Isolation of human DNA-unwinding elements as sites of DNA polymerase alpha/primase entry.
Human DNA libraries were screened for DNA synthesis activity in vitro using purified DNA polymerase alpha/primase and a viral DNA helicase (simian virus 40 large tumor antigen). Three clones exhibited a high activity distinguishable from the rest. The DNA synthesis was dependent on negative supertwisting and initiated at a unique region in the human DNA insert. Functional subclone DNA fragments which could be shortened to less than 1 kb are located in the initiation region. Binding with a single-stranded DNA-binding protein and digestion with nuclease P1 demonstrated that these DNAs have a highly single-stranded nature at a certain site in a closed circular plasmid. The minimal functional sequences coincide with the single-stranded region and contain a characteristic dinucleotide repeat sequence. These repeats have an extremely low free energy for DNA strand separation and are defined as DNA-unwinding elements, which are frequently observed at regions flanking replication origins in Escherichia coli and Saccharomyces cerevisiae chromosomes. We suggest that such a repeating sequence would have an important role during initiation of DNA replication and function as a site to recruit replication proteins.
Institute for Molecular and Cellular Biology, Osaka University, Japan.
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D001483:Base Sequence; D004265:DNA Helicases / Q000302:isolation & purification* / Q000378:metabolism; D019915:DNA Primase; D004261:DNA Replication; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid
D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004265:DNA Helicases