Abstract
Escherichia coli DinB (DNA polymerase IV) possesses an enzyme architecture resulting in specialized lesion bypass function and the potential for creating -1 frameshifts in homopolymeric nucleotide runs. We have previously shown that the mutagenic potential of DinB is regulated by the DNA damage response protein UmuD(2). In the current study, we employ a pre-steady-state fluorescence approach to gain a mechanistic understanding of DinB regulation by UmuD(2). Our results suggest that DinB, like its mammalian and archaeal orthologs, uses a template slippage mechanism to create single base deletions on homopolymeric runs. With 2-aminopurine as a fluorescent reporter in the DNA substrate, the template slippage reaction results in a prechemistry fluorescence change that is inhibited by UmuD(2). We propose a model in which DNA templates containing homopolymeric nucleotide runs, when bound to DinB, are in an equilibrium between non-slipped and slipped conformations. UmuD(2), when bound to DinB, displaces the equilibrium in favor of the non-slipped conformation, thereby preventing frameshifting and potentially enhancing DinB activity on non-slipped substrates.
Highlights
DNA polymerases of the Y family catalyze replication on damaged DNA templates, thereby providing cells with a mechanism to tolerate DNA damage by a process called translesion DNA synthesis (TLS)3 [1]
The 2-AP reporter positioned either at the 0- or Ϫ1-position within a run resulted in a rapid prechemistry fluorescence change, interpreted as a template slippage isomerization that causes a base within the duplex region to adopt an extrahelical, or unstacked, conformation
The clearest evidence supporting the template slippage model during the base-skipping reaction is provided by substrate II-G, having 2-AP at the Ϫ1-position, where we observe a fluorescence increase consistent with 2-AP being unstacked from its 5Ј and 3Ј neighbors in an extrahelical conformation
Summary
Materials—Native DinB was purified as described previously [6], except that the purified protein was stored in 20 mM TrisHCl, pH 7.5, 0.1 mM EDTA, 1 mM dithiothreitol, and 10% glycerol. Emission of 1 M annealed primer1⁄7template DNA, 6 –15 M DinB (as indicated) in DinB reaction buffer (20 mM TrisHCl, pH 7.5, 50 mM NaCl, and 4 mM MgCl2) was monitored for 50 s (65-l total volume), and extension was initiated by adding 2 l of dNTP (3 mM final), and emission was monitored for an additional Ն100 s. The final concentrations of components were 200 nM substrate DNA, 3 M DinB, 6 M RecA and 60 M UmuD, 2 mM dNTP, and 2 mM additional MgCl2. Rapid quench-flow (KinTek Corp., model RQF-3) and manual quench kinetic measurements were performed under singleturnover conditions at 22 °C with final concentrations of 10 nM annealed primer1⁄7template DNA and 1 M DinB in DinB reaction buffer. Reactions were quenched with 5 l of quench buffer
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