RecA stimulates AlkB-mediated direct repair of DNA adducts

Abstract

The Escherichia coli AlkB protein is a 2-oxoglutarate/Fe(II)-dependent demethylase that repairs alkylated single stranded and double stranded DNA. Immunoaffinity chromatography coupled with mass spectrometry identified RecA, a key factor in homologous recombination, as an AlkB-associated protein. The interaction between AlkB and RecA was validated by yeast two-hybrid assay; size-exclusion chromatography and standard pull down experiment and was shown to be direct and mediated by the N-terminal domain of RecA. RecA binding results AlkB–RecA heterodimer formation and RecA–AlkB repairs alkylated DNA with higher efficiency than AlkB alone.

A novel combination of Class-I fumarases and metabolites (α-ketoglutarate and fumarate) signal the DNA damage response inE. coli

“…To test whether fumarate and succinate inhibit AlkB demethylase activity in E. coli , we employed two previously published protocols (Shivange, Kodipelli, & Anindya, 2014; Shivange, Monisha, Nigam, Kodipelli, & Anindya, 2016) for in vitro measurement of DNA damage repair by AlkB in the presence and absence of succinate, fumarate and malate. Such an effect, if found could explain the increase in sensitivity of fum null mutants to MMS generated DNA damage.…”

“…The first approach was direct fluorescence-based formaldehyde detection using acetoacetanilide and ammonium acetate, which together form an enamine-type intermediate. This intermediate undergoes cyclodehydration to generate highly fluorescent dihydropyridine derivative, having maximum excitation at 365 nm and maximum emission at 465 nm [30]. A 70bp oligonucleotide (see materials and methods) was treated with MMS to allow methylation/alkylation, and was directly incubated with purified AlkB (2.5μM) to facilitate DNA repair.…”

“…AlkB demethylation activity was measured by two previously described methods (Shivange et al, 2014; Shivange et al, 2016). For both methods 40 µg of chemically synthesized oligonucleotides (IDT) that contains the restriction site for the methylation sensitive restriction enzyme MboI (underlined) was used 5’-GGATGCCTTC GACACCTAGC TTTGTTAGGT CTG GATC CTC GAAATACAAA GATTGTACTG AGAGTGCACC-3’).…”

“…This assay was performed as previously described (Shivange et al, 2016). Repair reactions (50 µl) were carried out at 37°C for 1 hour in the presence of 1 µM AlkB, 0.5 µg (1 µM) methylated oligonucleotide and reaction buffer (20 mM Tris–HCl pH 8.0, 200 µM indicated metabolite [αKG acid, succinic acid, fumaric acid and malic acid], 2 mM L-Ascorbate and 20 µM Fe(NH 4 ) 2 (SO 4 ) 2 ).…”

Novel Approaches for Efficient Antifungal Drug Action

“…They do not easily induce resistance as compared with conventional antibiotics due to their ability to bypass the common resistance mechanisms [74,76]. AMPs are composed of fewer than 100 amino acid residues commonly used for cell transfer and are rich in cationic and hydrophobic residues arranged in different groups depending on amino acid composition, size and morphology [74,77]. Although AMPs have been classified into several groups based on structures or sequences such as α-helical, cysteine-rich, glycine-rich and proline-rich peptides, AMPs generally have certain properties such as cationic charge and amphipathicity [78].…”