Construction of a circular single-stranded DNA template containing a defined lesion

We report a concise and efficient method to make a circular single-stranded DNA containing a defined DNA lesion. In this protocol, phagemid DNA containing Uracil is used as a template to synthesize a complementary DNA strand using T7 DNA polymerase and an oligonucleotide primer including a site-specific DNA lesion. The ligated lesion-containing strand can be recovered after the phage-derived template DNA is degraded by treatment with E. coli Uracil DNA glycosylase and Exonucleases I and III. The resulting product is a circular single-stranded DNA containing a defined DNA lesion suitable for in vitro translesion replication assays.This work was supported by funds from the NIH/NICHD Intramural Research Program. K.K. was also a recipient of a research fellowship from the Japan Society for the Promotion of Science. A.V. was supported by funds of the Programa Ramón y Cajal (Ministerio de Ciencia e Innovación, Spain).Peer reviewe

A RecA protein surface required for activation of DNA polymerase V.

DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3'-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3'-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA'2B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis

The crystal structure of the AAA domain of the ATP-dependent protease FtsH of Escherichia coli at 1.5 Å resolution

Eubacteria and eukaryotic cellular organelles have membrane-bound ATP-dependent proteases, which degrade misassembled membrane protein complexes and play a vital role in membrane quality control. The bacterial protease FtsH also degrades an interesting subset of cytoplasmic regulatory proteins, including σ32, LpxC, and λ CII. The crystal structure of the ATPase module of FtsH has been solved, revealing an α/β nucleotide binding domain connected to a four-helix bundle, similar to the AAA modules of proteins involved in DNA replication and membrane fusion. A sulfate anion in the ATP binding pocket mimics the β-phosphate group of an adenine nucleotide. A hexamer form of FtsH has been modeled, providing insights into possible modes of nucleotide binding and intersubunit catalysis

RumA′₂B can be activated by all RecA variants.

<p>Transactivation reaction was carried out on 3-nt overhang hairpin for UmuD′₂C (A) and RumA′₂B (B). RecA* (400 nM) was preformed on 30 nt ssDNA then mixed with UmuD′₂C or RumA′₂B (400 nM each) and incubated for 30 mins at 37°C. Unlike UmuD′₂C (A), RumA′₂B can be activated by RecA variants D112R, D112R N113R, and S117F (B).</p

RecA D112R and D112R N113R exhibit wild-type RecA binding affinities for pol V.

<p>(A) Location of investigated residues on the RecA protein surface. The D112 and N113 residues compose an acidic knob on the RecA surface. The RecA monomer represented in electrostatic coloring scheme (red = negative charged residues, blue = positive charged residues) was generated in Pymol (PDB 3CMU [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005066#pgen.1005066.ref067" target="_blank">67</a>]). In this illustration, the monomer shown is located at the 3' end of the ssDNA (the 3'-proximal RecA monomer), which is the monomer removed by pol V during the activation cycle. (B) Altering this acidic surface to basic residues does not affect the binding affinity for pol V. Equilibrium binding isotherms of wild-type RecA, RecA D112R, and RecA D112R titrated with pol V as monitored by fluorescence depolarization. All data are the average of at least three experiments. Error bars are one standard deviation from the mean.</p