RESUMEN
Initiation of DNA replication at oriC occurs bidirectionally both in vivo and in vitro. Although the proteins involved in establishing the replication forks are known, little is known about the events that ensure that initiation is bidirectional. We show here that in the absence of DNA gyrase, replication fork progression from oriC on a plasmid template in vitro is unidirectional, although both replication forks have formed at the origin. There was no bias in the release of one fork or the other, ruling out protein blockage of one fork as a possible reason for the asymmetric release. Timely release of both forks required the presence of either DNA gyrase or topoisomerase IV, suggesting that modulation of the topology of the origin region is the governing factor.
Asunto(s)
Proteínas Bacterianas , Replicación del ADN/genética , Replicación del ADN/fisiología , Western Blotting , Reactivos de Enlaces Cruzados/farmacología , Girasa de ADN/metabolismo , ADN Helicasas/metabolismo , AdnB Helicasas , Escherichia coli/enzimología , Escherichia coli/genética , Cinética , Modelos Genéticos , Unión Proteica , Factores de TiempoRESUMEN
Myb-related cdc5p is required for G(2)/M progression in the yeast Schizosaccharomyces pombe. We report here that all detectable cdc5p is stably associated with a multiprotein 40S complex. Immunoaffinity purification has allowed the identification of 10 cwf (complexed with cdc5p) proteins. Two (cwf6p and cwf10p) are members of the U5 snRNP; one (cwf9p) is a core snRNP protein. cwf8p is the apparent ortholog of the Saccharomyces cerevisiae splicing factor Prp19p. cwf1(+) is allelic to the prp5(+) gene defined by the S. pombe splicing mutant, prp5-1, and there is a strong negative genetic interaction between cdc5-120 and prp5-1. Five cwfs have not been recognized previously as important for either pre-mRNA splicing or cell cycle control. Further characterization of cwf1p, cwf2p, cwf3p, and cwf4p demonstrates that they are encoded by essential genes, cosediment with cdc5p at 40S, and coimmunoprecipitate with cdc5p. We further show that cdc5p associates with the U2, U5, and U6 snRNAs and that cells lacking cdc5(+) function are defective in pre-mRNA splicing. These data raise the possibility that the cdc5p complex is an intermediate in the assembly or disassembly of an active S. pombe spliceosome.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Proteínas Fúngicas/fisiología , Precursores del ARN/metabolismo , Empalme del ARN/genética , ARN de Hongos/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/fisiología , Schizosaccharomyces/genética , Ciclo Celular , Cromatografía de Afinidad , Regulación Fúngica de la Expresión Génica , Sustancias Macromoleculares , Peso Molecular , Complejos Multiproteicos , Proteínas de Unión al ARN , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe , Empalmosomas/químicaRESUMEN
Dimerization of simian virus 40 T-antigen hexamers (TAgH) into double hexamers (TAgDH) on model DNA replication forks has been found to greatly stimulate T-antigen DNA helicase activity. To explore the interaction of TAgDH with DNA during unwinding, we examined the binding of TAgDH to synthetic DNA replication bubbles. Tests of replication bubble substrates containing different single-stranded DNA (ssDNA) lengths indicated that efficient formation of a TAgDH requires >/=40 nucleotides (nt) of ssDNA. DNase I probing of a substrate containing a 60-nt ssDNA bubble complexed with a TAgDH revealed that T antigen bound the substrate with twofold symmetry. The strongest protection was observed over the 5' junction on each strand, with 5 bp of duplex DNA and approximately 17 nt of adjacent ssDNA protected from nuclease cleavage. Stimulation of the T-antigen DNA helicase activity by an increase in ATP concentration caused the protection to extend in the 5' direction into the duplex region, while resulting in no significant changes to the 3' edge of strongest protection. Our data indicate that each TAgH encircles one ssDNA strand, with a different strand bound at each junction. The process of DNA unwinding results in each TAgH interacting with a greater length of DNA than was initially bound, suggesting the generation of a more highly processive helicase complex.
Asunto(s)
Antígenos Transformadores de Poliomavirus/química , Antígenos Transformadores de Poliomavirus/metabolismo , Replicación del ADN/inmunología , Replicación del ADN/fisiología , Virus 40 de los Simios/inmunología , Virus 40 de los Simios/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , ADN Helicasas/metabolismo , ADN de Cadena Simple/síntesis química , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Dimerización , Técnicas In Vitro , Modelos Biológicos , Conformación ProteicaRESUMEN
Chromosomal DNA replication in higher eukaryotes takes place in DNA synthesis factories containing numerous replication forks. We explored the role of replication fork aggregation in vitro, using as a model the simian virus 40 (SV40) large tumor antigen (T antigen), essential for its DNA helicase and origin-binding activities. Previous studies have shown that T antigen binds model DNA replication forks primarily as a hexamer (TAgH) and to a lesser extent as a double hexamer (TAgDH). We find that DNA unwinding in the presence of ATP or other nucleotides strongly correlates with the formation of TAgDH-DNA fork complexes. TAgH- and TAgDH-fork complexes were isolated, and the TAgDH-bound fork was denatured at a 15-fold-higher rate during the initial times of unwinding. TAgDH bound preferentially to a DNA substrate containing a 50-nucleotide bubble, indicating the bridging of each single-stranded DNA/duplex DNA junction, and this DNA molecule was also unwound at a high rate. Both the TAgH- and TAgDH-fork complexes were relatively stable, with the half-life of the TAgDH-fork complex greater than 40 min. Our data therefore indicate that the linking of two viral replication forks serves to activate DNA replication.
Asunto(s)
Antígenos Transformadores de Poliomavirus/química , ADN Helicasas/química , ADN Helicasas/metabolismo , Replicación del ADN , Virus 40 de los Simios/genética , Antígenos Transformadores de Poliomavirus/metabolismo , Sistema Libre de Células , ADN Viral/biosíntesis , Dimerización , Cinética , Proteínas Recombinantes , Virus 40 de los Simios/enzimología , Relación Estructura-ActividadRESUMEN
Combination of chemical DNA synthesis and polymerase chain reactions has been used for obtaining DNA templates of preset structure coding for target mRNAs. The obtained DNA were 136 to 246 bp in length, contained promoters for RNA polymerases of phage T7 or E. coli and coded for 136-membered mRNA.