scholarly journals Differential Processing of Leading- and Lagging-Strand Ends at Saccharomyces cerevisiae Telomeres Revealed by the Absence of Rad27p Nuclease

Genetics ◽  
2002 ◽  
Vol 162 (4) ◽  
pp. 1583-1594
Author(s):  
Julie Parenteau ◽  
Raymund J Wellinger
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Double Mutant ◽  
Telomerase Activity ◽  
Differential Processing ◽  
Okazaki Fragment Processing ◽  
Mutant Strains ◽  
Leading Strand ◽  
Replication Intermediates ◽  
Lagging Strand

Abstract Saccharomyces cerevisiae strains lacking the Rad27p nuclease, a homolog of the mammalian FEN-1 protein, display an accumulation of extensive single-stranded G-tails at telomeres. Furthermore, the lengths of telomeric repeats become very heterogeneous. These phenotypes could be the result of aberrant Okazaki fragment processing of the C-rich strand, elongation of the G-rich strand by telomerase, or an abnormally high activity of the nucleolytic activities required to process leading-strand ends. To distinguish among these possibilities, we analyzed strains carrying a deletion of the RAD27 gene and also lacking genes required for in vivo telomerase activity. The results show that double-mutant strains died more rapidly than strains lacking only telomerase components. Furthermore, in such strains there is a significant reduction in the signals for G-tails as compared to those detected in rad27Δ cells. The results from studies of the replication intermediates of a linear plasmid in rad27Δ cells are consistent with the idea that only one end of the plasmid acquires extensive G-tails, presumably the end made by lagging-strand synthesis. These data further support the notion that chromosome ends have differential requirements for end processing, depending on whether the ends were replicated by leading- or lagging-strand synthesis.

scholarly journals Near-continuously synthesized leading strands inEscherichia coliare broken by ribonucleotide excision

2019 ◽  
Vol 116 (4) ◽  
pp. 1251-1260 ◽  
Author(s):  
Glen E. Cronan ◽  
Elena A. Kouzminova ◽  
Andrei Kuzminov
Keyword(s):  
Dna Replication ◽  
Excision Repair ◽  
Chromosomal Dna ◽  
E Coli ◽  
Okazaki Fragments ◽  
Leading Strand ◽  
Replication Intermediates ◽  
Lagging Strand

In vitro, purified replisomes drive model replication forks to synthesize continuous leading strands, even without ligase, supporting the semidiscontinuous model of DNA replication. However, nascent replication intermediates isolated from ligase-deficientEscherichia colicomprise only short (on average 1.2-kb) Okazaki fragments. It was long suspected that cells replicate their chromosomal DNA by the semidiscontinuous mode observed in vitro but that, in vivo, the nascent leading strand was artifactually fragmented postsynthesis by excision repair. Here, using high-resolution separation of pulse-labeled replication intermediates coupled with strand-specific hybridization, we show that excision-proficientE. coligenerates leading-strand intermediates >10-fold longer than lagging-strand Okazaki fragments. Inactivation of DNA-repair activities, including ribonucleotide excision, further increased nascent leading-strand size to ∼80 kb, while lagging-strand Okazaki fragments remained unaffected. We conclude that in vivo, repriming occurs ∼70× less frequently on the leading versus lagging strands, and that DNA replication inE. coliis effectively semidiscontinuous.

Calreticulin Mediates Anesthetic Sensitivity in Drosophila melanogaster 

2003 ◽  
Vol 99 (4) ◽  
pp. 867-875 ◽  
Author(s):  
Sumiko Gamo ◽  
Junya Tomida ◽  
Katsuyuki Dodo ◽  
Dai Keyakidani ◽  
Hitoshi Matakatsu ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Double Mutant ◽  
Developmental Stages ◽  
Northern Blotting ◽  
General Anesthetics ◽  
Wild Type ◽  
Mutant Strains ◽  
Low Expression

Background Various species, e.g., Caenorhabditis elegans, Drosophila melanogaster, and mice, have been used to explore the mechanisms of action of general anesthetics in vivo. The authors isolated a Drosophila mutant, ethas311, that was hypersensitive to diethylether and characterized the calreticulin (crc) gene as a candidate of altered anesthetic sensitivity. Methods Molecular analysis of crc included cloning and sequencing of the cDNA, Northern blotting, and in situ hybridization to accomplish the function of the gene and its mutation. For anesthetic phenotype assay, the 50% anesthetizing concentrations were determined for ethas311, revertants, and double-mutant strains (wild-type crc transgene plus ethas311). Results Expression of the crc 1.4-kb transcript was lower in the mutant ethas311 than in the wild type at all developmental stages. The highest expression at 19 h after pupation was observed in the brain of the wild type but was still low in the mutant at that stage. The mutant showed resistance to isoflurane as well as hypersensitivity to diethylether, whereas it showed the wild phenotype to halothane. Both mutant phenotypes were restored to the wild type in the revertants and double-mutant strains. Conclusion ethas311 is a mutation of low expression of the Drosophila calreticulin gene. The authors demonstrated that hypersensitivity to diethylether and resistance to isoflurane are associated with low expression of the gene. In Drosophila, calreticulin seems to mediate these anesthetic sensitivities, and it is a possible target for diethylether and isoflurane, although the predicted anesthetic targets based on many studies in vitro and in vivo are the membrane proteins, such as ion channels and receptors.

scholarly journals Yeast Est2p Affects Telomere Length by Influencing Association of Rap1p with Telomeric Chromatin

2008 ◽  
Vol 28 (7) ◽  
pp. 2380-2390 ◽  
Author(s):  
Hong Ji ◽  
Christopher J. Adkins ◽  
Bethany R. Cartwright ◽  
Katherine L. Friedman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomere Length ◽  
Specific Binding ◽  
Telomerase Activity ◽  
Negative Regulator ◽  
Wild Type ◽  
Telomeric Dna ◽  
Telomere Length Homeostasis

ABSTRACT In Saccharomyces cerevisiae, the sequence-specific binding of the negative regulator Rap1p provides a mechanism to measure telomere length: as the telomere length increases, the binding of additional Rap1p inhibits telomerase activity in cis. We provide evidence that the association of Rap1p with telomeric DNA in vivo occurs in part by sequence-independent mechanisms. Specific mutations in EST2 (est2-LT) reduce the association of Rap1p with telomeric DNA in vivo. As a result, telomeres are abnormally long yet bind an amount of Rap1p equivalent to that observed at wild-type telomeres. This behavior contrasts with that of a second mutation in EST2 (est2-up34) that increases bound Rap1p as expected for a strain with long telomeres. Telomere sequences are subtly altered in est2-LT strains, but similar changes in est2-up34 telomeres suggest that sequence abnormalities are a consequence, not a cause, of overelongation. Indeed, est2-LT telomeres bind Rap1p indistinguishably from the wild type in vitro. Taken together, these results suggest that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, implicating telomerase in roles both upstream and downstream of Rap1p in telomere length homeostasis.

scholarly journals The genetic control of direct-repeat recombination in Saccharomyces: the effect of rad52 and rad1 on mitotic recombination at GAL10, a transcriptionally regulated gene.

Genetics ◽  
1989 ◽  
Vol 123 (4) ◽  
pp. 725-738 ◽  
Author(s):  
B J Thomas ◽  
R Rothstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Control ◽  
Plasmid Dna ◽  
Double Mutant ◽  
Direct Repeat ◽  
Single Mutant ◽  
Plasmid Loss ◽  
Mutant Strains ◽  
Recombination Pathway

Abstract We have previously shown direct-repeat recombination events leading to loss of a plasmid integrated at the GAL10 locus in Saccharomyces cerevisiae are stimulated by transcription of the region. We have examined the role of two recombination- and repair-defective mutations, rad1 and rad52, on direct repeat recombination in transcriptionally active and inactive sequences. We show that the RAD52 gene is required for transcription-stimulated recombination events leading to loss of the integrated plasmid. Similarly, Gal+ events between the duplicated repeats that retain the integrated plasmid DNA (Gal+ Ura+ replacement events) are reduced 20-fold in the rad52 mutant in sequences that are constitutively expressed. In contrast, in sequences that are not expressed, the rad52 mutation reduces plasmid loss events by only twofold and Gal+ Ura+ replacements by fourfold. We also observe an increase in disome-associated plasmid loss events in the rad52 mutant, indicative of chromosome gain. This event is not affected by expression of the region. Plasmid loss events in rad1 mutant strains are reduced only twofold in transcriptionally active sequences and are not affected in sequences that are repressed. However, the rad1 and rad52 double mutant shows a decrease in plasmid loss events greater than the sum of the decreases in the rates of this event displayed by either single mutant in both constitutive and repressed DNA, indicating a synergistic interaction between these two genes. The synergism is limited to recombination since the rad1 rad52 double mutant is no more sensitive when compared with either single mutant in its ability to survive radiation damage. Finally, the recombination pathway that remains in the double mutant is positively affected by transcription of the region.

scholarly journals Generation of temperature-sensitive cbp1 strains of Saccharomyces cerevisiae by PCR mutagenesis and in vivo recombination: characteristics of the mutant strains imply that CBP1 is involved in stabilization and processing of cytochrome b pre-mRNA.

Genetics ◽  
1993 ◽  
Vol 135 (4) ◽  
pp. 981-991 ◽  
Author(s):  
R R Staples ◽  
C L Dieckmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome B ◽  
Mitochondrial Gene ◽  
Nuclear Gene ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
In Vivo Recombination ◽  
Mutant Strains ◽  
Precursor Rna

Abstract Mitochondrial biogenesis is dependent on both nuclearly and mitochondrially encoded proteins. Study of the nuclearly encoded mitochondrial gene products and their effect on mitochondrial genome expression is essential to understanding mitochondrial function. Mutations in the nuclear gene CBP1 of Saccharomyces cerevisiae result in degradation of mitochondrially encoded cytochrome b (cob) RNA; thus, the cells are unable to respire. Putative roles for the CBP1 protein include processing of precursor RNA to yield the mature 5' end of cob mRNA and/or physical protection of the mRNA from degradation by nucleases. To examine the activity of CBP1, we generated temperature-sensitive cbp1 mutant strains by polymerase chain reaction (PCR) mutagenesis and in vivo recombination. These temperature-sensitive cbp1 strains lack cob mRNA only at the nonpermissive temperature. Quantitative primer extension analyses of RNA from these strains and from a cbp1 deletion strain demonstrated that CBP1 is required for the stability of precursor RNAs in addition to production of the stable mature mRNA. Thus, CBP1 is not involved solely in the protection of mature cob mRNA from nucleases. Moreover, we found that mature mRNAs are undetectable while precursor RNAs are reduced only slightly at the nonpermissive temperature. Collectively, these data lead us to favor a hypothesis whereby CBP1 protects cob precursor RNAs and promotes the processing event that generates the mature 5' end of the mRNA.

scholarly journals Replication dynamics of recombination-dependent replication forks

2020 ◽  
Author(s):  
Karel Naiman ◽  
Eduard Campillo-Funollet ◽  
Adam T. Watson ◽  
Alice Budden ◽  
Izumi Miyabe ◽  
...  
Keyword(s):  
Monte Carlo Simulation ◽  
Monte Carlo ◽  
Homologous Recombination ◽  
Replication Fork ◽  
S Phase ◽  
Replication Forks ◽  
Leading Strand ◽  
The Stability ◽  
Lagging Strand

AbstractDNA replication fidelity is essential for maintaining genetic stability. Forks arrested at replication fork barriers can be stabilised by the intra-S phase checkpoint, subsequently being rescued by a converging fork, or resuming when the barrier is removed. However, some arrested forks cannot be stabilised and fork convergence cannot rescue in all situations. Thus, cells have developed homologous recombination-dependent mechanisms to restart persistently inactive forks. To understand HR-restart we use polymerase usage sequencing to visualize in vivo replication dynamics at an S. pombe replication barrier, RTS1, and model replication by Monte Carlo simulation. We show that HR-restarted forks synthesise both strands with Pol δ for up to 30 kb without maturing to a δ/ε configuration and that Pol α is not used significantly on either strand, suggesting the lagging strand template remains as a gap that is filled in by Pol δ later. We further demonstrate that HR-restarted forks progress uninterrupted through a fork barrier that arrests canonical forks. Finally, by manipulating lagging strand resection during HR-restart by deleting pku70, we show that the leading strand initiates replication at the same position, signifying the stability of the 3’ single strand in the context of increased resection.

scholarly journals Two isoforms of Saccharomyces cerevisiae glutaredoxin 2 are expressed in vivo and localize to different subcellular compartments

2002 ◽  
Vol 364 (3) ◽  
pp. 617-623 ◽  
Author(s):  
José R. PEDRAJAS ◽  
Pablo PORRAS ◽  
Emilia MARTÍNEZ-GALISTEO ◽  
C. Alicia PADILLA ◽  
Antonio MIRANDA-VIZUETE ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Exponential Phase ◽  
Start Codon ◽  
Immunochemical Analysis ◽  
Translation Product ◽  
Differential Processing ◽  
Reading Frame ◽  
Yeast Extract Peptone Dextrose ◽  
Long Form

Glutaredoxin (Grx)2 from Saccharomyces cerevisiae is a member of the two-cysteine (dithiol) subfamily of Grxs involved in the defence against oxidative stress in yeast. Recombinant yeast Grx2p, expressed in Escherichia coli, behaves as a ‘classical’ Grx that efficiently catalyses the reduction of hydroxyethyl disulphide by GSH. Grx2p also catalyses the reduction of GSSG by dihydrolipoamide with even higher efficiency. Western blot analysis of S. cerevisiae crude extracts identifies two isoforms of Grx2p of 15.9 and 11.9kDa respectively. The levels of these two isoforms reach a peak during the exponential phase of growth in normal yeast extract/peptone/dextrose ('YPD') medium, with the long form predominating over the short one. From immunochemical analysis of subcellular fractions, it is shown that both isoforms are present in mitochondria, but only the short one is detected in the cytosolic fraction. On the other hand, only the long form is prominent in microsomes. Mitochondrial isoforms should represent the processed and unprocessed products of an open reading frame (YDR513W), with a putative start codon 99bp upstream of the GRX2 start codon described thus far. These results indicate that GRX2 contains two in-frame start codons, and that translation from the first AUG results in a product that is targeted to mitochondria. The cytosolic form would result either by initiation from the second AUG, or by differential processing of one single translation product.

Senescence Mutants of Saccharomyces cerevisiae With a Defect in Telomere Replication Identify Three Additional EST Genes

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1399-1412 ◽  
Author(s):  
Thomas S Lendvay ◽  
Danna K Morris ◽  
Jeannie Sah ◽  
Bhuvana Balasubramanian ◽  
Victoria Lundblad
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomerase Activity ◽  
Gene Encoding ◽  
Epistasis Analysis ◽  
Mutant Strains ◽  
Primary Determinant ◽  
Complementation Groups ◽  
Telomere Replication ◽  
Senescence Phenotype ◽  
Novel Protein

The primary determinant for telomere replication is the enzyme telomerase, responsible for elongating the G-rich strand of the telomere. The only component of this enzyme that has been identified in Saccharomyces cermzsiae is the TLC1 gene, encoding the telomerase RNA subunit. However, a yeast strain defective for the EST1 gene exhibits the same phenotypes (progressively shorter telomeres and a senescence phenotype) as a strain deleted for TLC1, suggesting that EST1 encodes either a component of telomerase or some other factor essential for telomerase function. We designed a multitiered screen that led to the isolation of 22 mutants that display the same phenotypes as est1 and tlc1 mutant strains. These mutations mapped to four complementation groups: the previously identified EST1 gene and three additional genes, called EST2, EST3 and EST4. Cloning of the EST2 gene demonstrated that it encodes a large, extremely basic novel protein with no motifs that provide clues as to function. Epistasis analysis indicated that the four EST genes function in the same pathway for telomere replication as defined by the TLC1 gene, suggesting that the EST genes encode either components of telomerase or factors that positively regulate telomerase activity.

scholarly journals DNA Replication Through Strand Displacement During Lagging Strand DNA Synthesis in Saccharomyces cerevisiae

Genes ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 167 ◽  
Author(s):  
Michele Giannattasio ◽  
Dana Branzei
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerases ◽  
Experimental Results ◽  
Genome Integrity ◽  
Strand Displacement ◽  
Viral Dna ◽  
Okazaki Fragment Processing ◽  
Lagging Strand

This review discusses a set of experimental results that support the existence of extended strand displacement events during budding yeast lagging strand DNA synthesis. Starting from introducing the mechanisms and factors involved in leading and lagging strand DNA synthesis and some aspects of the architecture of the eukaryotic replisome, we discuss studies on bacterial, bacteriophage and viral DNA polymerases with potent strand displacement activities. We describe proposed pathways of Okazaki fragment processing via short and long flaps, with a focus on experimental results obtained in Saccharomyces cerevisiae that suggest the existence of frequent and extended strand displacement events during eukaryotic lagging strand DNA synthesis, and comment on their implications for genome integrity.

scholarly journals The Biochemical Activities of the Saccharomyces cerevisiae Pif1 Helicase Are Regulated by Its N-Terminal Domain

Genes ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 411 ◽  
Author(s):  
Nickens ◽  
Sausen ◽  
Bochman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomerase Activity ◽  
Full Length ◽  
Dna Unwinding ◽  
Genome Maintenance ◽  
Terminal Domain ◽  
Pif1 Helicase ◽  
Telomerase Regulation

: Pif1 family helicases represent a highly conserved class of enzymes involved in multiple aspects of genome maintenance. Many Pif1 helicases are multi-domain proteins, but the functions of their non-helicase domains are poorly understood. Here, we characterized how the N-terminal domain (NTD) of the Saccharomyces cerevisiae Pif1 helicase affects its functions both in vivo and in vitro. Removal of the Pif1 NTD alleviated the toxicity associated with Pif1 overexpression in yeast. Biochemically, the N-terminally truncated Pif1 (Pif1ΔN) retained in vitro DNA binding, DNA unwinding, and telomerase regulation activities, but these activities differed markedly from those displayed by full-length recombinant Pif1. However, Pif1ΔN was still able to synergize with the Hrq1 helicase to inhibit telomerase activity in vitro, similar to full-length Pif1. These data impact our understanding of Pif1 helicase evolution and the roles of these enzymes in the maintenance of genome integrity.

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