RESUMO
When challenged by DNA-damaging agents, Escherichia coli cells respond by inducing the SOS stress response, which leads to an increase in mutation frequency by two mechanisms: translesion replication, a process that causes mutations because of misinsertion opposite the lesions, and an inducible mutator activity, which acts at undamaged sites. Here we report that DNA polymerase V (pol V; UmuC), which previously has been shown to be a lesion-bypass DNA polymerase, was highly mutagenic during in vitro gap-filling replication of a gapped plasmid carrying the cro reporter gene. This reaction required, in addition to pol V, UmuD', RecA, and single-stranded DNA (ssDNA)-binding protein. pol V produced point mutations at a frequency of 2.1 x 10(-4) per nucleotide (2.1% per cro gene), 41-fold higher than DNA polymerase III holoenzyme. The mutational spectrum of pol V was dominated by transversions (53%), which were formed at a frequency of 1.3 x 10(-4) per nucleotide (1. 1% per cro gene), 74-fold higher than with pol III holoenzyme. The prevalence of transversions and the protein requirements of this system are similar to those of in vivo untargeted mutagenesis (SOS mutator activity). This finding suggests that replication by pol V, in the presence of UmuD', RecA, and ssDNA-binding protein, is the basis of chromosomal SOS untargeted mutagenesis.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Escherichia coli , Proteínas de Transporte de Monossacarídeos , Resposta SOS em Genética/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Transporte/genética , Dano ao DNA/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Mutação da Fase de Leitura , Proteínas Ligantes de Maltose , Dados de Sequência Molecular , Mutagênese , Mutação , Plasmídeos/genética , Mutação Puntual , Recombinases Rec A/metabolismo , Proteínas Recombinantes de Fusão/genéticaRESUMO
Replication of DNA lesions leads to the formation of mutations. In Escherichia coli this process is regulated by the SOS stress response, and requires the mutagenesis proteins UmuC and UmuD'. Analysis of translesion replication using a recently reconstituted in vitro system (Reuven, N. B., Tomer, G., and Livneh, Z. (1998) Mol. Cell 2, 191-199) revealed that lesion bypass occurred with a UmuC fusion protein, UmuD', RecA, and SSB in the absence of added DNA polymerase. Further analysis revealed that UmuC was a DNA polymerase (E. coli DNA polymerase V), with a weak polymerizing activity. Upon addition of UmuD', RecA, and SSB, the UmuC DNA polymerase was greatly activated, and replicated a synthetic abasic site with great efficiency (45% bypass in 6 min), 10-100-fold higher than E. coli DNA polymerases I, II, or III holoenzyme. Analysis of bypass products revealed insertion of primarily dAMP (69%), and to a lesser degree dGMP (31%) opposite the abasic site. The UmuC104 mutant protein was defective both in lesion bypass and in DNA synthesis. These results indicate that UmuC is a UmuD'-, RecA-, and SSB-activated DNA polymerase, which is specialized for lesion bypass. UmuC is a member of a new family of DNA polymerases which are specialized for lesion bypass, and include the yeast RAD30 and the human XP-V genes, encoding DNA polymerase eta.