An origin of bidirectional DNA replication is located within a CpG island at the 3′ end of the chicken lysozyme gene

• Published in: Nucleic acids research 1999-08, Vol.27 (15), p.3009-3017
• Main Authors: Loc, Phi-van, Strätling, Wolf H.
• Format: Article
• Language: English
• Subjects: Animals; Cell Line; Cells, Cultured; Chickens - blood; Chickens - genetics; Chromatin - genetics; Chromatin - metabolism; CpG Islands - genetics; Deoxyribonuclease I - metabolism; DNA - biosynthesis; DNA - metabolism; DNA Methylation; DNA Replication - genetics; Erythrocytes - metabolism; Introns - genetics; Muramidase - genetics; Physical Chromosome Mapping; Polymerase Chain Reaction; Replication Origin - genetics
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/27.15.3009

We previously identified a broad initiation zone of DNA replication at the chicken lysozyme gene locus. However, the existence of a highly preferred origin of bidirectional replication (OBR), often found in initiation zones, remained elusive. In order to re-examine this issue we used a competitive PCR assay to determine the abundance of closely spaced genomic segments in a 1 kb size fraction of nascent DNA. A sharp peak of nascent strand abundance occurred at the 3′ end of the gene, where initiation events were 17 times more frequent than upstream of the gene. This primary initiation site, active in lysozyme expressing myelo-monocytic HD11 cells and non-expressing hepatic DU249 cells, was found to reside within an unusually located CpG island. While most CpG islands are found at the 5′ end of genes, the lysozyme gene island extends from the 3′ end of the second intron and includes ∼1.2 kb of 3′ flanking DNA. As diagnosed by methylation-sensitive restriction enzymes, the island is largely non-methylated in HD11 cells, DU249 cells and inactive chicken erythrocytes. Furthermore, a DNase I hypersensitive site (HS) that is composed of two subsites separated by ∼100 bp, was localised very close to the segment with the highest initiation activity. Our results suggest that the non-methylated CpG island and the HS provide an accessible chromatin structure for the lysozyme gene origin of replication.