discovered that a minimal 11-base-pair sequence is essential for origin activity. Known as the "A" element, which appears common to all origins from Saccharomyces, the 11-base-pair essential element is always accompanied by "B" elements that stimulate origin activity. Last year, after an intense search by many labs for this protein, Stillman and Bell, also of the Cold Spring Harbor Laboratory, announced discovery of a multiprotein complex in yeast that could act as an initiator by binding the essential 11-base-pair element of the ARs.

The search required arduous testing of many proteins present in yeast cell extracts. What Stillman calls "molecular shrapnel" was shot at the yeast DNA, splitting it into random pieces. However, if the initiator protein is bound over the origin, it protects the DNA from being cut. With this information, a "footprint" of the initiator protein binding to a particular set of DNA sequences was pieced together, and, using special biochemical techniques, the protein was extracted and purified from the complex. This protein complex, it was found, bound to all of the yeast's replication origins. Moreover, although this initiation protein complex binds strongly to natural, wild-type ARs sequences, it does not bind to inactive DNA elements whose ARs have been tinkered with, and destroyed, by mutations.

Like the initiator protein T antigen, but unlike most other DNA binding proteins of viruses, this newly discovered protein complex needs the addition of energy from the compound adenosine triphosphate (ATP) to work properly. In yeast the initiator protein appears to bind the DNA by wrapping the DNA around itself "and is reminiscent of the pattern observed when the E. coli initiation protein dna A binds to its cognate origin," says Stillman. But the development of new reagents, such as antibodies against particular protein subunits and cloning of the genes encoding the multiple protein subunits, "will help us understand its many roles in initiating DNA replication," Stillman adds.

IMPLICATIONS FOR THE FUTURE

Discovery of a yeast initiator protein complex is significant for many reasons, as has been recognized by the editors of several leading scientific publications, who rushed commentaries on this finding into print. First, the presence of at least six protein subunits suggests that the triggering of DNA replication may be controlled in a complicated way. In fact, Stillman has noted that there is precedent for the use of multiple proteins to recognize an origin of DNA replication. In 1991 it was discovered by Michael Botchan and his colleagues at the University of



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