Efficient labeling of fission yeast Schizosaccharomyces pombe with thymidine and BUdR

Hodson, Jeffrey A.; Bailis, Julie M.; Forsburg, Susan L.

doi:10.1093/nar/gng134

Cited by 61 publications

(64 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The accumulation of XDbf4 during maturation could indicate that the XCdc7/Dbf4 complex is required for meiosis. Alternatively, the potential involvement of XCdc7/Dbf4 in sister chromatid cohesion, origin recognition complex localization, or gene regulation, as shown in different organisms, could explain why the complex binds to chromatin in a pre-RC independent manner (12,40,42,43). Additional studies will be needed to define the role of XCdc7/Dbf4 during the early embryonic cell cycle and determine whether a zygotic version of the XCdc7/Drf1 complex is also required for initiation of DNA replication during the somatic cell cycle.…”

Section: Discussionmentioning

confidence: 99%

“…Functional differences between family members have started to emerge but they need to be further characterized. In fission yeast the Spo4/Spo6 kinase complex is required for meiosis, whereas the other DDK, Hsk1/Dfp1, not only regulates initiation of DNA replication but also centromere cohesion mediated by heterochromatin (11,12). Two DDKs have recently been identified in humans and Xenopus laevis, the Cdc7/Dbf4 and Cdc7/Drf1 complexes (9,13,14).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Xenopus CDC7/DRF1 Complex Is Required for the Initiation of DNA Replication

Silva

Bradley

Gao

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

The Cdc7 kinase is essential for the initiation of DNA replication in eukaryotes. Two regulatory subunits of the Xenopus Cdc7 kinase have been identified: XDbf4 and XDrf1. In this study we determined the expression pattern of XDbf4 and XDrf1 and examined their involvement in DNA replication. We show that XDrf1 expression is restricted to oogenesis and early embryos, whereas XDbf4 is expressed throughout development. Immunodepletion from Xenopus egg extracts indicated that both proteins are only found in complexes with XCdc7 and there is a 5-fold molar excess of the XCdc7/Drf1 over SCdc7/Dbf4 complexes. Both complexes exhibit kinase activity and are differentially phosphorylated during the cell cycle. Depletion of the XCdc7/Drf1 from egg extracts inhibited DNA replication, whereas depletion of XCdc7/Dbf4 had little effect. Chromatin binding studies indicated that XCdc7/Drf1 is required for pre-replication complex activation but not their assembly. XCdc7/Dbf4 complexes bound to the chromatin in two steps: the first step was independent of pre-replication complex assembly and the second step was dependent on pre-replication complex activation. By contrast, binding of XCdc7/Drf1 complexes was entirely dependent on pre-replication complex assembly. Finally, we present evidence that the association of the two complexes on the chromatin is not regulated by ATR checkpoint pathways that result from DNA replication blocks. These data suggest that Cdc7/Drf1 but not Cdc7/Dbf4 complexes support the initiation of DNA replication in Xenopus egg extracts and during early embryonic development.In eukaryotes, initiation of DNA replication requires the assembly and activation of pre-replication complexes (pre-RCs) 5 on chromatin (1). Sequential binding to DNA of the origin recognition complex, Cdc6, Cdt1, and mini-chromosome maintenance proteins (Mcm2-7) lead to formation of pre-RCs. Pre-RC activation is under the control of two kinases, Cdk2 and Cdc7, and ultimately results in the loading of replication factors such as Cdc45 and the unwinding of replication origins by the MCM helicase complex (2-5).Cdc7 is a serine/threonine kinase that is conserved from yeast to human and is essential for cell proliferation and embryonic development (6). Like CDKs (cyclin-dependent kinases), Cdc7 activity is regulated by its association with a regulatory subunit, the Dbf4 protein. This complex is often referred to as DDK (Dbf4-dependent kinase). The existence in fission yeast of a Cdc7/Dbf4 complex paralog, Spo4/Spo6, and the recent discovery of multiple Dbf4-related molecules in animal cells suggest that they belong to a novel DDK protein kinase family (7-10

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Xenopus CDC7/DRF1 Complex Is Required for the Initiation of DNA Replication

Silva

Bradley

Gao

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…In contrast, mammalian cells possess both pathways. It is noteworthy that the two fungi most closely related to Pneumocystis, as assessed by comparing the amino acid sequences of homologous proteins, Schizosaccharomyces pombe and Saccharomyces cerevisiae, both lack thymidine kinase and the ability to efficiently take up exogenous deoxyribonucleosides, and thus cannot utilize exogenous thymidine (Hodson et al, 2003;Vernis et al, 2003). Both fungi have been genetically engineered to salvage thymidine and incorporate BrdU, which can be detected by immunofluorescent assays (Hodson et al, 2003;Vernis et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

“…However, Pneumocystis DNA is accessible to in situ hybridization by oligonucleotide probes in fixed tissues similar to those we utilized here (Edman et al, 1988), and BrdU incorporation has been detected by immunofluorescent staining in the ciliate Colpoda inflata (Gutierrez et al, 2000), and in Schizosaccharomyces pombe and Saccharomyces cerevisiae that have been genetically engineered to salvage thymidine (Hodson et al, 2003;Vernis et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

Inability of Pneumocystis organisms to incorporate bromodeoxyuridine suggests the absence of a salvage pathway for thymidine

Vestereng

Kovacs

2004

Microbiology

View full text Add to dashboard Cite

Because thymidine metabolism is a potential target for therapy of Pneumocystis pneumonia, it was investigated whether Pneumocystis organisms have a salvage pathway for thymidine by administering 5-bromo-29-deoxyuridine (BrdU) to mice and rats with Pneumocystis pneumonia. Although BrdU incorporation was detected in host cells, no incorporation was seen in Pneumocystis organisms infecting either rats or mice. This suggests that Pneumocystis organisms do not have a salvage pathway for thymidine, and that inhibitors of de novo synthesis, such as thymidylate synthase inhibitors, may be effective drugs for treating Pneumocystis pneumonia. INTRODUCTIONPneumocystis jirovecii is an atypical fungus that infects patients suffering from a variety of immunosuppressive diseases, such as AIDS and cancer (Kovacs et al., 2001a;Roblot et al., 2002). Although there has been a decrease in morbidity among AIDS patients due to the availability of potent anti-retroviral regimens, Pneumocystis pneumonia (PCP) continues to be the most common life-threatening opportunistic infection among HIV-infected patients. Therapy with the combination of trimethoprim and sulfamethoxazole (TMP/SMX) is the preferred approach for primary prophylaxis in susceptible patients (i.e. for AIDS patients with CD4 + count levels <200 mm 23 ) as well as for treatment of active PCP (Kovacs & Masur, 2000;Phair et al., 1990). These drugs target the enzymes dihydrofolate reductase and dihydropteroate synthase (DHPS), respectively. Other drugs including dapsone, atovaquone and pentamidine are alternatives to TMP/SMX, but they are either more toxic or less active against P. jirovecii (Hughes Hughes, 1998;Kovacs et al., 2001a;Sattler et al., 1988; Vasconcelles et al., 2000). Unfortunately, recent studies have identified mutations in the gene encoding DHPS suggesting the emergence of resistance in P. jirovecii to sulfa drugs (dapsone and SMX) (Helweg-Larsen et al., 1999;Kazanjian et al., 2000;Ma et al., 1999;Navin et al., 2001). Development of new drugs to treat or prevent PCP thus remains an important priority for research.P. jirovecii is closely related to Pneumocystis species that infect rats (Pneumocystis carinii) and mice (P. carinii f. sp. muris). A better understanding of the metabolic pathways of Pneumocystis species may lead to the identification of novel therapeutic targets. Thymidine metabolism has been partially characterized in Pneumocystis: thymidylate synthase has been identified, isolated, cloned and crystallized (Anderson et al., 2000; Edman et al., 1989;Kovacs et al., 1990;Santi et al., 1991). Thus, Pneumocystis organisms possess de novo synthetic pathways for thymidine, a target of chemotherapy in other infections. Therapies that target de novo thymidylate synthesis can potentially be bypassed using thymidine salvage pathways. To see if Pneumocystis organisms possessed a salvage pathway for thymidine, 5-bromo-29-deoxyuridine (BrdU) was administered for 14 days to Pneumocystis-infected rats and mice. BrdU is a thymidine analogue which can be incorpora...

show abstract

“…After transformation of the shuffle strains with pRS315-RPO21 or pRS315-rpb1-1, the resulting strains were streaked twice on 5-FOA plates and then on SC-Leu. Sp strain FY2317 h+, leu1-32ThENT1-leu1+(pJAH29) his7-366Thsv-tkhis7+(pJAH31) ura4-D18 ade6-M210 (Hodson et al 2003) was kindly provided by Susan Forsburg. YPD medium was inoculated with a single Sc colony.…”

Section: Yeast Strains and Growth Curvesmentioning

confidence: 99%

Comparative dynamic transcriptome analysis (cDTA) reveals mutual feedback between mRNA synthesis and degradation

Sun

Schwalb²,

Schulz³

et al. 2012

Genome Res.

277

377

View full text Add to dashboard Cite

To monitor eukaryotic mRNA metabolism, we developed comparative dynamic transcriptome analysis (cDTA). cDTA provides absolute rates of mRNA synthesis and decay in Saccharomyces cerevisiae (Sc) cells with the use of Schizosaccharomyces pombe (Sp) as an internal standard. cDTA uses nonperturbing metabolic labeling that supersedes conventional methods for mRNA turnover analysis. cDTA reveals that Sc and Sp transcripts that encode orthologous proteins have similar synthesis rates, whereas decay rates are fivefold lower in Sp, resulting in similar mRNA concentrations despite the larger Sp cell volume. cDTA of Sc mutants reveals that a eukaryote can buffer mRNA levels. Impairing transcription with a point mutation in RNA polymerase (Pol) II causes decreased mRNA synthesis rates as expected, but also decreased decay rates. Impairing mRNA degradation by deleting deadenylase subunits of the Ccr4-Not complex causes decreased decay rates as expected, but also decreased synthesis rates. Extended kinetic modeling reveals mutual feedback between mRNA synthesis and degradation that may be achieved by a factor that inhibits synthesis and enhances degradation.

show abstract

Efficient labeling of fission yeast Schizosaccharomyces pombe with thymidine and BUdR

Cited by 61 publications

References 34 publications

Xenopus CDC7/DRF1 Complex Is Required for the Initiation of DNA Replication

Xenopus CDC7/DRF1 Complex Is Required for the Initiation of DNA Replication

Inability of Pneumocystis organisms to incorporate bromodeoxyuridine suggests the absence of a salvage pathway for thymidine

Comparative dynamic transcriptome analysis (cDTA) reveals mutual feedback between mRNA synthesis and degradation

Contact Info

Product

Resources

About