Ku80 binds to human replication origins prior to the assembly of the ORC complex

Biochemistry. 2005 May 31;44(21):7885-96. doi: 10.1021/bi047327n.

Abstract

The Ku heterodimer, an abundant nuclear protein, binds DNA replication origins in a sequence-specific manner and promotes initiation. In this study, using HCT116 Ku80+/- haplo-insufficient and Orc2(delta/-) hypomorphic cells, the order of binding of Ku and the human origin recognition complex (HsORC) was determined. The nuclear expression of Ku80 was found to be decreased by 60% in Ku80+/- cells, while its general association with chromatin was decreased by 33%. Coimmunoprecipitation studies indicated that the Ku heterodimer associates specifically with the human HsOrc-2, -3, -4, and -6 subunits. Chromatin immunoprecipitation (ChIP) experiments, using cells synchronized to late G1, showed that the association of Ku80 with the lamin B2, beta-globin, and c-myc origins in vivo was decreased by 1.5-, 2.3-, and 2.5-fold, respectively, in Ku80+/- cells. The association of HsOrc-3, -4, and -6 was consistently decreased in all three origins examined in Ku80+/- cells, while that of HsOrc-2 showed no significant variation, indicating that the HsOrc-3, -4, and -6 subunits bind to the origins after Ku80. In Orc2(delta/-) cells, the association of HsOrc-2 with the lamin B2, beta-globin, and c-myc origins was decreased by 2.8-, 4.9-, and 2.8-fold, respectively, relative to wild-type HCT116 cells. Furthermore, nascent strand abundance at these three origins was decreased by 4.5-, 2.3-, and 2.6-fold in Orc2(delta/-) relative to HCT116 cells, respectively. Interestingly, the association of Ku80 with these origins was not affected in this hypomorphic cell line, indicating that Ku and HsOrc-2 bind to origins independently of each other.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Nuclear / biosynthesis
  • Antigens, Nuclear / genetics
  • Antigens, Nuclear / metabolism*
  • Cell Line, Tumor
  • Chromatin / metabolism
  • DNA Replication* / genetics
  • DNA-Binding Proteins / biosynthesis
  • DNA-Binding Proteins / deficiency
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Dimerization
  • Humans
  • Immunoprecipitation
  • Ku Autoantigen
  • Origin Recognition Complex
  • Protein Binding
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • Protein Processing, Post-Translational* / genetics
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Replication Origin*
  • Sequence Deletion

Substances

  • Antigens, Nuclear
  • Chromatin
  • DNA-Binding Proteins
  • ORC2 protein, human
  • Origin Recognition Complex
  • Protein Isoforms
  • Protein Subunits
  • Xrcc6 protein, human
  • Ku Autoantigen