Deletion analysis of minimal sequence requirements for autonomous replication of ors8, a monkey early-replicating DNA sequence.
We have generated a panel of deletion mutants of ors8 (483 bp), a mammalian autonomously replicating DNA sequence, previously isolated by extrusion of nascent monkey (CV-1) DNA from replication bubbles active at the onset of S phase. The deletion mutants were tested for replication function by the DpnI resistance assay, in vivo, after transfection into HeLa cells, and in vitro. An internal fragment of 186-bp that is required for autonomous replication function of ors8 was identified. This fragment, when subcloned into pBR322 and similarly tested, was capable of autonomous replication in vivo and in vitro. The 186-bp fragment contains several repeated sequence motifs, such as the ATTA and ATTTAT motifs, occurring three and five times, respectively, the sequences TAGG and TAGA, occurring three and seven times, respectively, two 5'-ATT-3' repeats, a 44-bp imperfect inverted repeat (IR) sequence, and an imperfect consensus binding element for the transcription factor Oct-1. A measurable sequence-directed DNA curvature was also detected, coinciding with the AT-rich regions of the 186-bp fragment.
Todd
A
A
McGill Cancer Centre, Department of Medicine, McGill University, Montreal, Quebec, Canada.
Landry
S
S
Pearson
C E
CE
Khoury
V
V
Zannis-Hadjopoulos
M
M
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D006367:HeLa Cells; D051863:Host Cell Factor C1; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D050811:Octamer Transcription Factor-1; D010957:Plasmids; D015183:Restriction Mapping; D016196:S Phase; D017384:Sequence Deletion; D013379:Substrate Specificity; D014157:Transcription Factors / Q000378:metabolism
D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; D014157:Transcription Factors; D004247:DNA; C020102:endodeoxyribonuclease DpnI; D015252:Deoxyribonucleases, Type II Site-Specific