Correct removal of RNA primers of Okazaki fragments during lagging-strand DNA synthesis is a critical process for the maintenance of genome integrity. Disturbance of this process has severe mutagenic consequences and could contribute to the development of cancer. The role of the mammalian nucleases RNase HI and FEN-1 in RNA primer removal has been substantiated by several studies. Recently, RNase H(35), the Saccharomyces cerevisiae homologue of mammalian RNase HI, was identified and its possible role in DNA replication was proposed (P. Frank, C. Braunshofer-Reiter, and U. Wintersberger, FEBS Lett. 421:23-26, 1998). This led to the possibility of moving to the genetically powerful yeast system for studying the homologues of RNase HI and FEN-1, i.e., RNase H(35) and Rad27p, respectively. In this study, we have biochemically defined the substrate specificities and the cooperative as well as independent cleavage mechanisms of S. cerevisiae RNase H(35) and Rad27 nuclease by using Okazaki fragment model substrates. We have also determined the additive and compensatory pathological effects of gene deletion and overexpression of these two enzymes. Furthermore, the mutagenic consequences of the nuclease deficiencies have been analyzed. Based on our findings, we suggest that three alternative RNA primer removal pathways of different efficiencies involve RNase H(35) and Rad27 nucleases in yeast.Replication of double-stranded DNA is an asymmetric process. While leading-strand synthesis proceeds continuously, lagging-strand synthesis takes place by synthesis, processing, and ligation of Okazaki fragments (39). These fragments, measuring about 200 nucleotides (nt) in eukaryotes, are primed by DNA polymerase alpha/primase with a short oligoribonucleotide of 7 to 14 residues. Before the nascent Okazaki fragments are ligated to form a continuous lagging strand, the short RNA primers must be removed by an enzyme exhibiting RNase H activity. Although several such enzymes from eukaryotes are known, the process of RNA primer hydrolysis is as yet not fully understood.An RNase H was first detected in calf thymus extracts (57). Subsequently, RNase H enzymatic activity was detected in all prokaryotes and eukaryotes examined as well as in a bacteriophage and in retroviruses as a part of reverse transcriptases (for reviews, see references 12 and 66). Generally, RNases H are defined as ribonucleotide-specific endonucleases, cleaving the RNA portion of RNA-DNA/DNA or RNA/DNA duplexes. Several RNases H implicated in RNA primer removal have been purified and/or cloned from diverse organisms ranging from bacteriophages to human cells (see, e.g., references 8, 9, 13, 14, 24, 26, 37, 51, and 52). Nevertheless, conclusive evidence of the involvement of these enzymes in primer removal is still lacking. In the budding yeast, Saccharomyces cerevisiae, three different RNases H were identified and partially characterized as RNase H(70), RNase H1, and RNase H(35) (17,32,34). These enzymes are evolutionarily related to prokaryotic as well as to mammalia...

We have cloned a new gene, SCP160, from Saccharomyces cerevisiae, the deduced amino acid sequence of which does not exhibit overall similarity to any known yeast protein. A weak resemblance between the C-terminal part of the Scp160 protein and regulatory subunits of cAMP-dependent protein kinases from eukaryotes as well as the pstB protein of Escherichia coli was observed. The SCP160 gene resides on the left arm of chromosome X and codes for a polypeptide of molecular weight around 160 kDa. By immunofluorescence microscopy the Scp160 protein appears to be localized to the nuclear envelope and to the endoplasmic reticulum (ER). However, no signal sequence or membrane-spanning region exists, suggesting that the Scp160 protein is attached to the cytoplasmic surface of the ER-nuclear envelope membranes. Disruption of the SCP160 gene is not lethal but results in cells of decreased viability, abnormal morphology and increased DNA content. This phenotype is not reversible by transformation with a plasmid carrying the wild-type gene. Crosses of SCP160 deletion mutant strains among each other or with unrelated strains lead to irregular segregation of genetic markers. Taken together the data suggest that the Scp160 protein is required during cell division for faithful partitioning of the ER-nuclear envelope membranes which in S. cerevisiae enclose the duplicated chromosomes.

Resting cells experience mutations without apparent external mutagenic in¯uences. Such DNA replicationindependent mutations are suspected to be a consequence of processing of spontaneous DNA lesions. Using experimental systems based on reversions of frameshift alleles in Saccharomyces cerevisiae, we evaluated the impact of defects in DNA double-strand break (DSB) repair on the frequency of replicationindependent mutations. The deletion of the genes coding for Ku70 or DNA ligase IV, which are both obligatory constituents of the non-homologous end joining (NHEJ) pathway, each resulted in a 50% reduction of replication-independent mutation frequency in haploid cells. Sequencing indicated that typical NHEJ-dependent reversion events are small deletions within mononucleotide repeats, with a remarkable resemblance to DNA polymerase slippage errors. Experiments with diploid and RAD52-or RAD54-de®cient strains con®rmed that among DSB repair pathways only NHEJ accounts for a considerable fraction of replication-independent frameshift mutations in haploid and diploid NHEJ non-repressed cells. Thus our results provide evidence that G 0 cells with unrepressed NHEJ capacity pay for a large-scale chromosomal stability with an increased frequency of small-scale mutations, a ®nding of potential relevance for carcinogenesis.

Act3p/Arp4, an essential actin-related protein of Saccharomyces cerevisiae located within the nucleus, is, according to genetic data, involved in transcriptional regulation. In addition to the basal core structure of the actin family members, which is responsible for ATPase activity, Act3p possesses two insertions, insertions I and II, the latter of which is predicted to form a loop-like structure protruding from beyond the surface of the molecule. Because Act3p is a constituent of chromatin but itself does not bind to DNA, we hypothesized that insertion II might be responsible for an Act3p-specific function through its interaction with some other chromatin protein. Far Western blot and two-hybrid analyses revealed the ability of insertion II to bind to each of the core histones, although with somewhat different affinities. Together with our finding of coimmunoprecipitation of Act3p with histone H2A, this suggests the in vivo existence of a protein complex required for correct expression of particular genes. We also show that a conditional act3 mutation affects chromatin structure of an episomal DNA molecule, indicating that the putative Act3p complex may be involved in the establishment, remodeling, or maintenance of chromatin structures.

Actin filaments provide the internal scaffold of eukaryotic cells; they are involved in maintenance of cell shape, cytokinesis, organelle movement, and cell motility. The major component of these filaments, actin, is one of the most well-conserved eukaryotic proteins. Recently genes more distantly related to the conventional actins were cloned from several organisms. In the budding yeast, Saccharomyces cerevisiae, one conventional actin gene, ACT) (coding for the filament actin), and a so-caled actin-like gene, ACT2 (of unknown function), have so far been identified. We report here the discovery of a third member of the actin gene family from this organism, which we named ACT3. The latter gene is essential for viability and codes for a putative polypeptide, Act3, of 489 amino acids (Mr = 54,831). The deduced amino acid sequence of Act3 is less related to conventional actins than is the deduced amino acid sequence of Act2, mainly because of three unique hydrophobic segments. These segments are found inserted into a part of the sequence corresponding to a surface loop of the known three-dimensional structure of the actin molecule. According to sequence comparison, the basal core structure of conventional actin may well be conserved in Act3.Our findings demonstrate that, unexpectedly, there exist three members of the diverse actin protein family in buddin yeast that obviously provide different essential functions for survival.Actin, one of the most highly conserved eukaryotic proteins, is the major component of thin filaments, which provide the internal scaffold of eukaryotic cells. The actin filaments participate in vital cellular functions like cytokinesis, maintenance of cell shape, cell locomotion, and organelle transport (1, 2). Performance and regulation of these actin functions involve a number of actin-binding proteins (3-6). Actin proteins isolated from a broad range of phyla are about 90% identical to each other (7). Recently, however, the isolation of three genes coding for proteins more distantly related to "conventional" actins was reported: Saccharomyces cerevisiae ACT2 (8), Schizosaccharomyces pombe act2 (9), and a gene coding for vertebrate centractin/actin-RPV (10, 11) are, respectively, 47%, 35-40%, and 54% identical to conventional actins (reviewed in ref. 7). Furthermore it was shown that the functionally diverse protein molecules, the actins, the hsp70 heat shock proteins, and the sugar kinases, all of which bind and hydrolyze ATP, as well as several prokaryotic cell cycle proteins, have similar three-dimensional structures (12, 13), thus forming a large superfamily to which two phosphatases were recently added (14).Here we report the detection of a previously unknown essential gene from S. cerevisiae that codes for another actin-related protein and that we, therefore, call ACT3.1 It represents the most distantly related member of the actin family, indicating that this protein family is more divergent than previously thought. Furthermore, we show that, most probably, the actin-related p...