DNA polymerase-α is essential for mating-type switching in fission yeast

Singh, Jagmohan; Klar, Amar J. S.

doi:10.1038/361271a0

Cited by 70 publications

(65 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The PCR product was digested with BamHI and EcoRI and cloned at the same sites in pRSETA (Invitrogen) or pGEX1 vector (Amersham Pharmacia Biotech). To express the MBPPol␣ fusion protein, the XbaI-PstI fragment of the pol␣ gene (25) was cloned into the pMALp2 vector (New England Biolabs) at the same sites. For constructing the hemagglutinin-tagged Swi6, PCR was performed using forward oligonucleotide ATGCGGCCGCTAGCCATTCT-GTACACC and reverse oligonucleotide CATGCGGCCGCCTTCATTT-TCACGGAACGTTAAG.…”

Section: Methodsmentioning

confidence: 99%

“…To check whether pol␣ interacts with Swi6 in vivo, we transformed a construct carrying (His) 6 -tagged pol␣ gene in the vector pART1 (20) into a strain carrying a disruption of the pol␣ gene, as descried earlier (25). The whole cell extract prepared from these cells was fractionated by Ni-NTA chromatography to purify the (His) 6 -tagged pol␣ protein by elution with 250 mM imidazole and immunoblotted.…”

Section: Direct Physical Interaction Between Wild Type But Not Mutantmentioning

confidence: 99%

“…Silencing Defect Is Displayed by Other pol␣ Mutants Localized in Conserved Regions of DNA Polymerase-To find out whether mutations in certain regions of pol␣ are required for silencing, we tested two other ts mutants of pol␣, namely, ␣ts11 and ␣ts13 (26) and the viable mutant of pol␣ namely swi7-1 (25). Interestingly, both ␣ts11 and ␣ts13 mutants also exhibited iodine staining (Fig.…”

Section: Figmentioning

confidence: 99%

See 2 more Smart Citations

Chromodomain Protein Swi6-mediated Role of DNA Polymerase α in Establishment of Silencing in Fission Yeast

Ahmed¹,

Saini²,

Arora³

et al. 2001

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Although DNA replication has been thought to play an important role in the silencing of mating type loci in Saccharomyces cerevisiae, recent studies indicate that silencing can be decoupled from replication. In Schizosaccharomyces pombe, mating type silencing is brought about by the trans-acting proteins, namely Swi6, Clr1-Clr4, and Rhp6, in cooperation with the cis-acting silencers. The latter contain an autonomous replication sequence, suggesting that DNA replication may be critical for silencing in S. pombe. To investigate the connection between DNA replication and silencing in S. pombe, we analyzed several temperature-sensitive mutants of DNA polymerase ␣. We find that one such mutant, swi7H4, exhibits silencing defects at mat, centromere, and telomere loci. This effect is independent of the checkpoint and replication defects of the mutant. Interestingly, the extent of the silencing defect in the swi7H4 mutant at the silent mat2 locus is further enhanced in absence of the cis-acting, centromere-proximal silencer. The chromodomain protein Swi6, which is required for silencing and is localized to mat and other heterochromatin loci, interacts with DNA polymerase ␣ in vivo and in vitro in wild type cells. However, it does not interact with the mutant pol␣ and is delocalized away from the silent mat loci in the mutant. Our results demonstrate a role of DNA polymerase ␣ in the establishment of silencing. We propose a recruitment model for the coupling of DNA replication with the establishment of silencing by the chromodomain protein Swi6, which may be applicable to higher eukaryotes.The well studied system of mating type silencing in the budding yeast Saccharomyces cerevisiae has served as a paradigm for developmental regulation of gene regulation. Although the mating type phenotype of a homothallic strain is dictated by the MAT locus depending on whether it harbors the a-or ␣-specific alleles, two copies of the same genetic information are located at distant sites on the same chromosome, namely HML and HMR, which harbor ␣ and a alleles, respectively. However, these alleles are transcriptionally silent. The silencing is achieved by the cis-acting sequences E (essential) and I (important) that flank both HML and HMR loci (1, 2). In addition, several genes encode factors named mating type regulator/silent information regulator (MAR/SIR) that function in trans through the cis-acting sequences in keeping the HML and HMR loci silent. Extensive studies in S. cerevisiae have suggested that DNA replication is important for repression of the silent mating type loci HML and HMR (see Refs. 1 and 2 for reviews). These findings include a requirement of passage through S phase, a functional autonomous replication sequence (ARS) flanking the silent locus HMR, and a functional origin recognition complex for silencing (reviewed in Ref. 2). However, the requirement of DNA replication for silencing is obviated if the SIR1 silencing protein is recruited by alternative means, although passage through S phase is still essential (3, 4)....

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Direct Physical Interaction Between Wild Type But Not Mutantmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

See 1 more Smart Citation

Chromodomain Protein Swi6-mediated Role of DNA Polymerase α in Establishment of Silencing in Fission Yeast

Ahmed¹,

Saini²,

Arora³

et al. 2001

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…The double mutants between mis3-224 (Takahashi et al e1994) and various mutations noted below were constructed; cdc2-33, cdc6-121, cdc13-117, cdc25-22, cdc10-129 (Nurse et al 1976), cdc2-3w (Enoch et al 1992), Dcyc17/cig2 (Obara-Ishihara & Okayama 1994), Dmik1 (Lundgren et al 1991), wee1-50 (Russell & Nurse 1987), cdc16-116 (Fankhauser et al 1993), cdc17-K42, cdc18-K46 (Nasmyth & Nurse 1981), cdc22-C1 (Fernandez Sarabia et al 1993), swi7-H4 (Singh & Klar 1993), mis5-268, mis11-453 (Takahashi et al 1994, Drad3, Drad9, Drad17, Drad24, Drad25, Dchk1 (Al-Khodairy et al 1994), cut5-T401 (Saka et al 1994), Dcds1 (Murakami & Okayama 1995), Ddis1 (Nabeshima et al 1995), dis3-54 (Kinoshita et al 1991), dis2-11, Ddis2 (Ohkura et al 1989), cut1-21, cut2-364 (Funabiki et al 1996, cut9 (Yamada et al 1997), Dppa1, Dppa2 (Kinoshita et al 1993),Dpck1 (Toda et al 1993), Dtop1, top2-191 (Uemura & Yanagida 1984), and Ddsk1 mutants (Takeuchi & Yanagida 1993). The Mis3-HA integrant was obtained by integrating the HA-tagged mis3 gene at the carboxyl-terminus to replace the genomic mis3 locus.…”

Section: Strains Genetical Methods and Macromolecular Analysismentioning

confidence: 99%

Mis3 with a conserved RNA binding motif is essential for ribosome biogenesis and implicated in the start of cell growth and S phase checkpoint

2000

View full text Add to dashboard Cite

show abstract

“…Furthermore, the imprint is installed only during the first-time synthesis of the Watson strand and only when replicated by the lagging-strand DNA replication complex (Singh and Klar 1993;Dalgaard and Klar 1999). The imprint creates a DNA fragile site in the chromosome Egel et al 1984;Nielsen and Egel 1989).…”

Section: Introductionmentioning

confidence: 99%

Unbiased segregation of fission yeast chromosome 2 strands to daughter cells

Klar

Bonaduce

2013

Chromosome Res

Self Cite

View full text Add to dashboard Cite

The base complementarity feature (Watson and Crick in Nature 171(4356): [737][738] 1953) and the rule of semi-conservative mode of DNA replication (Messelson and Stahl in Proc Natl Acad Sci U S A 44: [671][672][673][674][675][676][677][678][679][680][681][682] 1958) dictate that two identical replicas of the parental chromosome are produced during replication. In principle, the inherent strand sequence differences could generate nonequivalent daughter chromosome replicas if one of the two strands were epigenetically imprinted during replication to effect silencing/expression of developmentally important genes. Indeed, inheritance of such a strand-and sitespecific imprint confers developmental asymmetry to fission yeast sister cells by a phenomenon called mating/cell-type switching. Curiously, location of DNA strands with respect to each other at the centromere is fixed, and as a result, their selected segregation to specific sister chromatid copies occurs in eukaryotic cells. The yeast system provides a unique opportunity to determine the significance of such biased strand distribution to sister chromatids. We determined whether the cylindrical-shaped yeast cell distributes the specific chromosomal strand to the same cellular pole in successive cycles of cell division. By observing the pattern of recurrent mating-type switching in progenies of individual cells by microscopic analyses, we found that chromosome 2 strands are distributed by the random mode in successive cell divisions. We also exploited unusual "hotspot" recombination features of this system to investigate whether there is selective segregation of strands such that oldest Watsoncontaining strands co-segregate in the diploid cell at mitosis. Our data suggests that chromosome 2 strands are segregated independently to those of the homologous chromosome.

show abstract

DNA polymerase-α is essential for mating-type switching in fission yeast

Cited by 70 publications

References 26 publications

Chromodomain Protein Swi6-mediated Role of DNA Polymerase α in Establishment of Silencing in Fission Yeast

Chromodomain Protein Swi6-mediated Role of DNA Polymerase α in Establishment of Silencing in Fission Yeast

Mis3 with a conserved RNA binding motif is essential for ribosome biogenesis and implicated in the start of cell growth and S phase checkpoint

Unbiased segregation of fission yeast chromosome 2 strands to daughter cells

Contact Info

Product

Resources

About