Sequence-independent DNA binding and replication initiation by the human origin recognition complex

Sanjay Vashee; Christin Cvetic; Wenyan Lu; Pamela Simancek; Thomas J. Kelly; Johannes C. Walter

doi:10.1101/gad.1084203

Sequence-independent DNA binding and replication initiation by the human origin recognition complex

¹ Institute for Biological Energy Alternatives, Rockville, Maryland 20850, USA
² Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
³ Laboratory of Regulation of DNA Replication, Program of Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA

Abstract

We report that a highly purified human origin recognition complex (HsORC) has intrinsic DNA-binding activity, and that this activity is modestly stimulated by ATP. HsORC binds preferentially to synthetic AT-rich polydeoxynucleotides, but does not effectively discriminate between natural DNA fragments that contain known human origins and control fragments. The complex fully restores DNA replication to ORC-depleted Xenopus egg extracts, providing strong evidence for its initiator function. Strikingly, HsORC stimulates initiation from any DNA sequence, and it does not preferentially replicate DNA containing human origin sequences. These data provide a biochemical explanation for the observation that in metazoans, initiation of DNA replication often occurs in a seemingly random pattern, and they have important implications for the nature of human origins of DNA replication.

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Footnotes

Corresponding authors.
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1084203.
↵4 These authors contributed equally to this work.
↵5 E-MAIL johannes_walter{at}hms.harvard.edu; FAX (617) 738-0516.
↵6 E-MAIL tkelly{at}mskcc.org; FAX (646) 422-2189.
- Accepted May 29, 2003.
- Received February 12, 2003.
Cold Spring Harbor Laboratory Press