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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Unrepaired DNA double-strand breaks (DSBs) typically result in G(2) arrest. Cell cycle progression can resume following repair of the DSBs or through adaptation to the checkpoint, even if the damage remains unrepaired. We developed a screen for factors in the yeast Saccharomyces cerevisiae that affect checkpoint control and/or viability in response to a single, unrepairable DSB that is induced by HO endonuclease in a dispensable yeast artificial chromosome containing human DNA. SIR2, -3, or -4 mutants exhibit a prolonged, RAD9-dependent G(2) arrest in response to the unrepairable DSB followed by a slow adaptation to the persistent break, leading to division and rearrest in the next G(2). There are a small number of additional cycles before permanent arrest as microcolonies. Thus, SIR genes, which repress silent mating type gene expression, are required for the adaptation and the prevention of indirect lethality resulting from an unrepairable DSB in nonessential DNA. Rapid adaptation to the G(2) checkpoint and high viability were restored in sir(-) strains containing additional deletions of the silent mating type loci HML and HMR, suggesting that genes under mating type control can reduce the toleration of a single DSB. However, coexpression of MATa1 and MATalpha2 in Sir(+) haploid cells did not lead to lethality from the HO-induced DSB, suggesting that toleration of an unrepaired DSB requires more than one Sir(+) function.
Mol Cell Biol 2001 Aug
PMID:SIR functions are required for the toleration of an unrepaired double-strand break in a dispensable yeast chromosome. 1146 19

The Ty5 retrotransposons of Saccharomyces cerevisiae integrate preferentially into regions of silent chromatin at the telomeres and silent mating loci (HMR and HML). We define a Ty5-encoded targeting domain that spans 6 amino acid residues near the C terminus of integrase (LXSSXP). The targeting domain establishes silent chromatin when it is tethered to a weakened HMR-E silencer, and it disrupts telomeric silencing when it is overexpressed. As determined by both yeast two-hybrid and in vitro binding assays, the targeting domain interacts with the C terminus of Sir4p, a structural component of silent chromatin. This interaction is abrogated by mutations in the targeting domain that disrupt integration into silent chromatin, suggesting that recognition of Sir4p by the targeting domain is the primary determinant in Ty5 target specificity.
Mol Cell Biol 2001 Oct
PMID:Targeting of the yeast Ty5 retrotransposon to silent chromatin is mediated by interactions between integrase and Sir4p. 1153 48

Initiation of transcriptional silencing at mating type loci and telomeres in Saccharomyces cerevisiae requires the recruitment of a Sir2/3/4 (silent information regulator) protein complex to the chromosome, which occurs at least in part through its association with the silencer- and telomere-binding protein Rap1p. Sir3p and Sir4p are structural components of silent chromatin that can self-associate, interact with each other, and bind to the amino-terminal tails of histones H3 and H4. We have identified a small region of Sir3p between amino acids 455 and 481 that is necessary and sufficient for association with the carboxyl terminus of Rap1p but not required for Sir complex formation or histone binding. SIR3 mutations that delete this region cause a silencing defect at HMR and telomeres. However, this impairment of repression is considerably less than that displayed by Rap1p carboxy-terminal truncations that are defective in Sir3p binding. This difference may be explained by the ability of the Rap1p carboxyl terminus to interact independently with Sir4p, which we demonstrate by in vitro binding and two-hybrid assays. Significantly, the Rap1p-Sir4p two-hybrid interaction does not require Sir3p and is abolished by mutation of the carboxyl terminus of Rap1p. We propose that both Sir3p and Sir4p can directly and independently bind to Rap1p at mating type silencers and telomeres and suggest that Rap1p-mediated recruitment of Sir proteins operates through multiple cooperative interactions, at least some of which are redundant. The physical separation of the Rap1p interaction region of Sir3p from parts of the protein required for Sir complex formation and histone binding raises the possibility that Rap1p can participate directly in the maintenance of silent chromatin through the stabilization of Sir complex-nucleosome interactions.
Mol Cell Biol 2001 Dec
PMID:Multiple interactions in Sir protein recruitment by Rap1p at silencers and telomeres in yeast. 1168 98

In Saccharomyces cerevisiae, silencing at the HM loci depends on Sir proteins, which are structural components of silenced chromatin. To explore the structure and assembly of silenced chromatin, the associations of Sir proteins with sequences across the HMR locus were examined by chromatin immunoprecipitation. In wild-type cells, Sir2p, Sir3p, and Sir4p were spread throughout and coincident with the silenced region at HMR. Sir1p, in contrast, associated only with the HMR-E silencer, consistent with its role in establishment but not maintenance of silencing. Sir4p was required for the association of other Sir proteins with silencers. In contrast, in the absence of Sir2p or Sir3p, partial assemblies of Sir proteins could form at silencers, where Sir protein assembly began. Spreading across HMR required Sir2p and Sir3p, as well as the deacetylase activity of Sir2p. These data support a model for the spreading of silenced chromatin involving cycles of nucleosome deacetylation by Sir2p followed by recruitment of additional Sir2p, Sir3p, and Sir4p to the newly deacetylated nucleosome. This model suggests mechanisms for boundary formation, and for maintenance and inheritance of silenced chromatin. The principles are generalizable to other types of heritable chromatin states.
Mol Biol Cell 2002 Jul
PMID:Ordered nucleation and spreading of silenced chromatin in Saccharomyces cerevisiae. 1213 62

The Swi/Snf chromatin remodeling complex has been previously demonstrated to be required for transcriptional activation and repression of a subset of genes in Saccharomyces cerevisiae. In this work we demonstrate that Swi/Snf is also required for repression of RNA polymerase II-dependent transcription in the ribosomal DNA (rDNA) locus (rDNA silencing). This repression appears to be independent of both Sir2 and Set1, two factors known to be required for rDNA silencing. In contrast to many other rDNA silencing mutants that have elevated levels of rDNA recombination, snf2Delta mutants have a significantly decreased level of rDNA recombination. Additional studies have demonstrated that Swi/Snf is also required for silencing of genes near telomeres while having no detectable effect on silencing of HML or HMR.
Mol Cell Biol 2004 Sep
PMID:The Swi/Snf chromatin remodeling complex is required for ribosomal DNA and telomeric silencing in Saccharomyces cerevisiae. 1534 82

N(alpha) acetylation is one of the most abundant protein modifications in eukaryotes and is catalyzed by N-terminal acetyltransferases (NATs). NatA, the major NAT in Saccharomyces cerevisiae, consists of the subunits Nat1p, Ard1p, and Nat5p and is necessary for the assembly of repressive chromatin structures. Here, we found that Orc1p, the large subunit of the origin recognition complex (ORC), required NatA acetylation for its role in telomeric silencing. NatA functioned genetically through the ORC binding site of the HMR-E silencer. Furthermore, tethering Orc1p directly to the silencer circumvented the requirement for NatA in silencing. Orc1p was N(alpha) acetylated in vivo by NatA. Mutations that abrogated its ability to be acetylated caused strong telomeric derepression. Thus, N(alpha) acetylation of Orc1p represents a protein modification that modulates chromatin function in S. cerevisiae. Genetic evidence further supported a functional link between NatA and ORC: (i) nat1Delta was synthetically lethal with orc2-1 and (ii) the synthetic lethality between nat1Delta and SUM1-1 required the Orc1 N terminus. We also found Sir3p to be acetylated by NatA. In summary, we propose a model by which N(alpha) acetylation is required for the binding of silencing factors to the N terminus of Orc1p and Sir3p to recruit heterochromatic factors and establish repression.
Mol Cell Biol 2004 Dec
PMID:Dependence of ORC silencing function on NatA-mediated Nalpha acetylation in Saccharomyces cerevisiae. 1554 39

Inhibition of 20-hydroxyeicosatrienoic acid (20-HETE), by pretreatment with pharmacological inhibitors of cytochrome P450 (CYP) omega-hydroxylase, has been shown to reduce infarct size in canines when administered prior to ischemia. However, it is unknown whether these agents reduce infarct size when administered just prior to reperfusion and if the sarcolemmal and/or mitochondrial K(ATP) channels (sK(ATP) and mK(ATP)) contribute to cardioprotection. Therefore, we determined whether specific CYP inhibitors for epoxygenases and omega-hydroxylases are cardioprotective when given either prior to ischemia or prior to reperfusion and furthermore, if selective inhibition of the sK(ATP) by HMR-1098 or mK(ATP) by 5-hydroxydecanoic acid (5-HD) could abrogate this effect. Male Sprague-Dawley rats underwent 30 minutes of ischemia followed by 2 hours of reperfusion. Groups received either miconazole (MIC, non-selective CYP inhibitor, 3 mg/kg), 17-octadecynoic acid (17-ODYA, CYP omega-hydroxylase inhibitor, 0,3 or 3 mg/kg), N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS, CYP omega-hydroxylase inhibitor, 0,4 or 4 mg/kg), N-methanesulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH, CYP epoxygenase inhibitor, 3 mg/kg), or vehicle either 10 minutes prior to ischemia or 5 minutes prior to reperfusion. Rats also received either HMR-1098 (6 mg/kg) or 5-HD (10 mg/kg) 10 minutes prior to reperfusion, with subsets of rats also receiving either MIC or 17-ODYA 5 minutes prior to reperfusion. DDMS and 17-ODYA dose dependently reduced infarct size. Rats treated with MIC, 17-ODYA and DDMS, but not MS-PPOH, produced comparable reductions in infarct size when administered prior to ischemia or reperfusion compared to vehicle. HMR-1098, but not 5-HD, also blocked the infarct size reduction afforded by MIC and 17-ODYA. These data suggest a novel cardioprotective pathway involving CYP omega-hydroxylase inhibition and subsequent activation of the sK(ATP) channel during reperfusion.
J Mol Cell Cardiol 2004 Dec
PMID:Cytochrome P450 omega-hydroxylase inhibition reduces infarct size during reperfusion via the sarcolemmal KATP channel. 1557 55

ATP-sensitive K(+) (K(ATP)) channels are involved in ischemic cardioprotection induced by preconditioning (IPC), though the relative role of sarcolemmal (sK(ATP)) and mitochondrial (mitoK(ATP)) channels remains controversial. The sK(ATP)-selective sulphonylthiourea HMR 1098 has often been reported to be without effect on ischemic cardioprotection, suggesting minimal involvement of sK(ATP). Since some sulphonylureas show reduced potency under conditions of metabolic stress, we used patch clamp to assess the ability of HMR 1098 to block sK(ATP) currents of adult rat ventricular myocytes activated by metabolic inhibition (MI, NaCN+iodoacetate). In contrast to the prototype sulphonylurea glibenclamide, HMR 1098 (10 muM) was without effect on sK(ATP) currents, and also did not inhibit MI-induced action potential shortening. However, HMR 1098 blocked sK(ATP) current induced by the K(ATP) opener pinacidil (IC(50)=0.36+/-0.02 muM), and reversed pinacidil-induced action potential shortening. In inside-out patches, block by HMR 1098 was relieved by increasing MgADP concentrations (1-100 muM). HMR 1098 inhibited pinacidil-activated recombinant Kir6.2/SUR2A channels with a similar IC(50) (0.30+/-0.04 muM), but was less effective when channels were activated by low intracellular ATP. HMR 1098 displaced binding of the pinacidil analogue [(3)H]P1075 to native cardiac membranes with a biphasic inhibition curve. Our results show that HMR 1098 becomes a much less effective inhibitor of sK(ATP) during metabolic stress, and suggest that the lack of effect of HMR 1098 on ischemic cardioprotection reported in some studies may represent loss of block by the drug under these conditions rather than a lack of involvement of sK(ATP) channels.
J Mol Cell Cardiol 2005 Oct
PMID:Reduced effectiveness of HMR 1098 in blocking cardiac sarcolemmal K(ATP) channels during metabolic stress. 1609 67

The silenced HMR domain is restricted from spreading by barrier elements, and the right barrier is a unique t-RNA(THR) gene. We show that sequences immediately flanking the silenced domain were enriched in acetylated, but not methylated, histones, whereas the barrier element was associated with a nucleosome-free region. Surprisingly, the SAGA acetyltransferase resided across the entire region. We further demonstrate that a mutation in the barrier eliminated the nucleosome-free gap but only subtly altered the distribution of SAGA. Interestingly, neither reformation of the nucleosome nor mutations in chromatin-modifying enzymes alone led to an unrestricted spread of silenced chromatin. Double mutations in the t-RNA barrier and these complexes, on the other hand, led to a significant spread of Sir proteins. These results suggest two overlapping mechanisms function to restrict the spread of silencing: one of which involves a DNA binding element, whereas the other mechanism involves specific chromatin-modifying activities.
Mol Cell 2005 Sep 02
PMID:Barrier function at HMR. 1613 26

The possible role of hydrogen sulfide (H2S) in cardioprotection was investigated in isolated rat ventricular myocytes exposed to severe metabolic inhibition (MI) in glucose-free buffer containing 2-deoxy-D-glucose (2-DOG), an inhibitor of glycolysis. Pretreatment (30 min) with NaHS (a H2S donor) at concentrations of 10(-5) to 10(-4) mol/L caused a concentration related increase in cell viability and the ratio of rod-shaped cells. A time course study showed that NaHS-induced cardioprotection occurred in 2 time windows (approximately 1 h and 16-28 h). To observe whether endogenous H2S may be involved in the delayed cardioprotection response of IP, DL-propargylglycine (PAG) and beta-cyano-L-alanine (BCA; two inhibitors of H2S biosynthesis) were used. Both drugs significantly attenuated the cardioprotection produced by MI using cell viability, cellular injury index, and electrically-induced [Ca2+]i transients as end-points. These data suggest that endogenous H2S plays an important role in the cardioprotection following MI preconditioning. In an attempt to determine the mechanism of the cardioprotective effect of H2S, we examined the effect of blocking KATP channels with glibenclamide (a non-selective KATP channel blocker), 5-hydroxydecanoic acid (5-HD, a mitochondrial KATP blocker), and HMR-1098 (a sarcolemmal KATP blocker). The cardioprotective effects of NaHS were significantly attenuated by glibenclamide and HMR-1098 treatment but not by 5-HD. Inhibition of NO production with L-NG nitroarginine methyl ester (L-NAME) also attenuated the cardioprotection of NaHS. In conclusion, our findings provide the first evidence that H2S may protect the heart most probably by activating sarcolemmal KATP channels and/or provoking NO release and the cardioprotective effects of metabolic ischemic preconditioning is, at least partially, mediated by endogenous H2S.
J Mol Cell Cardiol 2006 Jan
PMID:Endogenous hydrogen sulfide contributes to the cardioprotection by metabolic inhibition preconditioning in the rat ventricular myocytes. 1632 98


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