Influence of the C-Terminal Tail of RecA Proteins from Alkaline pH-Resistant Bacterium Deinococcus Ficus

Abstract

Deinococcus ficus CC - FR2 - 10T , resistant to ultraviolet, ionizing radiation, and chemicals which may cause DNA damage, was identified in Taiwan. The expression level of D. ficus RecA, which has 92% sequence identity with Deinococcus radiodurans ( Dr. ) RecA, will be upregulated upon UV radiation. Multiple sequence alignment of RecA proteins from bacteria belonging to Escherichia coli and the Deinococcus genus reveals that the C-terminal tail of D. ficus RecA is shorter and contains less acidic residues than E. coli RecA. D. ficus RecA exhibits a higher ATPase activity toward single-stranded (ss) DNA and efficiently promotes DNA strand exchange that a filament is first formed on ssDNA, followed by uptake of the double-stranded (ds) substrate. Moreover, D. ficus RecA exhibits a pH-reaction profile for DNA strand exchange similar to E. coli Δ C17 RecA. Later, a chimera D. ficus C17 E. coli RecA with more acidic residues in the C-terminal tail was constructed and purified. Increased negativity in the C-terminal tail makes the pH reaction profile for Chimera D. ficus C17 E. coli RecA DNA strand exchange exhibit a reaction optimum similar to E. coli RecA. To sum up, D. ficus RecA exhibits reaction properties in substrate-dependent ATPase activity and DNA strand exchange similar to E. coli RecA. Our data indicate that the negativity in the C-terminal tail plays an important role in the regulation of pH-dependent DNA strand exchange activity.

The regulation mechanism of the C-terminus of RecA proteins during DNA strand-exchange process

“…A chimeric RecA, D. ficus C17 E. coli RecA (in which the C-terminus was replaced with the corresponding residues in the C-terminus of E. coli RecA), was constructed and purified. Interestingly, the pH-reaction profile of RecA-mediated strand exchange shifted to acidic conditions for D. ficus C17 E. coli RecA (29). These observations highlight the influence of the C-terminus of RecA proteins on the DNA strand-exchange reaction.…”

“…For E. coli RecA, the formation of nucleoprotein filaments is relatively slow and exhibits high proton dependence. The rate-limiting step of DNA strand exchange is the formation of nucleoprotein filaments for E. coli RecA, and the reaction optimum is near pH 7.0 (29,49). In a previous study, Lusetti et al reported that progressively removing C-terminal residues from E. coli RecA proteins will cause a progressive shift in the pH-reaction profile of RecA-induced DNA strand exchange and result in strong inhibition of DNA strand exchange %pH 7.0 (18).…”

“…GeneBank: WP_000963143.1) and used without further purification. D. ficus RecA (GeneBank: WP_022801718.1), E. coli DC17 RecA, and chimera D. ficus C17 E. coli RecA were expressed in BLR (DE3, recA À ; Novagen, Madison, WI) and purified with a modified protocol as previously described (18,19,29).…”

“…The dsDNA substrates were prepared by PCR with a pair of primers, one containing a 5 0 -digoxigenin label and the other a 5 0 -biotin label. Hybrid DNA substrates were generated using a previously described protocol (29,33,34). The homologous hybrid DNA substrate is a 427/352-nt DNA with 40-nt and 35-nt gaps at its 5 0 and 3 0 ends, respectively.…”

“…Here, comprehensive experiments were used to investigate the reaction properties of E. coli RecA (with seven acidic residues in the C-terminus), D. ficus RecA (with two acidic residues in the C-terminus), E. coli DC17 RecA (without acidic residues in the C-terminus), and chimera D. ficus C17 E. coli RecA (with seven acidic residues in the C-terminus) simultaneously. One fluorescence resonance energy transfer (FRET) experiment and three tethered particle motion (TPM) experiments were designed to investigate the RecA-mediated DNA strand exchange process, including filament formation, homologous sequence searching, and strand displacement, from pH 7.0 to pH 9.0, a range in which the secondary structure of E. coli RecA and D. ficus RecA exhibited no detectable changes (29,30). D. ficus RecA and E. coli DC17 RecA can efficiently nucleate with a shorter ssDNA and at higher pH than E. coli RecA and the chimera D. ficus C17 E. coli RecA.…”