Active mammalian replication origins are associated with a high-density cluster of mCpG dinucleotides.
ori-beta is a well-characterized origin of bidirectional replication (OBR) located approximately 17 kb downstream of the dihydrofolate reductase gene in hamster cell chromosomes. The approximately 2-kb region of ori-beta that exhibits greatest replication initiation activity also contains 12 potential methylation sites in the form of CpG dinucleotides. To ascertain whether DNA methylation might play a role at mammalian replication origins, the methylation status of these sites was examined with bisulfite to chemically distinguish cytosine (C) from 5-methylcytosine (mC). All of the CpGs were methylated, and nine of them were located within 356 bp flanking the minimal OBR, creating a high-density cluster of mCpGs that was approximately 10 times greater than average for human DNA. However, the previously reported densely methylated island in which all cytosines were methylated regardless of their dinucleotide composition was not detected and appeared to be an experimental artifact. A second OBR, located at the 5' end of the RPS14 gene, exhibited a strikingly similar methylation pattern, and the organization of CpG dinucleotides at other mammalian origins revealed the potential for high-density CpG methylation. Moreover, analysis of bromodeoxyuridine-labeled nascent DNA confirmed that active replication origins were methylated. These results suggest that a high-density cluster of mCpG dinucleotides may play a role in either the establishment or the regulation of mammalian replication origins.
Rein
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National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-2753, USA. theorein@box-t.nih.gov
Zorbas
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H
DePamphilis
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ML
D044503:5-Methylcytosine; D000818:Animals; D016466:CHO Cells; D018899:CpG Islands; D006224:Cricetinae; D003596:Cytosine / Q000031:analogs & derivatives / Q000032:analysis; D004247:DNA / Q000737:chemistry; D019175:DNA Methylation; D015226:Dinucleoside Phosphates / Q000032:analysis* / Q000378:metabolism; D008969:Molecular Sequence Data; D018741:Replication Origin / Q000235:genetics*; D012269:Ribosomal Proteins / Q000235:genetics; D013447:Sulfites
D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; D015226:Dinucleoside Phosphates; D012269:Ribosomal Proteins; D013447:Sulfites; C039747:ribosomal protein S14; C015772:CpG dinucleotide; C042345:hydrogen sulfite