The Recombination Function For Escherichia Coli Single-stranded DNA-binding Protein

Homologous recombination plays an essential housekeeping role for DNA damage repair and recombination among endogenous or alien DNA in all organisms.RecA homologous recombination system in Escherichia coli is the one of the most extensively studied homologous recombination system.RecA protein is essential in this system.recA mutation is devoid of homologous recombination.But several previous researchers reported that recA mutation of E.coli strain DH5a was capable of low efficient homologous recombination.The mechanism has not been defined.In present study,we constructed recA knockout mutant of DH5a using the homologous recombination methods.Plasmid and DNA fragments all which contained homology arms were transformated of DH5a and its recA knockout mutant,the recombination transformants were screened by using the modified double-plate method.The intramolecular and intermolecular homologous recombination dynamics of DH5a and its recA knockout mutant were explored.In additional,in vitro reconstitution experiments of RecA,RecBCD,SSB protein were performed.Combined with the above researches,the homologous recombination mechanism of E.coli recA mutant was investigated.The main results as follows:(1)double-layer plate method is an effective tool to study homologous recombination rate and homologous recombination dynamics.(2)E.coli recA mutant strain DH5a and its recA knockout mutant were able to plasmid intermolecule homologous recombination with the similar of homologous recombination efficiency.The mutated recA loci of DH5a strain lost all homologous recombination function.The homologous recombination function of DH5a should be compensated by the other gene.(3)The time courses of the recombinant processing of the recA knockout mutant of DH5a were accord with the enzymatic reaction curve.The transformants occured time was significantly negatively correlated with the concentration of DNA fragments.The relation ship between the recombination rate and the concentration of DNA fragments was conform to the Michaelis-Menten equation.The homologous recombination frequency was significantly positively correlated the DNA fragment concentration.However the homologous recombination frequency was considerably low.The relationship curve of the recombination rate of the recA knockout mutant between the homologous arms-length of the DNA fragments was accord with Logistic equation.When homologous arm length was 25.27 bp,the recombination rate increased fastest,the length exceeded it,the recombination rate decelerated.The recombination rate closed to the maximum value of 33.6116 transformants/h plate when the homology arms length was 40 bp.The relationship curve of homologous recombination frequency of the recA knockout mutant and the DNA fragments homologous arms-length was inverted N-shaped.When the 60 bp homology arms length,the homologous recombination frequency reached the maximum number of 40%.the results released that the recombinant processing in recA knockout mutant was an enzymatic reaction but with a lower homologous recombination frequency.(4)In vitro recombinant experiments results showed that that SSB had the catalytic recombinant function same as RecA,but the enzymatic reaction speed is only nearly half of the latter.The catalytic recombinant function of the two proteins all needs RecBCD.Only both of them did not catalyze recombinant reaction.In a summary,the homologous recombination mechanism of E.coli recA mutant strain DH5a strongly was proposed that SSB also had the recombinant function similar to RecA.The recombination rate catalyzed by SSB is much lower than that of the RecA,homologous recombination frequency was relatively lower than that of the RecA.