Role of the Putative Zinc Finger Domain of Saccharomyces cerevisiae DNA Polymerase ε in DNA Replication and the S/M Checkpoint Pathway

Dua, Rajiv; Levy, Daniel L.; Campbell, Judith L.

doi:10.1074/jbc.273.45.30046

Cited by 76 publications

(115 citation statements)

References 41 publications

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“…DPB3 and DPB4 encode the two nonessential components of DNA polymerase e (Pol e) and have been implicated in replication fork progression (Araki et al 1991;Dua et al 1998;Ohya et al 2000). In addition, previous studies have demonstrated roles for Dpb3 and Dpb4 in telomeric silencing (Tsubota et al 2006) and have shown that Dpb4 is also a component of the Isw2 complex (Iida and Araki 2004;McConnell et al 2004).…”

Section: Resultsmentioning

confidence: 99%

Alterations in DNA Replication and Histone Levels Promote Histone Gene Amplification in Saccharomyces cerevisiae

Libuda¹,

Winston

2010

Genetics

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Gene amplification, a process that increases the copy number of a gene or a genomic region to two or more, is utilized by many organisms in response to environmental stress or decreased levels of a gene product. Our previous studies in Saccharomyces cerevisiae identified the amplification of a histone H2A-H2B gene pair, HTA2-HTB2, in response to the deletion of the other H2A-H2B gene pair, HTA1-HTB1. This amplification arises from a recombination event between two flanking Ty1 elements to form a new, stable circular chromosome and occurs at a frequency higher than has been observed for other Ty1-Ty1 recombination events. To understand the regulation of this amplification event, we screened the S. cerevisiae nonessential deletion set for mutations that alter the amplification frequency. Among the deletions that increase HTA2-HTB2 amplification frequency, we identified those that either decrease DNA replication fork progression (rrm3D, dpb3D, dpb4D, and clb5D) or that reduce histone H3-H4 levels (hht2-hhf2D). These two classes are related because reduced histone H3-H4 levels increase replication fork pauses, and impaired replication forks cause a reduction in histone levels. Consistent with our mutant screen, we found that the introduction of DNA replication stress by hydroxyurea induces the HTA2-HTB2 amplification event. Taken together, our results suggest that either reduced histone levels or slowed replication forks stimulate the HTA2-HTB2 amplification event, contributing to the restoration of normal chromatin structure.

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Section: Resultsmentioning

confidence: 99%

Alterations in DNA Replication and Histone Levels Promote Histone Gene Amplification in Saccharomyces cerevisiae

Libuda¹,

Winston

2010

Genetics

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“…However, the requirement of Mec1p for replication of late-replicating regions is attributed to Mec1's role in the fork progression itself (54, 55) (through direct phosphorylation of replication machinery), not due to a defect in ATR signaling. Thus, it is compelling to hypothesize that in cdc14 mutants failure to recognize underreplication is not due to a checkpoint system defect, but rather is a function of the slow replication speed itself, as was shown for DNA polymerase epsilon mutants (56,57).…”

Section: Discussionmentioning

confidence: 96%

Essential global role of CDC14 in DNA synthesis revealed by chromosome underreplication unrecognized by checkpoints in cdc14 mutants

Dulev

Renty

Mehta

et al. 2009

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

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The CDC14 family of multifunctional evolutionarily conserved phosphatases includes major regulators of mitosis in eukaryotes and of DNA damage response in humans. The CDC14 function is also crucial for accurate chromosome segregation, which is exemplified by its absolute requirement in yeast for the anaphase segregation of nucleolar organizers; however the nature of this essential pathway is not understood. Upon investigation of the rDNA nondisjunction phenomenon, it was found that cdc14 mutants fail to complete replication of this locus. Moreover, other late-replicating genomic regions (10% of the genome) are also underreplicated in cdc14 mutants undergoing anaphase. This selective genome-wide replication defect is due to dosage insufficiency of replication factors in the nucleus, which stems from two defects, both contingent on the reduced CDC14 function in the preceding mitosis. First, a constitutive nuclear import defect results in a drastic dosage decrease for those replication proteins that are regulated by nuclear transport. Particularly, essential RPA subunits display both lower mRNA and protein levels, as well as abnormal cytoplasmic localization. Second, the reduced transcription of MBF and SBF-controlled genes in G1 leads to the reduction in protein levels of many proteins involved in DNA replication. The failure to complete replication of late replicons is the primary reason for chromosome nondisjunction upon CDC14 dysfunction. As the genome-wide slow-down of DNA replication does not trigger checkpoints [Lengronne A, Schwob E (2002) Mol Cell 9:1067-1078], CDC14 mutations pose an overwhelming challenge to genome stability, both generating chromosome damage and undermining the checkpoint control mechanisms.S uccessful chromosome segregation in mitosis requires the coordinated work of cellular regulatory circuits, which transmit cell cycle signals to chromatin and spindle proteins. One of such factors is the Cdc14 protein, an evolutionary conserved protein phosphatase (1-3). While in human cells a CDC14 ortholog has been recently shown to play a key role in DNA damage response (4), studies on S. cerevisiae were mostly focused on Cdc14p roles in anaphase regulation and in the exit from mitosis. The scope of Cdc14p activity in budding yeast is believed to be limited to anaphase, because Cdc14p is sequestered in the nucleolus (5) in apparently inactive form (6) at other cell cycle stages. Therefore, while Cdc14 can potentially dephosphorylate many substrates (7,8), the most studied physiological pathways are the anaphase pathways (FEAR and MEN), which are both dependent on the two sequential bursts of Cdc14 release (1, 9). The cdc14 mutations cause a mitotic exit block, but also display defects in nucleolar (10) and telomeric (11) segregation. The mechanisms of chromosome segregation defects (11-15) in cdc14 mutants are generally poorly understood. While condensin mutations phenocopy the cdc14 rDNA nondisjunction (11, 16) and Cdc14p is required for condensin loading to rDNA (14), it is unlikely that conden...

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“…2). In yeast, this area is very important for DNA replication and a checkpoint, its deletion resulting in sensitivity to methylmethane sulfonate (24,30). This region functions in protein/protein interaction and protein/DNA interaction.…”

Section: Discussionmentioning

confidence: 99%

“…2). This region was found to be essential for DNA replication and for an intact S/M cell cycle checkpoint in yeast (30). The subunit interaction region identified in DP2Pho(1255-1332) as shown above encompasses the putative zinc finger motif.…”

Section: The N-terminal 1-200 Domain Of Dp1pho Forms a Highly Stable mentioning

confidence: 91%

Subunit Interaction and Regulation of Activity through Terminal Domains of the Family D DNA Polymerase from Pyrococcus horikoshii

Shen

Tang

Matsui

2003

Journal of Biological Chemistry

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We also constructed and analyzed three internal deletion mutants and two site-directed mutants in the region of the putative zinc finger motif (cysteine cluster II) of DP2Pho at the COOH-terminal. We found that the internal region of the zinc finger motif is critical for the 3-5 exonuclease, but is dispensable for the DNA polymerization.Family D DNA polymerase (PolD) 1 is a recently found DNA polymerase that exists extensively in Euryarchaeota of Archaea, the third domain of life (1, 2 PolD is composed of a small subunit (DP1) and a large subunit (DP2). The interaction of DP1 and DP2 is essential for the stability and full activity of the enzyme (15, 16). DP1 or DP2 expressed in Escherichia coli is unstable and easily degraded. PolD possesses strong DNA polymerizing and 3Ј-5Ј exonuclease (proofreading) activities like other DNA replication polymerases, however, common motifs for the catalysis of other DNA polymerases are absent or not functional in PolD (17,19). Two invariant residues, Asp 1122 and Asp 1124 of DP2Pho, located in the most conserved region in the large subunit of PolD, were identified to be the catalytic residues for the DNA polymerization activities (17), indicating that the COOH-terminal of DP2 is involved in the formation of the catalytic center for DNA synthesis. A heterotetrameric structure for PolD from P. horikoshii (PolDPho) was proposed based on the result of gel filtration (17).Several lines of evidence indicate that family D DNA polymerase is likely the major DNA polymerase or replicase in Euryarchaeota, participating in DNA replication, repair, and recombination. In the genomes of the genus Pyrococcus, the genes coding the two subunits of PolD are adjacent to the replication origin and several essential genes for DNA metabolism (20). DP1 shows low but significant homology to the small subunit of eukaryote DNA polymerase ␣, ␦, and ⑀ (19, 21), and contains domain and catalytic motifs for the 3Ј-5Ј exonuclease, similar to those of Mre11, a nuclease involved in double strand DNA break repair (18,21,22). The amino acid sequences of DP2 contain a short region at the COOH-terminal, which shows homology to the catalytic subunit of yeast DNA polymerase ⑀ (this paper) around the COOH-terminal putative zinc finger motif (cysteine cluster II). This homologous region in yeast polymerase ⑀ is found to be vital for the survival of yeast (23,24). PolD interacts with many proteins related to DNA replication, repair, and recombination. It was found that DP1 from P. furiosus interacts specifically with archaeal RadB by yeast two-hybrid analysis and in vitro pull-down assays (25). DP2 directly interacts with proliferating cell nuclear antigen as * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Role of the Putative Zinc Finger Domain of Saccharomyces cerevisiae DNA Polymerase ε in DNA Replication and the S/M Checkpoint Pathway

Cited by 76 publications

References 41 publications

Alterations in DNA Replication and Histone Levels Promote Histone Gene Amplification in Saccharomyces cerevisiae

Alterations in DNA Replication and Histone Levels Promote Histone Gene Amplification in Saccharomyces cerevisiae

Essential global role of CDC14 in DNA synthesis revealed by chromosome underreplication unrecognized by checkpoints in cdc14 mutants

Subunit Interaction and Regulation of Activity through Terminal Domains of the Family D DNA Polymerase from Pyrococcus horikoshii

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