Ulrike Wintersberger

Saccharomyces cerevisiae RNase H(35) Functions in RNA Primer Removal during Lagging-Strand DNA Synthesis, Most Efficiently in Cooperation with Rad27 Nuclease

ABSTRACT 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 ofS. 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.

Non‐homologous end joining as an important mutagenic process in cell cycle‐arrested cells

Resting cells experience mutations without apparent external mutagenic influences. Such DNA replication‐independent 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 replication‐independent 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‐deficient strains confirmed 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 G0 cells with unrepressed NHEJ capacity pay for a large‐scale chromosomal stability with an increased frequency of small‐scale mutations, a finding of potential relevance for carcinogenesis.

Ribonucleases H of retroviral and cellular origin.

Ribonucleases H (RNases H) are enzymes which catalyse the hydrolysis of the RNA-strand of an RNA-DNA hybrid. Retroviral reverse transcriptases possess RNase H activity in addition to their RNA- as well as DNA-dependent DNA-polymerizing activity. These enzymes transcribe the viral single stranded RNA-genome into double stranded DNA, which then can be handled by the host cell like one of its own genes. Various, sometimes highly repeated, sequences related to retroviruses and like these encompassing two separate domains, one of which potentially codes for a DNA polymerizing, the other for an RNase H activity, are found in genomes of uninfected cells. In addition proteins coded for by cellular genes (e.g. from E. coli and from yeast) are known, which exhibit RNase H activity, the biological function of which is not fully understood. In the light of these facts the question of whether retroviral RNases H could be promising targets for antiviral drugs is discussed.

Cooperation between the INO80 Complex and Histone Chaperones Determines Adaptation of Stress Gene Transcription in the Yeast Saccharomyces cerevisiae

ABSTRACT In yeast, environmental stresses provoke sudden and dramatic increases in gene expression at stress-inducible loci. Stress gene transcription is accompanied by the transient eviction of histones from the promoter and the transcribed regions of these genes. We found that mutants defective in subunits of the INO80 complex, as well as in several histone chaperone systems, exhibit extended expression windows that can be correlated with a distinct delay in histone redeposition during adaptation. Surprisingly, Ino80 became associated with the ORFs of stress genes in a stress-specific way, suggesting a direct function in the repression during adaptation. This recruitment required elongation by RNA polymerase (Pol) II but none of the histone modifications that are usually associated with active transcription, such as H3 K4/K36 methylation. A mutant lacking the Asf1-associated H3K56 acetyltransferase Rtt109 or Asf1 itself also showed enhanced stress-induced transcript levels. Genetic data, however, suggest that Asf1 and Rtt109 function in parallel with INO80 to restore histone homeostasis, whereas Spt6 seems to have a function that overlaps that of the chromatin remodeler. Thus, chromatin remodeling by INO80 in cooperation with Spt6 determines the shape of the expression profile under acute stress conditions, possibly by an elongation-dependent mechanism.

Yeast RNase H(35) is the counterpart of the mammalian RNase HI, and is evolutionarily related to prokaryotic RNase HII1

We cloned the Saccharomyces cerevisiae homologue of mammalian RNase HI, which itself is related to the prokaryotic RNase HII, an enzyme of unknown function and previously described as having minor activity in Escherichia coli. Expression of the corresponding yeast 35 kDa protein (named by us RNase H(35)) in E. coli and immunological analysis proves a close evolutionary relationship to mammalian RNase HI. Deletion of the gene (called RNH35) from the yeast genome leads to an about 75% decrease of RNase H activity in preparations from the mutated, still viable cells. Sequence comparison discriminates this new yeast RNase H from earlier described yeast enzymes, RNase H(70) and RNase HI.

The nuclear actin-related protein Act3p/Arp4p of Saccharomyces cerevisiae is involved in transcription regulation of stress genes

A mutational analysis of the essential nuclear actin‐related protein of Saccharomyces cerevisiae, Act3p/Arp4p, was performed. The five residues chosen for substitution were amino acids conserved between actin and Act3p/Arp4p, the tertiary structure of which most probably resembles that of actin. Two thermosensitive (ts) mutants, a single and a double point mutant, and one lethal double point mutant were obtained. Both ts mutants were formamide‐sensitive which supports a structural relatedness of Act3p/Arp4p to actin; they were also hypersensitive against hydroxyurea and ultraviolet irradiation pointing to a possible role of Act3p/Arp4p in DNA replication and repair. Their ‘suppressor of Ty’ (SPT) phenotype, ob‐served with another ts mutant of Act3p/Arp4p before, suggested involvement of Act3p/Arp4p in transcription regulation. Accordingly, genome‐wide expression profiling revealed misregulated transcription in a ts mutant of a number of genes, among which increased expression of various stress‐responsive genes (many of them requiring Msn2p/Msn4p for induction) was the most salient result. This provides an explanation for the mutants enhanced resistance to severe thermal and oxidative stress. Thus, Act3p/Arp4p takes an important part in the repression of stress‐induced genes under non‐stress conditions.

Rifamycin insensitivity of RNA synthesis in yeast

Rifamycin and its derivatives are strong inhibitors of RNA synthesis in bacteria but not in the nuclei of animal cells [l-3] . It has been shown that the antibiotic binds to the bacterial DNA-dependent RNA polymerase, thereby interfering with the process of chain intiation [4-61 . The sensitivity or insensitivity of RNA synthesis towards rifamycin istherefore a property of the respective enzymes. This distinguishes rifamycin from other antibiotic inhibitors of RNA synthesis, such as actinomycin, which are known to react with the DNA template rather than with the polymerase. In this communication we report studies on the effect of rifampicin, a highly potent rifamycin deriva.tive, on the synthesis of RNA catalyzed by cellular extracts as well as by a partially purified preparation of DNA-dependent RNA polymerase from the yeast, Saccharomyces cerevisiae. In addition, we have examined the influence of the antibiotic on the RNA synthesis in isolated and highly purified yeast mitochondria. Rifamycin was found to be without effect on these reactions, even at concentrations as high as 50 pg/ml.

The nuclear actin-related protein Act3p/Arp4p is involved in the dynamics of chromatin-modulating complexes

Chromatin remodelling and histone‐modifying complexes govern the modulation of chromatin structure. While components of these complexes are diverse, nuclear actin‐related proteins (Arps) have been repeatedly found in these complexes from yeast to mammals. In most cases, Arps are required for functioning of the complexes, but the molecular mechanisms of nuclear Arps have as yet been largely unknown. The Arps and actin, sharing a common ancestor, are supposed to be highly similar in the three‐dimensional structure of their core regions, including the ATP‐binding pocket. The Arp Act3p/Arp4p of Saccharomyces cerevisiae exists within the nucleus, partly as a component of several high molecular mass complexes, including the NuA4 histone acetyltransferase (HAT) complex, and partly as uncomplexed molecules. We observed that mutations in the putative ATP‐binding pocket of Act3p/Arp4p increased its concentration in the high molecular mass complexes and, conversely, that an excess of ATP or ATPγS led to the release of wild‐type Act3p/Arp4p from the complexes. These results suggest a requirement of ATP binding by Act3p/Arp4p for its dissociation from the complexes. In accordance, a mutation in the putative ATP binding site of Act3p/Arp4p inhibited the conversion of the NuA4 complex into the smaller piccoloNuA4, which does not contain Act3p/Arp4p and exhibits HAT activity distinct from that of NuA4. Although the in vitro binding activity of ATP by recombinant Act3p/Arp4p was found to be rather weak, our observations, taken together, suggest that the ATP‐binding pocket of Act3p/Arp4p is involved in the function of chromatin modulating complexes by regulating their dynamics. Copyright

Replication-dependent and selection-induced mutations in respiration-competent and respiration-deficient strains of Saccharomyces cerevisiae

Abstract Adaptive or selection-induced mutations are defined as mutations that occur in non-dividing cells as a response to prolonged non-lethal selective pressure such as starvation for an essential amino acid. In the absence of DNA replication, the processing of endogenous DNA lesions by repair enzymes probably acts as a source of mutations. We are studying selection-induced reversions of frameshift alleles in the eukaryote Saccharomyces cerevisiae. Here we show that respiration-deficient strains, totally devoid of mitochondrial DNA, yield selection-induced mutants at slightly elevated frequencies compared to isonucleic respiration-competent strains. Therefore factors of mitochondrial origin such as reactive oxygen species or hypothetical recombinogenic DNA fragments are unlikely to be mediators of selection-induced nuclear frameshift mutation in yeast. Furthermore we compared sequence spectra of reversions of the +1 hom3-10 frameshift allele and found a strong preference for −1 deletions in mononucleotide repeats in selection-induced and replication-dependent revertants, indicating slippage errors during DNA repair synthesis as well as during DNA replication. Remarkably, a higher degree of variation in the site of the reverting frameshift and accompanying base substitutions was found among selection-induced revertants.

DNA COVALENTLY LINKED TO CARBOXYMETHYL-CELLULOSE AND ITS APPLICATION IN AFFINITY CHROMATOGRAPHY

Affinity chromatography on columns containing DNA has been successfully used in the isolation and purification of DNA dependent polymerases and other DNA-binding proteins as well as for the purification of nucleic acids hybridizable to the column bound DNA [l] . Two drawbacks of DNA column materials used so far have limited their application: firstly, in the most widely used column materials DNA-cellulose [2] and DNA-agarose [3] the nucleic acid is not bound covalently, imposing a restriction of the ionic strength of the buffers used to avoid a loss of DNA; secondly, in those instances where DNA was bound covalently to the matrix, the amount of bound DNA was very low [4,5]. We have therefore been interested in developing a column material containing covalently linked DNA in amounts comparable to those in DNA-agarose. In this paper we describe the preparation of an affinity chromatography material containing up to 3.6 mg DNA per ml bedvolume and its successful application in the purification of DNA dependent DNA polymerases from yeast nuclei and mitochondria. While this work was in progress Arndt-Jovin et al. [6] reported an improved method for the preparation of agarose containing similarly high amounts of covalently attached DNA.