Cell cycle expression of two replicative DNA polymerases alpha and delta from Schizosaccharomyces pombe.

Park, H.; Francesconi, Stefania; Wang, T. S.-F.

doi:10.1091/mbc.4.2.145

Cited by 39 publications

(27 citation statements)

References 66 publications

(66 reference statements)

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“…The Pol␣-primase complex was immunoprecipitated from cell lysates by using anti-Pol␣ antibody (27) immobilized on protein A-agarose. One milligram of total cell extract protein was mixed with 15 l of the anti-Pol␣ antibody beads in the cell lysis buffer described above.…”

Section: Methodsmentioning

confidence: 99%

Analysis of Fission Yeast Primase Defines the Checkpoint Responses to Aberrant S Phase Initiation

Tan

Wang

2000

Molecular and Cellular Biology

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To investigate the checkpoint response to aberrant initiation, we analyzed the cell cycle checkpoint response induced by mutations of Schizosaccharomyces pombe DNA primase. DNA primase has two subunits, Spp1 and Spp2 (S. pombe primases 1 and 2). Spp1 is the catalytic subunit that synthesizes the RNA primer, which is then extended by DNA polymerase ␣ (Pol␣) to synthesize an initiation DNA structure, and this catalytic function of Pol␣ is a prerequisite for generating the S-M phase checkpoint. Here we show that Spp2 is required for coupling the function of Spp1 to Pol␣. Thermosensitive mutations of spp2 ؉ destabilize the Pol␣-primase complex, resulting in an allele-specific S phase checkpoint defect. The mutant exhibiting a more severe checkpoint defect also has a higher extent of Pol␣-primase complex instability and deficiency in the hydroxyurea-induced Cds1-mediated intra-S phase checkpoint response. However, this mutant is able to activate the Cds1 response to S phase arrest induced by temperature. These findings suggest that the Cds1 response to the S-phase arrest signal(s) induced by a initiation mutant is different from that induced by hydroxyurea. Interestingly, a pol␣ts mutant with a defective S-M phase checkpoint and an spp2 mutant with an intact checkpoint have a similar Pol␣-primase complex stability, and the Cds1 response induced by hydroxyurea or by the mutant arrests at the restrictive temperature. Thus, the Cds1-mediated intra-S phase checkpoint response induced by hydroxyurea can also be distinguished from the S-M phase checkpoint response that requires the initiation DNA synthesis by Pol␣.

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Section: Methodsmentioning

confidence: 99%

Analysis of Fission Yeast Primase Defines the Checkpoint Responses to Aberrant S Phase Initiation

Tan

Wang

2000

Molecular and Cellular Biology

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“…3C, left bottom panel). Due to the weak reactivity of anti-Pol␣ antibody (32) in detecting Pol␣/p180 protein in crude cell extract, Pol␣ was detected in spb70-U03 crude cell extract in poor quality (data not shown). Despite having a substantially lower level of Spb70 protein in the spb70-U03 mutant, Ni beads were able to pull down the Spb70 mutant protein after 4 h at 36°C from whole-cell extracts of the mutant.…”

Section: Vol 24 2004 Role Of the B-subunit Of Pol␣-primase In Replimentioning

confidence: 99%

The B-Subunit of DNA Polymerase α-Primase Associates with the Origin Recognition Complex for Initiation of DNA Replication

Uchiyama

Wang

2004

Molecular and Cellular Biology

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The B-subunit (p70/Pol12p) of the DNA polymerase ␣-primase (Pol␣-primase) complex is thought to have a regulatory role in an early stage of S phase. We generated a panel of fission yeast thermosensitive mutants of the B-subunit (termed Spb70) to investigate its role in initiation of DNA replication by genetic and biochemical approaches. Here, we show that the fission yeast Spb70 genetically interacts and coprecipitates with origin recognition complex proteins Orp1/Orc1 and Orp2/Orc2 and primase coupling subunit Spp2/p58. A fraction of Spb70 associates with Orp2 on chromatin throughout the cell cycle independent of the other subunits of Pol␣-primase. Furthermore, primase Spp2/p58 subunit preferentially associates with the unphosphorylated Orp2, and the association requires Spb70. Mutations in orp2؉ that abolish or mimic the Cdc2 phosphorylation of Orp2 suppress or exacerbate the thermosensitivity of the spb70 mutants, respectively, indicating that an unphosphorylated Orp2 promotes an Spb70-dependent replication event. Together, these results indicate that the chromatin-bound B-subunit in association with origin recognition complex mediates recruiting Pol␣-primase complex onto replication origins in G 1 pre-Start through an interaction with primase Spp2/p58 subunit. Our results thus suggest a role for the recruited Pol␣-primase in the initiation of both leading and lagging strands at the replication origins.Initiation of eukaryotic chromosome replication requires the assembly of multiprotein complexes onto the chromosomal replication origins. Upon activation of the multiprotein complexes by two types of kinases, cyclin-dependent kinases (CDK) and Cdc7/Dbf4 kinase (DDK), cells transit from prereplication stage in G 1 to replication in S phase (7). A principal player in the replication complex for initiation of replication is DNA polymerase ␣-primase (Pol␣-primase). Pol␣-primase is a heterotetrameric enzyme complex, which is unique among the replicative polymerases for its ability to initiate de novo DNA synthesis of a short RNA primer on the leading and lagging strand templates by the primase activity and to extend RNA primer into a 35-nucleotide RNA-DNA primer, termed initiator DNA, by the DNA polymerase activity (6,42,44). The largest subunit of Pol␣-primase complex is of 180 kDa (p180), and it contains the DNA polymerase catalytic activity. The primase activity resides in two smaller subunits of 58 (p58) and 49 (p49) kDa. The p49 is the primase catalytic subunit that synthesizes the RNA primer. The p58 subunit is the coupling subunit of the p49 to p180 and is thought to play a role in regulating the length of RNA primer synthesis (3, 11, 37). The 70-kDa subunit, also named B-subunit, has no detectable enzymatic activity. B-subunit is essential for budding yeast (Saccharomyces cerevisiae) cell viability and has been proposed to execute a critical function in an initial stage of S phase prior to the hydroxyurea (HU) arrest point. Thus, B-subunit has been proposed to play a regulatory role in initiation of S phase ...

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“…p125 is the catalytic subunit for both DNA polymerase activity and a proofreading, 3 -5 exonuclease activity. cDNAs for each subunit have been cloned (175)(176)(177)(178)(179)(180)(181)(182)(183). Moreover, an active human dimeric complex has been reconstituted using proteins expressed in recombinant baculovirus-infected insect cells (184).…”

Section: Dna Polymerases δ and εmentioning

confidence: 99%

The Dna Replication Fork in Eukaryotic Cells

Waga

Stillman

1998

Annu. Rev. Biochem.

739

653

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Replication of the two template strands at eukaryotic cell DNA replication forks is a highly coordinated process that ensures accurate and efficient genome duplication. Biochemical studies, principally of plasmid DNAs containing the Simian Virus 40 origin of DNA replication, and yeast genetic studies have uncovered the fundamental mechanisms of replication fork progression. At least two different DNA polymerases, a single-stranded DNA-binding protein, a clamp-loading complex, and a polymerase clamp combine to replicate DNA. Okazaki fragment synthesis involves a DNA polymerase-switching mechanism, and maturation occurs by the recruitment of specific nucleases, a helicase, and a ligase. The process of DNA replication is also coupled to cell-cycle progression and to DNA repair to maintain genome integrity. CONTENTS

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Cell cycle expression of two replicative DNA polymerases alpha and delta from Schizosaccharomyces pombe.

Cited by 39 publications

References 66 publications

Analysis of Fission Yeast Primase Defines the Checkpoint Responses to Aberrant S Phase Initiation

Analysis of Fission Yeast Primase Defines the Checkpoint Responses to Aberrant S Phase Initiation

The B-Subunit of DNA Polymerase α-Primase Associates with the Origin Recognition Complex for Initiation of DNA Replication

The Dna Replication Fork in Eukaryotic Cells

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