The RecJ exonuclease from Escherichia coli: DNA binding characteristics, reaction products, and conserved amino acids

RecJ from Escherichia coli is a magnesium dependent, single-stranded DNA exonuclease with 5'-3' polarity. Our efforts have revealed information concerning the specificity of RecJ for single-stranded DNA and the reaction products of its activity. Furthermore, we investigated the importance of conserved amino acids with regards to RecJ function. These findings, together with recent structural information, offer insight into RecJ's biochemistry.; DNA substrates containing 5' single-stranded tails of varying lengths, adjacent to a double-stranded junction, were created. By excluding Mg2+ in vitro, RecJ could bind DNA without degradation. Subsequent mobility shift analysis on native polyacrylamide gels demonstrated that RecJ will bind tails of seven nucleotides in length or longer. Binding capability decreases when the single-stranded region is shorter.; By adding Mg2+, nuclease activity was initiated and reaction products could be analyzed. We observed that a double-stranded junction inhibits degradation. Still, with stable DNA association in the presence of Mg 2+, the reaction degraded the tail until the junction was reached, leaving a blunt, double-stranded product. After resolving degradation products by thin layer chromatography, we found that RecJ degrades DNA to mononucleotides. Furthermore, this exonuclease activity was enhanced by the incorporation of single-stranded DNA binding protein.; In structural analysis, we found that conserved amino acid residues are readily apparent upon sequence alignment with other eubacterial species. Mutation of many of these residues is detrimental to activity. Furthermore, a crystal structure of the Thermus thermophilus RecJ shows that these same residues are located within the reactive cleft. We presume these amino acids play a role in coordinating catalytic metal ions or binding DNA. To investigate this, we have purified MBP fusion constructs of D236A and R401A mutant RecJ forms. Neither purified mutant is active as a nuclease, being reduced in nuclease activity by over 8,000 fold when compared to the wild type MBP-RecJ fusion. With binding analysis, D236A binds DNA while R401A appears binding deficient.