Discrete functional elements required for initiation activity of the Chinese hamster dihydrofolate reductase origin beta at ectopic chromosomal sites.
The Chinese hamster dihydrofolate reductase (DHFR) DNA replication initiation region, the 5.8 kb ori-beta, can function as a DNA replicator at random ectopic chromosomal sites in hamster cells. We report a detailed genetic analysis of the DiNucleotide Repeat (DNR) element, one of several sequence elements necessary for ectopic ori-beta activity. Deletions within ori-beta identified a 132 bp core region within the DNR element, consisting mainly of dinucleotide repeats, and a downstream region that are required for ori-beta initiation activity at non-specific ectopic sites in hamster cells. Replacement of the DNR element with Xenopus or mouse transcriptional elements from rDNA genes restored full levels of initiation activity, but replacement with a nucleosome positioning element or a viral intron sequence did not. The requirement for the DNR element and three other ori-beta sequence elements was conserved when ori-beta activity was tested at either random sites or at a single specific ectopic chromosomal site in human cells. These results confirm the importance of specific cis-acting elements in directing the initiation of DNA replication in mammalian cells, and provide new evidence that transcriptional elements can functionally substitute for one of these elements in ori-beta.
Gray
Steven J
SJ
Department of Biological Sciences, Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, TN 37235-1634, USA.
Liu
Guoqi
G
Altman
Amy L
AL
Small
Lawrence E
LE
Fanning
Ellen
E
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002877:Chromosomes, Human / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers / Q000235:genetics; D018900:Dinucleotide Repeats; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D018741:Replication Origin; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014981:Xenopus
D017931:DNA Primers; D014157:Transcription Factors; D013762:Tetrahydrofolate Dehydrogenase