An initiation zone of chromosomal DNA replication located upstream of the c-myc gene in proliferating HeLa cells.

AUTOR(ES)

Vassilev, L

RESUMO

Studies on origins of DNA replication in mammalian cells have long been hampered by a lack of methods sensitive enough for the localization of such origins in chromosomal DNA. We have employed a new method for mapping origins, based on polymerase chain reaction amplification of nascent strand segments, to examine replication initiated in vivo near the c-myc gene in human cells. Nascent DNA, pulse-labeled in unsynchronized HeLa cells, was size fractionated and purified by immunoprecipitation with anti-bromodeoxyuridine antibodies. Lengths of the nascent strands that allow polymerase chain reaction amplification were determined by hybridization to probes homologous to amplified segments and used to calculate the position of the origin. We found that DNA replication through the c-myc gene initiates in a zone centered approximately 1.5 kilobases upstream of exon I. Replication proceeds bidirectionally from the origin, as indicated by comparison of hybridization patterns for three amplified segments. The initiation zone includes segments of the c-myc locus previously reported to drive autonomous replication of plasmids in human cells.

Documentos Relacionados

• DNA replication initiates non-randomly at multiple sites near the c-myc gene in HeLa cells.
• Evidence that a triplex-forming oligodeoxyribonucleotide binds to the c-myc promoter in HeLa cells, thereby reducing c-myc mRNA levels.
• An initiation site of DNA replication with transcriptional enhancer activity present upstream of the c-myc gene.
• Opposite replication polarity of the germ line c-myc gene in HeLa cells compared with that of two Burkitt lymphoma cell lines.
• DNA replication origin and transcriptional enhancer in c-myc gene share the c-myc protein binding sequences.