In Schizosaccharomyces pombe, the RNA interference (RNAi) machinery converts pericentromeric transcripts into small interfering RNAs (siRNAs) and is required for assembly of heterochromatin. Here we describe a mutation in second largest subunit polymerase II (RNAPII). Both wild-type mutant RNAPII localized to pericentromere. However, resulted loss heterochromatic histone modifications accumulation transcripts, accompanied by siRNAs. This phenotype resembles mutants RNAi suggests that couples...

We have previously identified a protein factor, PEBP2 (polyomavirus enhancer-binding protein), in the nuclear extract from mouse NIH 3T3 cells which binds to sequence motif, PEA2, located within polyomavirus enhancer A element. Upon cellular transformation with activated oncogene c-Ha-ras, this factor frequently undergoes drastic molecular modifications into an altered form having considerably reduced size. In study, form, PEBP3, was purified near homogeneity. The PEBP3 comprised two sets of...

Unlike ubiquitin, the ubiquitin-like protein modifier SUMO-1 and its budding yeast homologue Smt3p have been shown to be more important for posttranslational modification than degradation. Here we describe identification of fission yeast, which show required a number nuclear events including control telomere length chromosome segregation. A disruption pmt3(+) gene, Schizosaccharomyces pombe SMT3, was not lethal, but mutant cells carrying disrupted gene grew slowly. The pmt3Delta showed...

Certain noncoding RNAs (ncRNAs) implicated in the regulation of chromatin structure associate with chromatin. During formation RNAi‐directed heterochromatin fission yeast, ncRNAs transcribed from are thought to recruit RNAi machinery for heterochromatin; however, molecular details this association not clear. Here, using RNA immunoprecipitation assay, we showed that heterochromatic ncRNA was associated via a DNA–RNA hybrid and bound RNA‐induced transcriptional silencing (RITS) complex. The...

Abstract The heterochromatin protein 1 (HP1) family recognizes lysine 9‐methylated histone H3 (H3K9me) and recruits other transacting factors to establish higher order chromatin structures. In the fission yeast Schizosaccharomyces pombe ( S. ), two HP1 proteins, Swi6 Chp2, play distinct roles in recruiting factors: primarily Epe1, a Jumonji C domain‐containing involved H3K9 demethylation, whereas Chp2 Mit1, component of Snf2/Hdac Repressive Complex. However, detailed mechanisms how multiple...

Heterochromatin is a functionally important chromosomal component, especially at centromeres. In fission yeast, conserved heterochromatin-specific modifications of the histone H3 tail, involving deacetylation Lys 9 and 14 subsequent methylation 9, promote recruitment heterochromatin protein, Swi6, homolog Drosophila protein 1. However, primary determinants positioning are still unclear. The yeast proteins Abp1, Cbh1, Cbh2 homologs human CENP-B that bind to centromeric α-satellite DNA...

Heterochromatin protein 1 (HP1) recruits various effectors to heterochromatin for multiple functions, but its regulation is unclear. In fission yeast, a HP1 homolog Swi6 SHREC, Epe1, and cohesin, which are involved in transcriptional gene silencing (TGS), activation, sister chromatid cohesion, respectively. We found that casein kinase II (CK2) phosphorylated Swi6. Loss of CK2-dependent phosphorylation alleviated heterochromatic TGS without affecting structure. This was due the inhibited...

Abstract The tail of histone H3 is an ideal medium for storing epigenetic information because displacement heavily restricted during transcription. To maintain the locus-specific modifications H3, molecules should be retained locally at original position through multiple rounds Here, we found that fission yeast Spt6, a highly conserved RNA polymerase II-interacting H3–H4 chaperone, essential maintenance Lys-4 and Lys-9 methylation in euchromatin heterochromatin, respectively. In euchromatin,...

Inner nuclear membrane proteins interact with chromosomes in the nucleus and are important for chromosome activity. Lem2 Man1 conserved members of LEM-domain protein family. Mutations associated laminopathy, but their cellular functions remain unclear. Here, we report that maintains genome stability fission yeast Schizosaccharomyces pombe. S. pombe cells disrupted lem2(+) gene (lem2∆) showed slow growth increased rate minichromosome loss. These phenotypes were prominent rich culture medium,...

In budding yeast, Set2 catalyzes di- and trimethylation of H3K36 (H3K36me2 H3K36me3) via an interaction between its Set2-Rpb1 (SRI) domain C-terminal repeats RNA polymerase II (Pol2) phosphorylated at Ser2 Ser5 (CTD-S2,5-P). H3K36me2 is sufficient for recruitment the Rpd3S histone deacetylase complex to repress cryptic transcription from transcribed regions. fission also responsible methylation, which represses a subset RNAs including heterochromatic subtelomeric RNAs, least in part Clr6 II,...

DNA primase-dependent synthesis of oligoribonucleotides 10-15 nucleotides long was observed in the presence ATP, UTP, GTP, and CTP by using purified components simian virus 40 (SV40) replication system. The primase-catalyzed reaction required SV40 large tumor antigen (T antigen), polymerase alpha (pol-alpha), three-subunit human single-stranded binding protein (HSSB), topoisomerase I. small RNAs unaffected addition activator 1, proliferating cell nuclear antigen, delta, proteins that can...

Polyomavirus (Py) DNA replication is regulated by its enhancer, which contains an AP1 site, c-Jun and c-Fos, the products of nuclear protooncogenes c-jun c-fos, form heterodimeric transcriptional activating factor AP1. Overexpression c-fos genes strongly stimulated Py from origin as well transcription early promoter through binding site. The cAMP response element (CRE)-binding protein CREB only transcription, not replication, CRE under similar conditions. results indicate that functions a...

ABSTRACT Heterochromatin protein 1 (HP1) is a highly conserved, canonical factor involved in heterochromatin formation. HP1 has been shown to interact with proteins other than silencing factors and effectors. In fission yeast, the loss of homolog Swi6 disrupts structure affects mating type switching at mat locus, where exists; however, cell growth unaffected. this study, we focused on dimerization domain, which provides binding surface for various interactors. We isolated distinctive...

In response to nutritional starvation, living cells sensitively regulate the production rates of molecules required for survival. Under glucose a facultative heterochromatinization ribosomal DNA (rDNA) is considered RNA (rRNA) production. However, molecular mechanism still unclear. Here, we report novel function CENP-B homologue Abp1 in forming heterochromatin at rDNA repeats. We find that loss induces an ectopic nucleosome assembly Interestingly, two mutually exclusive changes repeats:...

The functional interaction of simian virus 40 (SV40) large tumor antigen (T antigen) with DNA polymerase alpha (pol alpha)-primase complex, human single-stranded binding protein (HSSB), and delta delta) holoenzyme, which includes pol delta, activator I (also called replication factor C), proliferating cell nuclear antigen, at the fork was examined using purified components that support SV40 replication. Dilution reaction mixtures during RNA primer synthesis revealed T remained associated...

The influence of DNA polymerase (pol) alpha and primase on SV40 replication was examined in both the monopolymerase dipolymerase systems. synthesis oligoribonucleotides systems, followed by pulse labeling with deoxynucleoside triphosphates, yielded short Okazaki fragments approximately 35 nucleotides length that were chased into full-length time. In presence activator 1 proliferating cell nuclear antigen (PCNA), but no pol delta, these hardly increased size similar (approximately...

The activated c-Ha-ras oncogene induced AP1-site DNA-binding activity in F9 cells. This induction appeared to be due, at least part, the of c-jun transcription. Both and differentiation cells endoderm-like Thus, AP1 appears play a key role initial stage cell differentiation.

The ATP-dependent binding of simian virus 40 (SV40) large tumor antigen (T antigen) to the SV40 origin replication is an essential step in initiation DNA synthesis.Previous studies indicated that complex consists a double hexamer T at origin.The reaction and subsequent unwinding duplex from were examined using gel mobility shift assay.T bound core cooperatively presence ATP.In human single-stranded DNA-binding protein (HSSB), antigen, complexed origin, started DNA.At low concentrations ATP,...

Autonomously replicating sequence (ARS) elements of the fission yeast Schizosaccharomyces pombe contain multiple imperfect copies consensus reported by Maundrell et al. [Maundrell K., Hutchison, A. & Shall, S. (1988) EMBO J. 7, 2203-2209]. When cell free extracts were incubated with a dimer or tetramer an oligonucleotide containing ARS sequence, several complexes detected using gel mobility-shift assay. The proteins forming these also bind ars3002, which is most active origin in ura4...

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine effects mutations within or adjacent core origin (ori) region vitro. The analysis plasmid DNAs containing deletions early-gene side ori indicated that sequences between nucleotides 41 57 define early boundary replication is consistent with previously published studies on...

The mini-F plasmids pSC138, pKP1013, and pKV513 were unable to transform Escherichia coli cells with a dnaA-defective mutation under nonpermissive conditions. dnaA defect was suppressed for host chromosome replication either by the simultaneous presence of rnh-199 (amber) or prophage P2 sig5 integrated at attP2II locus on chromosome, both providing new origins independent function. mutations tested dnaA17, dnaA5, dnaA46. dnaA5 dnaA46 are missense mutations. dnaA17 is an amber whose activity...

Chromatin assembly factor-1 (CAF1) is a well-conserved histone chaperone that loads the H3-H4 complex onto newly synthesized DNA in vitro through interaction with replication factor PCNA. CAF1 considered to be involved heterochromatin maintenance several organisms, but evidence circumstantial and functional details have not been established. We identified fission yeast CAF-1 (spCAF1), which interacts PCNA S phase. Depletion of spCAF1 caused defects silencing at centromeric mating locus...

In fission yeast, the RNAi pathway is required for centromeric heterochromatin assembly. siRNAs derived from transcripts are incorporated into RNA-induced transcriptional silencing (RITS) complex and direct it to nascent homologous transcripts. The silencing-bound further recruit RNA-directed RNA polymerase (RDRC) promote dsRNA synthesis siRNA production. Heterochromatin coated with Swi6/Heterochromain Protein 1 then formed following recruitment of chromatin modification machinery. Swi6 also...