Accumulation of Single-Stranded DNA and Destabilization of Telomeric Repeats in Yeast Mutant Strains Carrying a Deletion of <i>RAD27</i>

Parenteau, Julie; Wellinger, Raymund J.

doi:10.1128/mcb.19.6.4143

Cited by 122 publications

(115 citation statements)

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“…Mutations in genes involved in replication of the lagging strand (Pol␣, RAD27) cause an increase in the amount of telomeric single-stranded DNA (ssDNA) (24,36). We examined the telomeric ssDNA in rad24, ctf18, rfc1, and elg1 mutants and found no accumulation of ssDNA (data not shown).…”

Section: Resultsmentioning

confidence: 99%

ELG1 , a regulator of genome stability, has a role in telomere length regulation and in silencing

Smolikov

Mazor

Krauskopf

2004

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

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Telomeres, the natural ends of eukaryotic chromosomes, prevent the loss of chromosomal sequences and preclude their recognition as broken DNA. Telomere length is kept under strict boundaries by the action of various proteins, some with negative and others with positive effects on telomere length. Recently, data have been accumulating to support a role for DNA replication in the control of telomere length, although through a currently poorly understood mechanism. Elg1p, a replication factor C (RFC)-like protein of yeast, contributes to genome stability through a putative replication-associated function. Here, we show that Elg1p participates in negative control of telomere length and in telomeric silencing through a replication-mediated pathway. We show that the telomeric function of Elg1 is independent of recombination and completely dependent on an active telomerase. Additionally, this function depends on yKu and DNA polymerase. We discuss alternative models to explain how Elg1p affects telomere length.DNA replication ͉ replication factor C ͉ Saccharomyces cerevisiae R eplication of a linear DNA molecule is carried out by DNA polymerases and additional proteins that duplicate the DNA by moving coordinately on both the lagging and leading strands (1). DNA attrition at the ends of chromosomes due to the ''end replication problem'' is avoided by the action of the specialized reverse transcriptase, called telomerase (2, 3). Telomerase adds G-rich DNA repeats termed telomeres onto the ends of chromosomes. In addition to their role in preventing loss of chromosomal sequences, telomeres serve as a boarding pad for a multiprotein complex that is implicated in several important cellular functions. Among these functions is capping telomeres, thus avoiding the recognition of the natural ends of chromosomes as broken DNA. This capping in turn prevents cell cycle arrest and chromosomal fusions, allowing proper chromosomal segregation (4).Many proteins, most of which are part of the telomeric complex, regulate telomere length in both positive and negative fashions (5). The best-studied pathway of telomerase-negative regulation is the Rap1p pathway in yeast. Rap1p is bound throughout the telomeric sequence and interacts with two proteins, Rif1p and Rif2p (6, 7). The resulting complex is thought to prevent telomerase from accessing the telomeric DNA substrate by a yet unknown mechanism. Additional factors were shown to be involved in telomere length regulation. Tel1p and the yKu70p-yKu80p heterodimer act as positive regulators of telomerase (8-10). Tel1p is proposed to act by recruiting the MRX (Mer11͞Rad50͞Xrs2) complex (11). The yKu70p͞yKu80p heterodimer influences telomere length by protecting telomere ends from degradation. It also has additional functions in telomeric silencing (through recruiting Sir proteins to the telomeres), telomeric nuclear localization, and telomere replication timing (12-15). Cdc13p binds the telomeric single strand, recruits telomerase to the telomere, and prevents degradation (16,17). By these fu...

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Section: Resultsmentioning

confidence: 99%

ELG1 , a regulator of genome stability, has a role in telomere length regulation and in silencing

Smolikov

Mazor

Krauskopf

2004

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

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“…Suggesting that telomeric localization implies a telomeric function for Dna2, we have shown that dna2-2 est1⌬ mutants, lacking telomerase activity, senesce much more rapidly than est1⌬ mutants, and dna2-2 est1⌬ mutants show altered pathways of telomerase-independent survival (14). dna2 mutants have normal or slightly long telomeres, but overproduction of DNA2 leads to increased occurrence of single-stranded regions at telomeres (17,39). Figure 6 compares telomere length in wild-type, pif1⌬, and dna2⌬ pif1⌬ strains.…”

Section: Resultsmentioning

confidence: 99%

Evidence Suggesting that Pif1 Helicase Functions in DNA Replication with the Dna2 Helicase/Nuclease and DNA Polymerase δ

Budd

Reis

Smith

et al. 2006

Molecular and Cellular Biology

191

261

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The precise machineries required for two aspects of eukaryotic DNA replication, Okazaki fragment processing (OFP) and telomere maintenance, are poorly understood. In this work, we present evidence that Saccharomyces cerevisiae Pif1 helicase plays a wider role in DNA replication than previously appreciated and that it likely functions in conjunction with Dna2 helicase/nuclease as a component of the OFP machinery. In addition, we show that Dna2, which is known to associate with telomeres in a cell-cycle-specific manner, may be a new component of the telomere replication apparatus. Specifically, we show that deletion of PIF1 suppresses the lethality of a DNA2-null mutant. The pif1⌬ dna2⌬ strain remains methylmethane sulfonate sensitive and temperature sensitive; however, these phenotypes can be suppressed by further deletion of a subunit of pol ␦, POL32. Deletion of PIF1 also suppresses the cold-sensitive lethality and hydroxyurea sensitivity of the pol32⌬ strain. Dna2 is thought to function by cleaving long flaps that arise during OFP due to excessive strand displacement by pol ␦ and/or by an as yet unidentified helicase. Thus, suppression of dna2⌬ can be rationalized if deletion of POL32 and/or PIF1 results in a reduction in long flaps that require Dna2 for processing. We further show that deletion of DNA2 suppresses the long-telomere phenotype and the high rate of formation of gross chromosomal rearrangements in pif1⌬ mutants, suggesting a role for Dna2 in telomere elongation in the absence of Pif1.Yeast Pif1 is the founding member of the Pif1 subfamily of superfamily 1 DNA helicases (3). While other organisms, such as Caenorhabditis elegans and Homo sapiens, have only one identified Pif1 family member, in yeast, there is a second, closely related, protein, Rrm3p (3). In yeast, neither of these helicases is essential and mutants lacking both are viable and repair proficient. Both yeast proteins have 5Ј-to-3Ј DNA helicase activity (21,30,31). The region of similarity between Pif1 and Rrm3 is limited to the seven helicase motifs, which exhibit 40% identity and 60% similarity (3). This may indicate that the two helicases have structurally similar DNA substrates. Nevertheless, the two helicases differ in their biological functions, and these differences are likely mediated not only by the helicase domain but also by the divergent N termini, which are not required for helicase activity (4). To date, it has been impossible to determine which helicase, Rrm3 or Pif1, is the functional homolog of the single ortholog in other eukaryotes.One difference between Rrm3 and Pif1 is in their function at the rRNA gene. In rrm3 mutants, there is an increase in replisome pausing at the Fob1 protein-bound replication fork barrier (RFB) in the rRNA gene (22). The hypothesis is that Rrm3 is required to remove proteins that block the fork at that point, since Rrm3 is required for promoting fork movement at over 1,400 loci in the yeast genome, in addition to the RFB. In contrast to Rrm3, Pif1 seems to be required for pausing at the rRNA...

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“…In support of this, the involvement of Dna2 with regard to telomere homeostasis was demonstrated in S. cerevisiae (51) and S. pombe (52). In addition, it was reported that Fen1 is also involved in telomere homeostasis (32). Therefore, it is likely that Mph1 is capable of stimulating both nucleases functions in telomere homeostasis.…”

Section: Discussionmentioning

confidence: 99%

The MPH1 Gene of Saccharomyces cerevisiae Functions in Okazaki Fragment Processing

Kang

Kim

et al. 2009

Journal of Biological Chemistry

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Saccharomyces cerevisiae MPH1 was first identified as a gene encoding a 3 to 5 DNA helicase, which when deleted leads to a mutator phenotype. In this study, we isolated MPH1 as a multicopy suppressor of the dna2K1080E helicase-negative lethal mutant. Purified Mph1 stimulated the endonuclease activities of both Fen1 and Dna2, which act faithfully in the processing of Okazaki fragments. This stimulation required neither ATP hydrolysis nor the helicase activity of Mph1. Multicopy expression of MPH1 also suppressed the temperature-sensitive growth defects in cells expressing dna2⌬405N, which lacks the N-terminal 405 amino acids of Dna2. However, Mph1 did not stimulate the endonuclease activity of the Dna2⌬405N mutant protein. The stimulation of Fen1 by Mph1 was limited to flap-structured substrates; Mph1 hardly stimulated the 5 to 3 exonuclease activity of Fen1. Mph1 binds to flapstructured substrate more efficiently than to nicked duplex structures, suggesting that the stimulatory effect of Mph1 is exerted through its binding to DNA substrates. In addition, we found that Mph1 reversed the inhibitory effects of replication protein A on Fen1 activity. Our biochemical and genetic data indicate that the in vivo suppression of Dna2 defects observed with both dna2K1080E and dna2⌬405N mutants occur via stimulation of Fen1 activity. These findings suggest that Mph1 plays an important, although not essential, role in processing of Okazaki fragments by facilitating the formation of ligatable nicks.Lagging strand DNA synthesis requires the orchestrated actions of many proteins and can be divided into several distinct enzymatic steps (1-4). First, the polymerase (pol) 2 ␣-primase complex synthesizes RNA-DNA primers on the template DNA that are recognized by replication factor C. This complex loads proliferating cell nuclear antigen onto DNA, which acts as a processivity factor tethering pol ␦ to primer ends (5-7). This series of reactions leads to a polymerase switch in which the pol ␣-primase complex at primer ends is displaced and replaced by pol ␦. Okazaki fragments are then elongated by pol ␦ until they encounter downstream Okazaki fragments (2,(8)(9)(10). Pol ␦ continues to synthesize DNA by displacing the 5Ј termini of downstream Okazaki fragments, which generate 5Ј RNA-DNA flap structures (11). These flaps are then cleaved by structure-specific nucleases that lead to the generation of ligatable nicks, which are sealed by DNA ligase converting the noncontiguous lagging strands to a contiguous DNA chain (12, 13).

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Accumulation of Single-Stranded DNA and Destabilization of Telomeric Repeats in Yeast Mutant Strains Carrying a Deletion of RAD27

Cited by 122 publications

References 59 publications

ELG1 , a regulator of genome stability, has a role in telomere length regulation and in silencing

ELG1 , a regulator of genome stability, has a role in telomere length regulation and in silencing

Evidence Suggesting that Pif1 Helicase Functions in DNA Replication with the Dna2 Helicase/Nuclease and DNA Polymerase δ

The MPH1 Gene of Saccharomyces cerevisiae Functions in Okazaki Fragment Processing

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