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.

Normal terC-region of the Bacillus subtilis chromosome acts in a polar manner to arrest the clockwise replication fork.

A procedure is described for relocating a functional terC-region to various sites on the Bacillus subtilis chromosome, and in alternative orientations. The relocated terC-region comprised the IRR-rtp portion of the chromosome contained within a 1.75 x 10(3) base-pair segment of DNA. This segment was first cloned into the Tn 917 vector pTV20 in both orientations, and the two new plasmids used for inserting the terC-region into chromosomal copies of Tn 917. When relocated to the pyr and metD loci (139 degrees and 100 degrees positions on the 360 degrees map) it was found that clockwise replication fork arrest occurred only when the IRR-rtp (or terC-) region was oriented, in relation to the direction of approach of the fork, in the same way as in the wild-type strain. Thus, the complete IRR when located in the chromosome, and apparently made up of opposing terminators which might enable it to function in both orientations, is polar in its action. Of the two inverted repeats present in the IRR, it appears that IRI is functional in the chromosome, but not IRII.