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
en 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.
Buscar en Google
Base de datos:
MEDLINE
Asunto principal:
Saccharomyces cerevisiae
/
ADN de Hongos
/
Ciclo Celular
/
Origen de Réplica
/
Replicación del ADN
Idioma:
En
Revista:
Genes Dev
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
1997
Tipo del documento:
Article