Expression, purification and biochemical characterization of Listeria monocytogenes single stranded DNA binding protein 1

Add time:09/04/2019    Source:sciencedirect.com

Single-stranded DNA binding proteins play an important role in DNA metabolic processes including replication, recombination, and repair. Here, we report the identification and biochemical characterization of the SSB1 protein from the foodborne pathogen Listeria monocytogenes. The L. monocytogenes SSB1 share 33% identity and 50.5% similarity with the prototype E. coli SSB protein. The electrophoretic mobility shift assay revealed that the purified L. monocytogenes SSB1 protein binds to single stranded DNA, including the M13 circular single stranded DNA and oligonucleotide, with high affinity. The plasmid based strand transfer activity showed that, in the absence of the SSB protein, L. monocytogenes RecA fails to catalyze the reaction whereas, the E. coli RecA protein has shown nicked DNA formation. Interestingly the addition of SSB1 protein stimulates both L. monocytogenes and E. coli RecA strand transfer activities however, it is sensitive to the order of addition of SSB1 protein. L. monocytogenes RecA fails to catalyze the reaction when SSB1 is added prior to RecA; nevertheless, it readily catalyzes the reaction when added after the RecA filament formation. These results suggest that the interaction among of gene product between RecA and SSB1 is required to promote optimum strand exchange activities. Altogether, these studies provide the first functional characterization of the L. monocytogenes SSB1 protein and give insights into DNA repair and recombination processes in the gram-positive foodborne pathogen L. monocytogenes.

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