CpG methylation of DNA restricts prereplication complex assembly in Xenopus egg extracts.
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 approximately 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.
Harvey
Kevin J
KJ
Division of Biology, University of California, San Diego, La Jolla, California 92093, USA.
Newport
John
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D019175:DNA Methylation; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D051738:Origin Recognition Complex; D011485:Protein Binding; D013997:Time Factors; D014981:Xenopus / Q000378:metabolism*
D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051738:Origin Recognition Complex; D000255:Adenosine Triphosphate