Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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 alpha (Polalpha) to synthesize an initiation DNA structure, and this catalytic function of Polalpha is a prerequisite for generating the S-M phase checkpoint. Here we show that Spp2 is required for coupling the function of Spp1 to Polalpha. Thermosensitive mutations of spp2(+) destabilize the Polalpha-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 Polalpha-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 polalphats mutant with a defective S-M phase checkpoint and an spp2 mutant with an intact checkpoint have a similar Polalpha-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 Polalpha.
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PMID:Analysis of fission yeast primase defines the checkpoint responses to aberrant S phase initiation. 1102 57

The B-subunit (p70/Pol12p) of the DNA polymerase alpha-primase (Polalpha-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 Polalpha-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 Polalpha-primase complex onto replication origins in G1 pre-Start through an interaction with primase Spp2/p58 subunit. Our results thus suggest a role for the recruited Polalpha-primase in the initiation of both leading and lagging strands at the replication origins.
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PMID:The B-subunit of DNA polymerase alpha-primase associates with the origin recognition complex for initiation of DNA replication. 1531 53