All three SOS-inducible DNA polymerases (Pol II, Pol IV and Pol V) are involved in induced mutagenesis

Most organisms contain several members of a recently discovered class of DNA polymerases (umuC/dinB superfamily) potentially involved in replication of damaged DNA. In Escherichia coli, only Pol V (umuDC) was known to be essential for base substitution mutagenesis induced by UV light or abasic sites. Here we show that, depending upon the nature of the DNA damage and its sequence context, the two additional SOS-inducible DNA polymerases, Pol II (polB) and Pol IV (dinB), are also involved in error-free and mutagenic translesion synthesis (TLS). For example, bypass of N:-2-acetylaminofluorene (AAF) guanine adducts located within the NAR:I mutation hot spot requires Pol II for -2 frameshifts but Pol V for error-free TLS. On the other hand, error-free and -1 frameshift TLS at a benzo(a)pyrene adduct requires both Pol IV and Pol V. Therefore, in response to the vast diversity of existing DNA damage, the cell uses a pool of 'translesional' DNA polymerases in order to bypass the various DNA lesions.     

Rapid and efficient degradation of endogenous proteins in vivo identifies stage-specific roles of RNA Pol II pausing in mammalian development

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RecFOR proteins are essential for Pol V-mediated translesion synthesis and mutagenesis

When the replication fork moves through the template DNA containing lesions, daughter-strand gaps are formed opposite lesion sites. These gaps are subsequently filled-in either by translesion synthesis (TLS) or by homologous recombination. RecA filaments formed within these gaps are key intermediates for both of the gap-filling pathways. For instance, Pol V, the major lesion bypass polymerase in Escherichia coli, requires a functional interaction with the tip of the RecA filament. Here, we show that all three recombination mediator proteins RecFOR are needed to build a functionally competent RecA filament that supports efficient Pol V-mediated TLS in the presence of ssDNA-binding protein (SSB). A positive contribution of RecF protein to Pol V lesion bypass is demonstrated. When Pol III and Pol V are both present, Pol III imparts a negative effect on Pol V-mediated lesion bypass that is counteracted by the combined action of RecFOR and SSB. Mutations in recF, recO or recR gene abolish induced mutagenesis in E. coli.     

Effect of SOS-induced Pol II, Pol IV, and Pol V DNA polymerases on UV-induced mutagenesis and MFD repair in Escherichia coli cells

Irradiation of organisms with UV light produces genotoxic and mutagenic lesions in DNA. Replication through these lesions (translesion DNA synthesis, TSL) in Escherichia coli requires polymerase V (Pol V) and polymerase III (Pol III) holoenzyme. However, some evidence indicates that in the absence of Pol V, and with Pol III inactivated in its proofreading activity by the mutD5 mutation, efficient TSL takes place. The aim of this work was to estimate the involvement of SOS-inducible DNA polymerases, Pol II, Pol IV and Pol V, in UV mutagenesis and in mutation frequency decline (MFD), a mechanism of repair of UV-induced damage to DNA under conditions of arrested protein synthesis. Using the argE3-->Arg(+) reversion to prototrophy system in E. coli AB1157, we found that the umuDC-encoded Pol V is the only SOS-inducible polymerase required for UV mutagenesis, since in its absence the level of Arg(+) revertants is extremely low and independent of Pol II and/or Pol IV. The low level of UV-induced Arg(+) revertants observed in the AB1157mutD5DumuDC strain indicates that under conditions of disturbed proofreading activity of Pol III and lack of Pol V, UV-induced lesions are bypassed without inducing mutations. The presented results also indicate that Pol V may provide substrates for MFD repair; moreover, we suggest that only those DNA lesions which result from umuDC-directed UV mutagenesis are subject to MFD repair.     

Coping with replication 'train wrecks' in Escherichia coli using Pol V, Pol II and RecA proteins

DNA replication machineries tend to stall when confronted with damaged DNA template sites, causing the biochemical equivalent of a major 'train wreck'. A newly discovered bacterial DNA polymerase, Escherichia coli Pol V, acting in conjunction with the RecA protein, can exchange places with the stalled replicative Pol III core and catalyse 'error-prone' translesion synthesis. In contrast to Pol V-catalysed 'brute-force, sloppier copying', another SOS-induced DNA polymerase, Pol II, plays a pivotal role in an 'error-free', replication-restart DNA repair pathway and probably involves RecA-mediated homologous recombination.     

Density and substrata are important in lung type II cell transdifferentiation in vitro.

Morphological techniques and metabolic cell marker assays were used to study the transdifferentiation of pulmonary type II epithelial cells to type I-like cells in vitro. In the lung this process is important during remodelling and alveolar repair. Type II cell phenotype was best maintained over eight days when densely packed cells were plated out on a commercially available extracellular matrix. Such cells retained type II cell characteristics (lamellar bodies, high activities of gamma glutamyl transpeptidase and alkaline phosphatase) but expressed low levels of rT1(40) a surface protein marker of type I cells. In contrast, low density cultures, irrespective of substratum, exhibited rapid cell spreading, loss of lamellar bodies, loss of type II cell enzyme markers and expressed high levels or rT1(40). Conditions have been described whereby the same isolate of type II cells can be used to produce differential epithelial phenotypes and use can be made of this for further characterisation or to investigate the effect of toxins on different lung cell types in vitro.