Stimulation of SV40 DNA replication and transcription by Alu family sequence.
The sequence motif GGAGGC (Alu core) is present in the Alu family repeats, where it is required for RNA polymerase III promoter function. This motif is also found in the SV40 origin (ori) of replication. Here, an oligonucleotide containing the Alu sequence was inserted into pSV2CAT, a plasmid composed of the SV40 enhancer/promoter/ori linked to the bacterial chloramphenicol acetyltransferase gene (CAT), to see the effect of the Alu sequence on SV40 DNA replication and transcription. Results of transfection experiments in human HeLa cells showed that the Alu sequence stimulated sequence-specifically replication and transcription in the SV40 system. Stimulation effects on DNA replication were observed when the Alu sequence was placed upstream of enhancer/promoter/ori in either orientation, while effects on transcription were detected only when it was inserted in the normal orientation. These effects correlate with sequence-specific binding of two proteins (40 kDa and 120 kDa) to this motif. In fact, binding was abolished by a mutation in the cognate sequence that disrupted stimulation of replication and transcription. Both proteins bind duplex DNA, while the 40 kDa one also binds the minus strand with high affinity.
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Y
Y
Saëgusa
M
M
Sato
I
I
Galli
T
T
Nakagawa
N
N
Ono
S M
SM
Iguchi-Ariga
H
H
Ariga
D001483:Base Sequence; D002352:Carrier Proteins / Q000032:analysis; D004252:DNA Mutational Analysis; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D012091:Repetitive Sequences, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics*; D014158:Transcription, Genetic
D002352:Carrier Proteins; D004279:DNA, Viral