ORC-dependent and origin-specific initiation of DNA replication at defined foci in isolated yeast nuclei.
Genes Dev
; 11(12): 1504-18, 1997 Jun 15.
Article
em En
| MEDLINE
| ID: mdl-9203578
ABSTRACT
We describe an in vitro replication assay from yeast in which the addition of intact nuclei to an S-phase nuclear extract results in the incorporation of deoxynucleotides into genomic DNA at spatially discrete foci. When BrdUTP is substituted for dTTP, part of the newly synthesized DNA shifts to a density on CsCl gradients, indicative of semiconservative replication. Initiation occurs in an origin-specific manner and can be detected in G1- or S-phase nuclei, but not in G2-phase or mitotic nuclei. The S-phase extract contains a heat- and 6-DMAP-sensitive component necessary to promote replication in G1-phase nuclei. Replication of nuclear DNA is blocked at the restrictive temperature in an orc2-1 mutant, and the inactive Orc2p cannot be complemented in trans by an extract containing wild-type ORC. The initiation of DNA replication in cln-deficient nuclei blocked in G1 indicates that the ORC-dependent prereplication complex is formed before Start. This represents the first nonviral and nonembryonic replication system in which DNA replication initiates in an ORC-dependent and origin-specific manner in vitro.
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Base de dados:
MEDLINE
Assunto principal:
Saccharomyces cerevisiae
/
DNA Fúngico
/
Ciclo Celular
/
Origem de Replicação
/
Replicação do DNA
Idioma:
En
Ano de publicação:
1997
Tipo de documento:
Article