RESUMEN
The clamp-loader-helicase interaction is an important feature of the replisome. Although significant biochemical and structural work has been carried out on the clamp-loader-clamp-DNA polymerase alpha interactions in Escherichia coli, the clamp-loader-helicase interaction is poorly understood by comparison. The tau subunit of the clamp-loader mediates the interaction with DnaB. We have recently characterised this interaction in the Bacillus system and established a tau(5)-DnaB(6) stoichiometry. Here, we have obtained atomic force microscopy images of the tau-DnaB complex that reveal the first structural insight into its architecture. We show that despite the reported absence of the shorter gamma version in Bacillus, tau has a domain organisation similar to its E.coli counterpart and possesses an equivalent C-terminal domain that interacts with DnaB. The interaction interface of DnaB is also localised in its C-terminal domain. The combined data contribute towards our understanding of the bacterial replisome.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas , ADN Helicasas/química , ADN Helicasas/ultraestructura , Microscopía de Fuerza Atómica , Subunidades de Proteína/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/ultraestructura , Bacillus subtilis/genética , Secuencia de Bases , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/metabolismo , Replicación del ADN , AdnB Helicasas , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/químicaRESUMEN
Cells exposed to DNA damaging agents in their natural environment do not undergo continuous cycles of replication but are more frequently engaged in gene transcription. Luciferase gene expression analysis with DNA templates containing uracil or 8-oxoguanine, placed at a defined position, indicated that in nondividing Escherichia coli cells, efficient mutagenic lesion bypass does occur in vivo during transcription. Sequence analyses of the transcript population revealed that RNA polymerase inserts adenine opposite to uracil, and adenine or cytosine opposite to 8-oxoguanine. Surprisingly, deletions were also detected for 8-oxoguanine-containing templates, indicating RNA polymerase slippage over this lesion. Genetic analyses showed that, in E. coli, 8-oxoguanine is subject to transcription-coupled repair. Consequently, DNA damages alter transcription fidelity in vivo, which may lead to the production of mutant proteins that have the potential to change the phenotype of nondividing cells.
Asunto(s)
Daño del ADN/genética , Escherichia coli/genética , Guanina/análogos & derivados , Guanina/farmacología , Mutagénesis/genética , Transcripción Genética/genética , Uracilo/farmacología , Secuencia de Bases/efectos de los fármacos , Secuencia de Bases/genética , Reparación del ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/genética , Mutagénesis/efectos de los fármacos , Fenotipo , Transcripción Genética/efectos de los fármacos , Uracilo/metabolismoRESUMEN
We have previously reported the synthesis of vinylphosphonate-linked thymidine dimers and their incorporation into synthetic oligonucleotides to create vinylphosphonate internucleotide linkages in the DNA. Such linkages have a profound effect on DNA backbone rotational flexibility, and we have shown that the PcrA helicase, which requires such flexibility, is inhibited when it encounters these linkages on the translocating strand. In this study, we have investigated the effects of these linkages on the dsDNA specific exonuclease III and on the ssDNA specific mung bean nuclease to establish whether our modification confers resistance to nucleases making it suitable for antisense therapy applications. We also investigated the effect on DNA polymerase I to establish whether we could in the future use this enzyme to incorporate these linkages in the DNA. Our results show that a single modification does not affect the activity of DNA polymerase I, but four vinylphosphonate linkages in tandem inhibit its activity. Furthermore, such linkages do not confer significant nuclease resistance to either exonuclease III or mung bean nuclease, but unexpectedly, they alter the cleavage specificity of exonuclease III.