STRUCTURE-FUNCTION ANALYSIS OF THE SACCHAROMYCES-CEREVISIAE G(1) CYCLIN CLN2

We have generated 50 new alleles of the yeast CLN2 gene by using site- directed mutagenesis. With the recently obtained crystal structure of cyclin A as a guide, a peptide linker sequence was inserted at 13 site s within the cyclin box of Cln2 to determine if the architecture of Cl n2 is similar to that of cyclin A. Linkers inserted in what are predic ted to be helices 1, 2, 3, and 5 of the cyclin box resulted in nonfunc tional Cln2 molecules. Linkers inserted between these putative helix s ites and in the region believed to contain a fourth helix did not have significant effects upon Cln2 function. A series of deletions in the region between the third and fifth helices indicate that the putative fourth helix may lie at the C-terminal end of this region yet is not e ssential for function. Two residues that are predicted to form a burie d salt bridge important for interaction of two helices of the cyclin b ox were also mutated, and an additional set of 31 mutant alleles was g enerated by clustered-charge-to-alanine scanning mutagenesis. All of t he mutant CLN2 alleles made in this study were tested in a variety of genetic and functional assays previously demonstrated to differentiate specific cyclin functions. Some alleles demonstrated restricted patte rns of defects, suggesting that these mutations may interfere with spe cific aspects of Cln2 function.