No abstract

In Saccharomyces cerevisiae, DNA polymerase delta (POLIII), the product of the CDC2 (POL3) gene, possesses, in its N‐terminal half, the well conserved 3‐domain 3′ to 5′ exonuclease site. Strains selectively mutagenized in this site display a mutator phenotype detected as a drastically increased spontaneous forward mutation rate to canavanine resistance or as an elevated reversion rate to lysine prototrophy. Assays on a partially purified extract of the mutant giving the largest mutator effect indicate that the 3′ to 5′ exonuclease activity is reduced below the detection limit whereas the DNA polymerizing activity has wild‐type level. Therefore, our results provide experimental support for the hypothesis that the exonucleolytic proofreading activity associated with DNA polymerase delta resides on the DNA polymerase delta subunit and enhances the fidelity of DNA replication in yeast.

Proteins involved in mitochondrial splicing but encoded by nuclear genes have been characterized in Saccharomyces and Neurospora. The role in splicing of these proteins is largely unknown. Here we report that mutations in the nuclear gene MSS116 directly affect the splicing of several introns of the cytochrome b (cob) and cytochrome c oxidase subunit I (cox1) primary transcripts. This implies that the MSS116 protein (pMSS116) is an important component of the mitochondrial splicing machinery. The sequence of the cloned MSS116 gene shows that its protein product is homologous to the translation eIF-4A factor and the human nuclear protein p68. We show further that these proteins share several conserved amino-acid blocks with DNA helicases and related proteins. This suggests that pMSS116 has an RNA helicase activity. RNA helicases may be involved in many different processes including translation and splicing.

Induction of c-los by cyclic AMP in NIH-3T3 cells is distinct from induction by serum. Whereas induction by serum is mediated by the serum response element (SRE1, induction by cAMP does not require this element. In fact, no single sequence element in the c-fos promoter/enhancer is stringently required for the cAMP response. Rather, multiple sequence elements in the c-los promoter/enhancer can mediate induction by cAMP independently. These elements are: (1} the region from -72 to -54, which contains a binding site for a cellular activating transcription factor (ATF); (2) the region from -225 to -99; (3) the region from -303 to -281, which is homologous to the consensus binding site for the transcription factor AP1; and (4) the region from -317 to -298, which contains the SRE. These sequence elements convey cAMP inducibility when fused to the cAMP-inresponsive 'minimal' los promoter (-53 to + 42). In addition, the c-los regions from -700 to -63 and from -71 to -48 can confer cAMP inducibility to a heterologous promoter.

Saccharomyces cerevisiae cdc2 mutants arrest in the S‐phase of the cell cycle when grown at the non‐permissive temperature, implicating this gene product as essential for DNA synthesis. The CDC2 gene has been cloned from a yeast genomic library in vector YEp13 by complementation of a cdc2 mutation. An open reading frame coding for a 1093 amino acid long protein with a calculated mol. wt of 124,518 was determined from the sequence. This putative protein shows significant homology with a class of eukaryotic DNA polymerases exemplified by human DNA polymerase alpha and herpes simplex virus DNA polymerase. Fractionation of extracts from cdc2 strains showed that these mutants lacked both the polymerase and proofreading 3′‐5′ exonuclease activity of DNA polymerase III, the yeast analog of mammalian DNA polymerase delta. These studies indicate that DNA polymerase III is an essential component of the DNA replication machinery.