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 ac tivity distinguishable from the rest. The DNA synthesis was dependent on negative supertwisting and initiated at a unique region in the huma n DNA insert. Functional subclone DNA fragments which could be shorten ed to less than 1 kb are located in the initiation region. Binding wit h 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 se quences coincide with the single-stranded region and contain a charact eristic dinucleotide repeat sequence. These repeats have an extremely low free energy for DNA strand separation and are defined as DNA-unwin ding elements, which are frequently observed at regions flanking repli cation origins in Escherichia coli and Saccharomyces cerevisiae chromo somes. We suggest that such a repeating sequence would have an importa nt role during initiation of DNA replication and function as a site to recruit replication proteins.