RESUMO
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.
Assuntos
DNA Fúngico/química , RNA Fúngico/biossíntese , Sequência de Aminoácidos , Autorradiografia , Sequência de Bases , Clonagem Molecular , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Fagos T/enzimologia , Moldes Genéticos , Transcrição GênicaRESUMO
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.
Assuntos
Antígenos Transformantes de Poliomavirus/química , DNA Helicases/química , DNA Helicases/metabolismo , Replicação do DNA , Vírus 40 dos Símios/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Sistema Livre de Células , DNA Viral/biossíntese , Dimerização , Cinética , Proteínas Recombinantes , Vírus 40 dos Símios/enzimologia , Relação Estrutura-AtividadeRESUMO
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.