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Molecular and Cellular Biology, October 2003, p. 6769-6779, Vol. 23, No. 19
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.19.6769-6779.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

CpG Methylation of DNA Restricts Prereplication Complex Assembly in Xenopus Egg Extracts

Kevin J. Harvey and John Newport*

Division of Biology, University of California, San Diego, La Jolla, California 92093

Received 17 April 2003/ Returned for modification 6 June 2003/ Accepted 30 June 2003

In a Xenopus egg replication system, the origin recognition complex (ORC) does not bind to CpG methylated DNA and DNA replication is inhibited. Insertion of low density CpG DNA of at least 1.2 kb into methylated plasmids rescues both replication and ORC binding. Using this pseudo-origin, we find that ORC binding is restricted to low-CpG-density DNA; however, MCM is loaded onto both weakly and highly methylated DNA and occupies at least ~2 kb of DNA. Replication initiates coincident with MCM, and even the most distally bound MCM is associated with sites of replication initiation. These results suggest that in metazoans MCM is loaded onto and initiates replication over a large region distant from ORC.

* Corresponding author. Mailing address: Division of Biology, University of California, San Diego, Bonner Hall 4202 (M/C 0349), 9500 Gilman Dr., La, Jolla, CA 92093. Phone: (858) 534-3423. Fax: (858) 822-3531. E-mail: jnewport{at}ucsd.edu.

Molecular and Cellular Biology, October 2003, p. 6769-6779, Vol. 23, No. 19
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.19.6769-6779.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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