Franz Klein

Purification, sequence, and cellular localization of a novel chromosomal protein that binds to Methylated DNA

Methylation of mammalian DNA can lead to repression of transcription and alteration of chromatin structure. Recent evidence suggests that both effects are the result of an interaction between the methylated sites and methyl-CpG-binding proteins (MeCPs). MeCP1 has previously been detected in crude nuclear extracts. Here we report the identification, purification, and cDNA cloning of a novel MeCP called MeCP2. Unlike MeCP1, the new protein is able to bind to DNA that contains a single methyl-CpG pair. By staining with an antibody, we show that the distribution of MeCP2 along the chromosomes parallels that of methyl-CpG. In mouse, for example, MeCP2 is concentrated in pericentromeric heterochromatin, which contains a large fraction (about 40%) of all genomic 5-methylcytosine.

A central role for cohesins in sister chromatid cohesion, formation of axial elements, and recombination during yeast meiosis.

A multisubunit complex, called cohesin, containing Smc1p, Smc3p, Scc1p, and Scc3p, is required for sister chromatid cohesion in mitotic cells. We show here that Smc3p and a meiotic version of Scc1p called Rec8p are required for cohesion between sister chromatids, for formation of axial elements, for reciprocal recombination, and for preventing hyperresection of double-strand breaks during meiosis. Both Rec8p and Smc3p colocalize with chromosome cores independently of synapsis during prophase I and largely disappear from chromosome arms after pachytene but persist in the neighborhood of centromeres until the onset of anaphase II. The eukaryotic cells cohesion apparatus is required both for the repair of recombinogenic lesions and for chromosome segregation and therefore appears to lie at the heart of the meiotic process.

A screen for genes required for meiosis and spore formation based on whole-genome expression

BACKGROUND Meiosis is the process by which gametes are generated with half the ploidy of somatic cells. This reduction is achieved by three major differences in chromosome behavior during meiosis as compared to mitosis: the production of chiasmata by recombination, the protection of centromere-proximal sister chromatid cohesion, and the monoorientation of sister kinetochores during meiosis I. Mistakes in any of these processes lead to chromosome missegregation. RESULTS To identify genes involved in meiotic chromosome behavior in Saccharomyces cerevisiae, we deleted 301 open reading frames (ORFs) which are preferentially expressed in meiotic cells according to microarray gene expression data. To facilitate the detection of chromosome missegregation mutants, chromosome V of the parental strain was marked by GFP. Thirty-three ORFs were required for the formation of wild-type asci, eight of which were needed for proper chromosome segregation. One of these (MAM1) is essential for the monoorientation of sister kinetochores during meiosis I. Two genes (MND1 and MND2) are implicated in the recombination process and another two (SMA1 and SMA2) in prospore membrane formation. CONCLUSIONS Reverse genetics using gene expression data is an effective method for identifying new genes involved in specific cellular processes.

Meiotic chromosome synapsis in a haploid yeast.

An extensive synaptonemal complex (SC) is found at pachytene in whole mount spread preparations of a haploid yeast, Saccharomyces cerevisiae, strain. Whereas unsynapsed axial elements are present only in a few nuclei, in others non-homologous synapsis involves virtually the whole chromosome set. This suggests that homology is not an indispensable precondition for SC formation in yeast but that chromosomes engage in non-homologous synapsis if no homologous partner is available. Recent evidence that in the sporulation deficient yeast mutants rad50 and mer1 axial elements do form but remain unsynapsed in the majority of nuclei is discussed in the light of the above findings.

YDL142c encodes cardiolipin synthase (Cls1p) and is non-essential for aerobic growth of Saccharomyces cerevisiae

The unassigned open reading frame YDL142c was identified to code for cardiolipin synthase, Cls1p. A cls1 deletion strain is viable on glucose, galactose, ethanol, glycerol and lactate containing media, although the growth rate on non‐fermentable carbon sources is decreased. Mitochondria of the cls1 mutant are devoid of cardiolipin but accumulate the cardiolipin precursor phosphatidylglycerol when grown on non‐fermentable carbon sources. Specific activity of phosphatidylglycerolphosphate synthase in cls1 is reduced to 30–75% of the wild‐type level. Amounts of mitochondrial cytochromes and activity of cytochrome c oxidase, however, are not affected in the cls1 deletion strain. Collectively, these data indicate that cardiolipin is not essential for aerobic growth of Saccharomyces cerevisiae.

Transferring the concept of multinuclearity to ruthenium complexes for improvement of anticancer activity.

Multinuclear platinum anticancer complexes are a proven option to overcome resistance of established anticancer compounds. Transferring this concept to ruthenium complexes led to the synthesis of dinuclear Ru(II)-arene compounds containing a bis(pyridinone)alkane ligand linker. A pronounced influence of the spacer length on the in vitro anticancer activity was found, which is correlated to the lipophilicity of the complexes. IC(50) values in the same dimension as for established platinum drugs were found in human tumor cell lines. No cross-resistance to oxoplatin, a cisplatin prodrug, was observed for the most active complex in three resistant cell lines; in fact, a 10-fold reversal of sensitivity in two of the oxoplatin-resistant lines was found. (Bio)analytical characterization of the representative examples showed that the ruthenium complexes hydrolyze rapidly, forming predominantly diaqua species that exhibit affinity toward transferrin and DNA, indicating that both proteins and nucleobases are potential targets.

B-type cyclins CLB5 and CLB6 control the initiation of recombination and synaptonemal complex formation in yeast meiosis

BACKGROUND The life cycle of most eukaryotic organisms includes a meiotic phase, in which diploid parental cells produce haploid gametes. During meiosis a single round of DNA replication is followed by two rounds of chromosome segregation. In the first, or reductional, division (meiosis I), which is unique to meiotic cells, homologous chromosomes segregate from one another, whereas in the second, or equational, division (Meiosis II) sister centromeres disjoin. Meiotic DNA replication precedes the initiation of recombination by programmed Spo11-dependent DNA double-strand breaks. Recent reports that meiosis-specific cohesion is established during meiotic S phase and that the length of S phase is modified by recombination factors (Spo11 and Rec8) raise the possibility that replication plays a fundamental role in the recombination process. RESULTS To address how replication influences the initiation of recombination, we have used mutations in the B-type cyclin genes CLB5 and CLB6, which specifically prevent premeiotic replication in the yeast Saccharomyces cerevisiae. We find that clb5 and clb5 clb6 but not clb6 mutants are defective in DSB induction and prior associated changes in chromatin accessibility, heteroallelic recombination, and SC formation. The severity of these phenotypes in each mutant reflects the extent of replication impairment. CONCLUSIONS This assemblage of phenotypes reveals roles for CLB5 and CLB6 not only in DNA replication but also in other key events of meiotic prophase. Links between the function of CLB5 and CLB6 in activating meiotic DNA replication and their effects on subsequent events are discussed.

Genetic and morphological approaches for the analysis of meiotic chromosomes in yeast

Publisher Summary Meiosis is a specialized cell division in which the chromosome number is reduced to half, generating haploid gametes for sexual reproduction. Chromosome behavior during meiosis is complex, comprising a single round of replication followed by two successive rounds of segregation. The meiosis I division is unique in that homologous chromosomes disjoin from each other; at meiosis II, such as mitosis, sister chromatids segregate from one another. Although some details differ for different organisms, the fundamental process of meiotic chromosome segregation is conserved. Therefore, meiosis can be studied in the yeast Saccharomyces cerevisiae, taking advantage of its powerful genetics. During prophase of meiosis I, homologous chromosomes pair, synapse along the meiosis-specific organelle called “the synaptonemal complex (SC),” undergo recombination. These processes are essential for the correct alignment of the homologous chromosomes on the metaphase plate and subsequent segregation. Genetic, biochemical, and cytological analyses of meiosis in yeast led to the identification of key regulatory molecules and structural components of the SC and the meiotic recombination machinery.

A novel plant gene essential for meiosis is related to the human CtIP and the yeast COM1/SAE2 gene

Obligatory homologous recombination (HR) is required for chiasma formation and chromosome segregation in meiosis I. Meiotic HR is initiated by DNA double‐strand breaks (DSBs), generated by Spo11, a homologue of the archaebacterial topoisomerase subunit Top6A. In Saccharomyces cerevisiae, Rad50, Mre11 and Com1/Sae2 are essential to process an intermediate of the cleavage reaction consisting of Spo11 covalently linked to the 5′ termini of DNA. While Rad50 and Mre11 also confer genome stability to vegetative cells and are well conserved in evolution, Com1/Sae2 was believed to be fungal‐specific. Here, we identify COM1/SAE2 homologues in all eukaryotic kingdoms. Arabidopsis thaliana Com1/Sae2 mutants are sterile, accumulate AtSPO11‐1 during meiotic prophase and fail to form AtRAd51 foci despite the presence of unrepaired DSBs. Furthermore, DNA fragmentation in AtCom1 is suppressed by eliminating AtSPO11‐1. In addition, AtCOM1 is specifically required for mitomycin C resistance. Interestingly, we identified CtIP, an essential protein interacting with the DNA repair machinery, as the mammalian homologue of Com1/Sae2, with important implications for the molecular role of CtIP.

Mnd1 is required for meiotic interhomolog repair

BACKGROUND While double-strand break (DSB) repair is vital to the survival of cells during both meiosis and mitosis, the preferred mechanism of repair differs drastically between the two types of cell cycle. Thus, during meiosis, it is the homologous chromosome rather than the sister chromatid that is used as a repair template. RESULTS Cells attempting to undergo meiosis in the absence of Mnd1 arrest in prophase I due to the activation of the Mec1 DNA-damage checkpoint accumulating hyperresected DSBs and aberrant synapsis. Sporulation of mnd1Delta strains can be restored by deleting RED1 or HOP1, which permits repair of DSBs by using the sister chromatid as a repair template. Mnd1 localizes to chromatin as foci independently of DSB formation, axial element (AE) formation, and synaptonemal complex (SC) formation and does not colocalize with Rad51. Mnd1 does not preferentially associate with hotspots of recombination. CONCLUSIONS Our results suggest that Mnd1 acts specifically to promote DSB repair by using the homologous chromosome as a repair template. The presence of Rec8, Red1, or Hop1 renders Mnd1 indispensable for DNA repair, presumably through the establishment of interhomolog (IH) bias. Localization studies suggest that Mnd1 carries out this function without being specifically recruited to the sites of DNA repair. We propose a model in which Mnd1 facilitates chromatin accessibility, which is required to allow strand invasion in meiotic chromatin.