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Cellular and Molecular Life Sciences

, Volume 68, Issue 22, pp 3741–3756 | Cite as

Different roles of the human Orc6 protein in the replication initiation process

  • Andreas W. Thomae
  • Jens Baltin
  • Dagmar Pich
  • Manuel J. Deutsch
  • Máté Ravasz
  • Krisztina Zeller
  • Manfred Gossen
  • Wolfgang Hammerschmidt
  • Aloys Schepers
Research Article

Abstract

In eukaryotes, binding of the six-subunit origin recognition complex (ORC) to DNA provides an interactive platform for the sequential assembly of pre-replicative complexes. This process licenses replication origins competent for the subsequent initiation step. Here, we analyze the contribution of human Orc6, the smallest subunit of ORC, to DNA binding and pre-replicative complex formation. We show that Orc6 not only interacts with Orc1–Orc5 but also with the initiation factor Cdc6. Biochemical and imaging experiments reveal that this interaction is required for licensing DNA replication competent. Furthermore, we demonstrate that Orc6 contributes to the interaction of ORC with the chaperone protein HMGA1a (high mobility group protein A1a). Binding of human ORC to replication origins is not specified at the level of DNA sequence and the functional organization of origins is poorly understood. We have identified HMGA1a as one factor that might direct ORC to AT-rich heterochromatic regions. The systematic analysis of the interaction between ORC and HMGA1a revealed that Orc6 interacts with the acidic C-terminus of HMGA1a and also with its AT-hooks. Both domains support autonomous replication if targeted to DNA templates. As such, Orc6 functions at different stages of the replication initiation process. Orc6 can interact with ORC chaperone proteins such as HMGA1a to facilitate chromatin binding of ORC and is also an essential factor for pre-RC formation.

Keywords

Origin recognition complex Orc6 Chromatin HMGA1a Replication initiation 

Abbreviations

BiFC

Bimolecular fluorescent complementation

ChIP

Chromatin immunoprecipitation

DS

Dyad symmetry element

EBV

Epstein–Barr virus

FR

Family of repeats

HMG

High mobility group

ORC

Origin recognition complex

pre-RC

Prereplicative complex

tetR

Tetracyclin repressor

Notes

Acknowledgments

We thank Christoph-Erik Mayer and Stefanie Fülöp for discussions and suggestions and J.F.X. Diffley for the protocol of the plasmid-binding system. We gratefully acknowledge R. Knippers for ideas and advice. This work was supported by the following institutional grants: Deutsche Forschungsgemeinschaft grants SFB646 (A.S.), SFB/TR05 (A.S.), SPP1230 (A.S. and W.H.), and NIH grant CA70723 (W.H.).

Supplementary material

18_2011_675_MOESM1_ESM.doc (2.9 mb)
Supplementary material 1 (DOC 3012 kb)

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Copyright information

© Springer Basel AG 2011

Authors and Affiliations

  • Andreas W. Thomae
    • 1
    • 2
  • Jens Baltin
    • 1
  • Dagmar Pich
    • 1
  • Manuel J. Deutsch
    • 1
  • Máté Ravasz
    • 1
  • Krisztina Zeller
    • 1
  • Manfred Gossen
    • 3
  • Wolfgang Hammerschmidt
    • 1
  • Aloys Schepers
    • 1
  1. 1.Department of Gene VectorsHelmholtz Zentrum München, German Research Center for Environmental HealthMunichGermany
  2. 2.Adolf-Butenandt-InstitutLudwig-Maximilians-UniversityMunichGermany
  3. 3.Berlin-Brandenburg Center for Regenerative Therapies, Charité, BCRTBerlinGermany

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