De novo synthesis of budding yeast DNA polymerase alpha and POL1 transcription at the G1/S boundary are not required for entrance into S phase.

Muzi-Falconi, Marco; Piseri, Anna; Ferrari, Marina G.; Lucchini, Giovanna; Plevani, Paolo; Foiani, Marco

doi:10.1073/pnas.90.22.10519

Cited by 49 publications

(47 citation statements)

References 44 publications

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“…This result suggests that the cellular polymerase activity of this mutant is higher than the amount required for cell growth, which is consistent with the report that wild type pol ␣ concentration is in excess of the amount required for cell growth in yeast (55). However, these data suggest that Y951P pol ␣ causes genome instability, because its activity is lower than wild type pol ␣.…”

Section: Fig 3 Amino Acid Substitutions In Active Clones In Yeast (supporting

confidence: 66%

Distinct Function of Conserved Amino Acids in the Fingers of Saccharomyces cerevisiae DNA Polymerase α

Ogawa

Limsirichaikul

Niimi

et al. 2003

Journal of Biological Chemistry

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Structural differences between class A and B DNA polymerases suggest that the motif B region, a wall of the catalytic pocket, may have evolved differentially in the two polymerase families. This study examines the function of the motif B residues in Saccharomyces cerevisiae DNA polymerase ␣ (pol ␣). Effects of the mutations were determined by biochemical analysis and genetic complementation of a yeast strain carrying a temperature-sensitive pol ␣ mutant. Many conserved residues were viable with a variety of substitutions. Among them, mutations at Asn-948 or Tyr-951 conferred up to 8-fold higher colony formation frequency in a URA3 forward mutation assay, and 79-fold higher trp1 reversion frequency was observed for Y951P in yeast. Purified Y951P was as accurate as wild type in DNA synthesis but ϳ6-fold less processive and 22-fold less active in vitro. Therefore, Y951P may increase the frequency of mutant colony formation because of its low level of DNA polymerase activity in yeast. Mutations at Lys-944 or Gly-952 were not viable, which is consistent with the observation that mutants with substitutions at Gly-952 have strongly reduced catalytic activity in vitro. Gly-952 may provide a space for the nascent base pair and thus may play an essential function in S. cerevisiae DNA pol ␣. These results suggest that class B DNA polymerases have a unique structure in the catalytic pocket, which is distinct from the corresponding region in class A DNA polymerases.During cell division, a mother cell divides to generate two identical daughter cells, each of which receives a complete genetic complement. DNA replication is a critical step, which duplicates the parental chromosomes prior to cell division in the cell cycle. In eukaryotic cells, at least three DNA polymerases participate in replication of nuclear DNA: DNA polymerases ␣ (pol ␣), 1 ␦, and ⑀, all of which belong to class B (pol ␣) DNA polymerases (1). The pol ␣/DNA primase complex plays a key role in the initiation phase of DNA replication; pol ␦ and pol ⑀ play roles primarily in the elongation phase. During initiation of DNA synthesis, DNA primase synthesizes a short RNA chain (ϳ10-mer), and pol ␣ extends the RNA primer by ϳ30 deoxyribonucleotides. Pol ⑀ and pol ␦ are highly processive DNA polymerases that carry out bidirectional DNA synthesis during the elongation phase of DNA replication (2).Class B family DNA polymerases share structures and catalytic mechanism with class A DNA polymerases such as Taq DNA pol I (3, 4). Motif B is an essential region conserved in class A DNA polymerases. A Lys residue is essential in motif B in Taq pol I (Lys-663) and Klenow fragment (Lys-758) of Escherichia coli pol I (5-7). Phe-667 in Taq (and the equivalent residues in Klenow and T7 pol) are also critical for polymerase activity, as well as discrimination of 2Ј,3Ј-dideoxyribonucleotides (5, 8, 9). Tyr-766 in Klenow fragment (Tyr-671 in Taq pol I) plays a role in fidelity of DNA synthesis (10, 11). Recently, Ponamarev et al. (12) reported that a human pol ␥ mutant in which a T...

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Section: Fig 3 Amino Acid Substitutions In Active Clones In Yeast (supporting

confidence: 66%

Distinct Function of Conserved Amino Acids in the Fingers of Saccharomyces cerevisiae DNA Polymerase α

Ogawa

Limsirichaikul

Niimi

et al. 2003

Journal of Biological Chemistry

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“…This is of particular interest, given that Cdc17 levels seem to be tightly controlled in the cell. For example, in budding yeast, Cdc17/Pol1 overexpression results in immediate protein degradation, whereas the endogenous protein has a very low protein turnover rate in vivo (35). The same is true for p180, the mammalian counterpart for Cdc17/Pol1 (36).…”

mentioning

confidence: 56%

“…However, Cdc17 protein levels were restored quantitatively when Mcm10-3HA was expressed for 6 h prior to the temperature shift (Fig. 4C) (35). However, when Cdc17 is overexpressed, it is rapidly degraded (35), suggesting that a factor required to stabilize Cdc17 is absent in stoichiometric amounts.…”

Section: Exogenous Overexpression Of Mcm10 Restores Cdc17 Levels In Tmentioning

confidence: 97%

“…4C) (35). However, when Cdc17 is overexpressed, it is rapidly degraded (35), suggesting that a factor required to stabilize Cdc17 is absent in stoichiometric amounts. Given the role of Mcm10 as a nuclear chaperone for Cdc17, we hypothesized that Mcm10 levels were insufficient for stabilizing Cdc17 when the latter was overexpressed.…”

Section: Exogenous Overexpression Of Mcm10 Restores Cdc17 Levels In Tmentioning

confidence: 99%

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A Conserved Hsp10-like Domain in Mcm10 Is Required to Stabilize the Catalytic Subunit of DNA Polymerase-α in Budding Yeast

Ricke

Bielinsky

2006

Journal of Biological Chemistry

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Initiation of DNA replication is a strictly regulated process in eukaryotic cells. In order to prepare replication origins for initiation of DNA replication, a prereplicative complex assembles at origins during the G 1 phase of the cell cycle (1). The prereplicative complex is composed of the six-subunit initiator origin recognition complex, Cdc6, Cdt1, and the putative replicative helicase, the minichromosome maintenance 2-7 (Mcm2-7) complex (reviewed in Ref.2). All of these factors are conserved from yeast to humans. Once the Mcm2-7 complex is loaded (3, 4), the origin is licensed for a single round of DNA synthesis. Following loading of Mcm2-7, Mcm10 associates with replication origins (5-7), and biochemical evidence suggests that its addition results in activating the Cdc7-Dbf4 kinase (8). Cdc7-Dbf4 kinase and S phasespecific cyclin-dependent kinase activities increase at the G 1 /S phase transition, and their activation triggers the initiation of DNA replication. One likely target of the Cdc7-Dbf4 kinase is the Mcm2-7 complex (9). Phosphorylation of the Mcm2-7 complex precedes the loading of other replication factors, such as Cdc45 (10) and the four-subunit GINS complex (11,12). After origin unwinding, the single-stranded regions are coated by replication protein A (RPA) 2 (13,14), facilitating the recruitment of DNA polymerase-␣ (pol-␣)⅐primase (7,14). Pol-␣⅐pri-mase synthesizes a short RNA-DNA primer before being replaced by a processive DNA polymerase, such as pol-␦ or pol-⑀ (15). After initiation of DNA replication, the Mcm2-7 complex (4), Mcm10 (7), Cdc45 (16), the GINS complex (11,12), and the replicative DNA polymerases (16) migrate with the replication fork to promote DNA elongation.Since pol-␣ is the only enzyme in eukaryotic cells capable of initiating DNA synthesis de novo, its recruitment to replication origins is essential for the initiation of DNA replication. The largest subunit, Cdc17/Pol1 in budding yeast, contains the catalytic activity of pol-␣ (17). Cdc17/Pol1 binds the B-subunit of pol-␣, Pol12 (18 -20), and although Pol12 has no known catalytic activity, it is phosphorylated in a cell cycle-specific manner in budding yeast and in human cells (21,22). This may serve a regulatory role as cells progress through S phase. More recently, Pol12 has been shown to be associated with the origin recognition complex in fission yeast (23). In addition to Pol12, Cdc17/Pol1 also interacts with Pri2, which regulates the primase activity of the catalytic subunit, Pri1 (24).We and others have recently demonstrated that chromatin association of pol-␣⅐primase requires Mcm10 (7,25). Mcm10 is a conserved eukaryotic DNA replication factor that is essential for S-phase progression, since it serves a critical role in coordinating replication fork assembly (7). Mcm10 was initially identified in two independent genetic screens in Saccharomyces cerevisiae (26,27). In addition to pol-␣, Mcm10 has been shown to interact with several replication factors, including the Mcm2-7 complex, origin recognition complex, RPA,...

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“…Trichloroacetic acid (TCA) protein extracts 22 were separated by SDS-PAGE in 10% acrylamide gels. Western blotting was performed with α-HA (12CA5) and α-Rad53 antibodies, using standard techniques.…”

Section: Methodsmentioning

confidence: 99%

Alk1 and Alk2 are Two New Cell Cycle-Regulated Haspin-Like Proteins in Budding Yeast

Nespoli

Vercillo²,

Nola³

et al. 2006

Cell Cycle

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De novo synthesis of budding yeast DNA polymerase alpha and POL1 transcription at the G1/S boundary are not required for entrance into S phase.

Cited by 49 publications

References 44 publications

Distinct Function of Conserved Amino Acids in the Fingers of Saccharomyces cerevisiae DNA Polymerase α

Distinct Function of Conserved Amino Acids in the Fingers of Saccharomyces cerevisiae DNA Polymerase α

A Conserved Hsp10-like Domain in Mcm10 Is Required to Stabilize the Catalytic Subunit of DNA Polymerase-α in Budding Yeast

Alk1 and Alk2 are Two New Cell Cycle-Regulated Haspin-Like Proteins in Budding Yeast

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