO Journal·M P LongheseP Plevani
Oct 6, 1998·Annual Review of Biochemistry·S Waga, B Stillman
Dec 19, 1998·The EMBO Journal·R G MartinhoN J Bentley
Jan 16, 1999·Current Biology : CB·A BlasinaC H McGowan
Mar 31, 1999·Proceedings of the National Academy of Sciences of the United States of America·A L BrownJ H Chung
Apr 24, 1999·Biochimie·T Caspari, A M Carr
Feb 5, 2000·Current Biology : CB·C M GreenN F Lowndes
Feb 19, 2000·Current Opinion in Genetics & Development·N F Lowndes, J R Murguia
Jun 15, 2000·The Journal of Biological Chemistry·M A BurtelowL M Karnitz
Jul 8, 2000·The Journal of Biological Chemistry·M RauenL M Karnitz

❮ Previous
Next ❯

Citations

Nov 27, 2008·Cell Biochemistry and Biophysics·Magda Budzowska, Roland Kanaar
Dec 4, 2003·Mutation Research·Yves PommierKurt W Kohn
Nov 6, 2003·Toxicology·Markus ChristmannBernd Kaina
Nov 13, 2003·Genomics·Vanessa M DufaultLarry M Karnitz
Mar 23, 2005·Cell Research·Richard Y Zhao, Robert T Elder
Aug 9, 2002·Genes to Cells : Devoted to Molecular & Cellular Mechanisms·Yasushi ShiomiToshiki Tsurimoto
Dec 12, 2001·Molecular Biology of the Cell·R KaurT Enoch
Jul 12, 2011·Nucleic Acids Research·Darius Kazlauskas, Ceslovas Venclovas
Feb 5, 2002·Genes & Development·Eun-Jin Erica Hong, G Shirleen Roeder
Sep 19, 2002·Protein Science : a Publication of the Protein Society·Ceslovas VenclovasMichael P Thelen
Dec 15, 2004·Journal of Virology·Jaana JurvansuuPeter Beard
Jun 11, 2004·Annual Review of Biochemistry·Aziz SancarStuart Linn
Dec 22, 2009·BMC Molecular Biology·Caroline CanfieldArrigo De Benedetti
May 22, 2007·BMC Molecular Biology·Kazuhiro IshikawaKeiichi Ichimura
Jan 1, 2012·ISRN Molecular Biology·Arrigo De Benedetti
Nov 1, 2001·Proceedings of the National Academy of Sciences of the United States of America·S PostE Y Lee
Feb 13, 2003·Proceedings of the National Academy of Sciences of the United States of America·Vladimir P BermudezAziz Sancar
May 16, 2002·Proceedings of the National Academy of Sciences of the United States of America·Keziban Unsal-KaçmazAziz Sancar
Aug 12, 2003·Proceedings of the National Academy of Sciences of the United States of America·Shay Ben-AroyaMartin Kupiec
Oct 23, 2008·DNA Repair·Gulshan Sunavala-Dossabhoy, Arrigo De Benedetti
Sep 11, 2008·Biochemical and Biophysical Research Communications·Shuhong LuYulong Shen
May 12, 2007·Protein Expression and Purification·Vinay Kumar SinghZongchao Jia
Jul 29, 2004·DNA Repair·Edgardo R Parrilla-CastellarLarry Karnitz
Jul 4, 2006·Biochemical and Biophysical Research Communications·Patrick D BrandtRobert A Bambara
Nov 14, 2002·Annual Review of Genetics·Kara A NybergTed A Weinert

❮ Previous
Next ❯

Related Concepts

Related Feeds

ASBMB Publications

The American Society for Biochemistry and Molecular Biology (ASBMB) includes the Journal of Biological Chemistry, Molecular & Cellular Proteomics, and the Journal of Lipid Research. Discover the latest research from ASBMB here.

Cell Checkpoints & Regulators

Cell cycle checkpoints are a series of complex checkpoint mechanisms that detect DNA abnormalities and ensure that DNA replication and repair are complete before cell division. They are primarily regulated by cyclins, cyclin-dependent kinases, and the anaphase-promoting complex/cyclosome. Here is the latest research.