RecJ exonuclease: substrates, products and interaction with SSB.
The RecJ exonuclease from Escherichia coli degrades single-stranded DNA (ssDNA) in the 5'-3' direction and participates in homologous recombination and mismatch repair. The experiments described here address RecJ's substrate requirements and reaction products. RecJ complexes on a variety of 5' single-strand tailed substrates were analyzed by electrophoretic mobility shift in the absence of Mg2+ ion required for substrate degradation. RecJ required single-stranded tails of 7 nt or greater for robust binding; addition of Mg2+ confirmed that substrates with 5' tails of 6 nt or less were poor substrates for RecJ exonuclease. RecJ is a processive exonuclease, degrading approximately 1000 nt after a single binding event to single-strand DNA, and releases mononucleotide products. RecJ is capable of degrading a single-stranded tail up to a double-stranded junction, although products in such reactions were heterogeneous and RecJ showed a limited ability to penetrate the duplex region. RecJ exonuclease was equally potent on 5' phosphorylated and unphosphorylated ends. Finally, DNA binding and nuclease activity of RecJ was specifically enhanced by the pre-addition of ssDNA-binding protein and we propose that this specific interaction may aid recruitment of RecJ.
Han
Eugene S
ES
Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA.
Cooper
Deani L
DL
Persky
Nicole S
NS
Sutera
Vincent A
VA
Whitaker
Richard D
RD
Montello
Melissa L
ML
Lovett
Susan T
ST
D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D004247:DNA; D005090:Exodeoxyribonucleases; C489467:RecJ protein, E coli