Abstract
BackgroundThe MucA’ and MucB proteins comprise the core of DNA polymerase RI which is a strong mutator utilized in mutagenicity assays such as the standard Ames test. A close relative DNA polymerase V, composed of the homologous UmuD’ and UmuC proteins, is considered to be an ortholog of the mammalian DNA polymerase η. The catalytic subunits of these polymerases belong to the Y-family which specializes in the translesion DNA synthesis across various DNA adducts to rescue stalled chromosomal replication at the expense of mutations. Based on genetic evidence, DNA polymerase RI possesses the greatest ability to induce various types of mutations among all so far characterized members of the Y-superfamily. The exceptionally high mutagenic potential of MucA’B has been taken advantage of in numerous bacterial mutagenicity assays incorporating the conjugative plasmid pKM101 carrying the mucAB operon such as the Ames Test.ResultsWe established new procedures for the purification of MucB protein as well as its accessory protein MucA’ using the refolding techniques. The purified MucA’ protein behaved as a molecular dimer which was fully stable in solution. The soluble monomeric form of MucB protein was obtained after refolding on a gel-filtration column and remained stable in a nondenaturing buffer containing protein aggregation inhibitors. Using the surface plasmon resonance technique, we demonstrated that the purified MucA’ and MucB proteins interacted and that MucB protein preferentially bound to single-stranded DNA. In addition, we revealed that MucB protein interacted with the β-subunit of DNA polymerase III holoenzyme of E. coli.ConclusionThe MucA’ and MucB proteins can be isolated from inclusion bodies and solubilized in vitro. The refolded MucB protein interacts with its MucA’ partner as well as with DNA what suggests it retains biological activity. The interaction of MucB with the processivity subunit of DNA polymerase III may imply the role of the subunit as an accessory protein to MucB during the translesion DNA synthesis.
Highlights
In Escherichia coli, mutagenesis by ultraviolet light and most chemicals requires the expression of the umuDC operon [1, 2]
Bovine serum albumin (BSA), RecA and single-stranded DNA binding protein (SSB) proteins were purchased from Pharmacia, Sweden, and their purity was greater than 95% as judged by SDS-PAGE analysis
Since neither decreasing the expression rate, stimulating the culture into overproduction of chaperones nor the expression of engineered N-terminal HisTag- or GST-MucB fusions led to the production of soluble MucB protein, we focused on the development of a suitable refolding buffer which would inhibit the aggregation of MucB protein upon its dilution from the denaturant
Summary
In Escherichia coli, mutagenesis by ultraviolet light and most chemicals requires the expression of the umuDC operon [1, 2]. The umuDC operon is located at about 26 min on the E. coli chromosome and encodes the 15.1- and 47.7-kDa proteins UmuD and UmuC, respectively [3, 4]. It has been shown that the UmuC protein has an intrinsic DNA polymerase activity dependent on the accessory proteins UmuD’, RecA*, Ssb and β, γ complex [20, 21]. This DNA polymerase has been named Pol V next to another homologous previously characterized DNA polymerase Pol IV encoded by the dinB gene and involved in untargeted mutagenesis [20, 22]. The exceptionally high mutagenic potential of MucA’B has been taken advantage of in numerous bacterial mutagenicity assays incorporating the conjugative plasmid pKM101 carrying the mucAB operon such as the Ames Test
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