A Yeast Replicative Helicase, Dna2 Helicase, Interacts with Yeast FEN-1 Nuclease in Carrying Out Its Essential Function

Budd, Martin E.; Campbell, Judith L.

doi:10.1128/mcb.17.4.2136

Cited by 204 publications

(204 citation statements)

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“…RAD27, the homolog of the mammalian FEN-1, is a structure-specific nuclease implicated in Okazaki fragment maturation during DNA replication. These genetic interactions suggested that at least one specific role for Dna2p is also in the maturation of the 5Ј ends of Okazaki fragments (Budd and Campbell, 1997), and in vitro reconstitution studies indicate that Dna2 is required for efficient RNA removal and ligation when FEN-1 activity is impaired (Bae and Seo, 2000;Ayyagari et al, 2003; Kao, Campbell, and Bambara, unpublished data). With respect to aging, it is interesting that the human BLM gene, encoding a RecQ helicase defective in Bloom syndrome, can suppress the replication defect of dna2 mutants (Imamura and Campbell, 2003).…”

Section: Introductionmentioning

confidence: 99%

The Transcriptome of Prematurely Aging Yeast Cells Is Similar to That of Telomerase-deficient Cells

Lesur

Campbell

2004

MBoC

116

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To help define the pathologies associated with yeast cells as they age, we analyzed the transcriptome of young and old cells isolated by elutriation, which allows isolation of biochemical quantities of old cells much further advanced in their life span than old cells prepared by the biotin-streptavidin method. Both 18-generation-old wild-type yeast and 8-generation-old cells from a prematurely aging mutant (dna2-1), with a defect in DNA replication, were evaluated. Genes involved in gluconeogenesis, the glyoxylate cycle, lipid metabolism, and glycogen production are induced in old cells, signifying a shift toward energy storage. We observed a much more extensive generalized stress response known as the environmental stress response (ESR), than observed previously in biotin-streptavidin-isolated cells, perhaps because the elutriated cells were further advanced in their life span. In addition, there was induction of DNA repair genes that fall in the so-called DNA damage "signature" set. In the dna2-1 mutant, energy production genes were also induced. The response in the dna2-1 strain is similar to the telomerase delete response, genes whose expression changes during cellular senescence in telomerase-deficient cells. We propose that these results suggest, albeit indirectly, that old cells are responding to genome instability. INTRODUCTIONMuch aging research is aimed at identifying the processes that lead to the generation of age-related cellular damage and understanding exactly how and where this damage occurs. Recently, there has been renewed focus on model organisms, such as the budding yeast Saccharomyces cerevisiae, for experimental work on aging. Yeast provides a model for contributions to aging made by tissues and organs that consist of constantly proliferating cells, replicative aging. Yeast has many advantages for replicative aging studies, such as its short life span, completely sequenced genome, and well-characterized biology. Individual yeast cells are mortal, and their life span is measured by the number of times they divide, i.e., the number of daughter cells produced by a single mother (Mortimer and Johnston, 1959;Muller et al., 1980). These daughter cells have the potential for a full life span, whereas the mother cell increases in age with each cell division; thus, yeast aging is asymmetric. The probability that an individual yeast cell will produce daughters declines exponentially as a function of its age in cell divisions or generations (Jazwinski et al., 1998). As yeast cells age, their size increases (Mortimer and Johnston, 1959;Nestelbacher, 2000), their cell cycle slows down (Mortimer and Johnston, 1959), their shape is altered (Pichova et al., 1997), their nucleolus tends to be larger and/or more fragmented, and they become sterile (Guarente, 1997). In wild-type cells, extrachromosomal ribosomal DNA circles (ERCs) also accumulate . Cells in the second half of their life span also show a switch to a state inducing increase loss of heterozygosity (LOH) (McMurray and Gottschling, 2003). Although...

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Section: Introductionmentioning

confidence: 99%

The Transcriptome of Prematurely Aging Yeast Cells Is Similar to That of Telomerase-deficient Cells

Lesur

Campbell

2004

MBoC

116

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“…Although Fen1 acts most efficiently as a flap endonuclease, it is likely that similar enzymatic mechanisms are involved for its exonuclease activity (Lieber, 1997). Fen1 interacts with several proteins at the replication fork, such as PCNA (Li et al, 1995;Wu et al, 1996;Jo´nsson et al, 1998), Dna2 helicase/endonuclease (Budd and Campbell, 1997), Werner syndrome helicase/exonuclease (WRN) (Brosh Jr et al, 2001) and replication protein A (Biswas et al, 1997). The Dna2 helicase/endonuclease can physically and genetically interact with RAD27 in budding yeast (Budd and Campbell, 1997), and both play a direct role in Okazaki fragment maturation Kang et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

“…Fen1 interacts with several proteins at the replication fork, such as PCNA (Li et al, 1995;Wu et al, 1996;Jo´nsson et al, 1998), Dna2 helicase/endonuclease (Budd and Campbell, 1997), Werner syndrome helicase/exonuclease (WRN) (Brosh Jr et al, 2001) and replication protein A (Biswas et al, 1997). The Dna2 helicase/endonuclease can physically and genetically interact with RAD27 in budding yeast (Budd and Campbell, 1997), and both play a direct role in Okazaki fragment maturation Kang et al, 2000). Moreover, since WRN protein and Fen1 physically interact, an alternative pathway during DNA replication and/or repair has been proposed (Brosh Jr et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Phosphorylation of human Fen1 by cyclin-dependent kinase modulates its role in replication fork regulation

2003

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Cyclin-dependent kinase (Cdk) Cdk1-Cyclin A can phosphorylate Flap endonuclease 1 (Fen1), a key-enzyme of the DNA replication machinery, in late S phase. Cdk1-cyclin A forms a complex in vitro and in vivo with Fen1. Furthermore, Fen1 phosphorylation is detected in vivo and depends upon Cdks activity. As a functional consequence of phosphorylation by Cdk1-Cyclin A in vitro, endo-and exonuclease activities of Fen1 are reduced whereas its DNA binding is not affected. Moreover, phosphorylation of Fen1 by Cdk1-Cyclin A abrogates its proliferating cell nuclear antigen (PCNA) binding thus preventing stimulation of Fen1 by PCNA. Concomitantly, human cells expressing the S187A mutant defective for Cdk1-Cyclin A phosphorylation accumulate in S phase consistent with a failure in cell cycle regulation through DNA replication. Our results suggest a novel regulatory role of Cdks onto the end of S phase by targeting directly a key enzyme involved in DNA replication.

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“…DNA2 is essential and is required for the elongation stage of DNA replication in vivo and in vitro (11,(15)(16)(17)(18). High-copy-number suppression of the temperature-sensitive growth defect of a yeast rad27⌬ strain by DNA2 (and vice versa), synthetic lethality of the dna2-1 allele with a rad27⌬ mutation and with a DNA ligase mutant, and biochemical evidence for an interaction between Dna2 and the RAD27 gene product FEN1 suggest that Dna2 may be involved in Okazaki fragment maturation (19)(20)(21)(22). Interaction of DNA2 with lagging strand-replication functions has also been shown in Schizosaccharomyces pombe (23).…”

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confidence: 99%

The human Bloom syndrome gene suppresses the DNA replication and repair defects of yeastdna2mutants

Imamura

Campbell²

2003

Proc. Natl. Acad. Sci. U.S.A.

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Bloom syndrome is a disorder of profound and early cancer predisposition in which cells become hypermutable, exhibit high frequency of sister chromatid exchanges, and show increased micronuclei. BLM, the gene mutated in Bloom syndrome, has been cloned previously, and the BLM protein is a member of the RecQ family of DNA helicases. Many lines of evidence suggest that BLM is involved either directly in DNA replication or in surveillance during DNA replication, but its specific roles remain unknown. Here we show that hBLM can suppress both the temperature-sensitive growth defect and the DNA damage sensitivity of the yeast DNA replication mutant dna2-1. The dna2-1 mutant is defective in a helicase-nuclease that is required either to coordinate with the crucial Saccharomyces cerevisiae (sc) FEN1 nuclease in Okazaki fragment maturation or to compensate for scFEN1 when its activity is impaired. We show that human BLM interacts with both scDna2 and scFEN1 by using coimmunoprecipitation from yeast extracts, suggesting that human BLM participates in the same steps of DNA replication or repair as scFEN1 and scDna2. E ukaryotic genomes encode hundreds of genes with homology to DNA helicases. Many of these show functional overlap, making it difficult to assign the precise DNA structures that are unwound by the respective gene products during DNA replication, recombination, and repair. One class of human helicase, the RecQ family, has received special attention in the past few years, because mutations in three different RecQ helicase family members give rise to three distinct human disorders leading to cancer predisposition and͞or segmental premature aging, Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome (1-3). The genes affected in the diseases, BLM, WRN, and RECQ4, have a single, sequence-specific homolog in yeast called SGS1 (4,5). Both hBLM and hWRN have been shown to be biologically active in yeast by demonstrating the effects of their expression on sgs1⌬ mutants. mWRN suppresses the slow-growth phenotype and the inhibition of type II recombination at telomeres observed in est2⌬sgs1⌬ survivors (6). Both hBLM and hWRN suppress the excessive illegitimate and homologous recombination phenotypes of sgs1⌬ mutants (7). However, hBLM and hWRN are not completely interchangeable in yeast. hBLM, but not hWRN, restores slow growth in the sgs1⌬top3 mutants (7-10). Similarly, hBLM, but not hWRN, suppresses the premature aging phenotype of yeast sgs1⌬ mutants (8). Differential function in yeast is consistent with structural and biochemical differences between BLM and WRN. BLM and WRN are homologous mainly in the helicase domain but are divergent in N-terminal and C-terminal domains. WRN, but not BLM, contains an intrinsic nuclease activity in addition to helicase activity (see ref. 2 for review). There are also clear differences in physiological function, because the symptoms of Werner and Bloom syndromes and types of cellular damage in humans differ. Taken together, the sgs1 suppression studies clarify the function of ...

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A Yeast Replicative Helicase, Dna2 Helicase, Interacts with Yeast FEN-1 Nuclease in Carrying Out Its Essential Function

Cited by 204 publications

References 32 publications

The Transcriptome of Prematurely Aging Yeast Cells Is Similar to That of Telomerase-deficient Cells

The Transcriptome of Prematurely Aging Yeast Cells Is Similar to That of Telomerase-deficient Cells

Phosphorylation of human Fen1 by cyclin-dependent kinase modulates its role in replication fork regulation

The human Bloom syndrome gene suppresses the DNA replication and repair defects of yeastdna2mutants

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