Fig 4.

PrgJ DNA binding in vitro. Purified PrgJ-His₆ and PrgJ.K471E-His₆ (see the text) were analyzed by EMSAs for binding to the 3≪-end DIG-labeled double-stranded (A) and single-stranded (B) DNA substrates listed below the gels. (A) (Left) PrgJ binding to a 186-bp sequence encompassing the pCF10 oriT sequence. Lane 1, DNA probe only. Lanes 2 to 7, DNA probe plus the PrgJ proteins shown at the following concentrations: lane 2, 76 nM; lane 3, 152 nM; lane 4, 458 nM; lanes 5 to 7, 610 nM. Lanes 6 and 7, protein was preincubated with unlabeled 186-bp dsDNA competitor DNA at concentrations 10- and 100-fold higher than that of the DIG-labeled substrate. (Right) Binding of PrgJ proteins to a 39-bp nonspecific dsDNA substrate. Lanes: 1, DNA probe only; 2, DNA probe plus PrgJ (458 nM); 3, same as lane 2, except PrgJ was preincubated with unlabeled competitor DNA. (B) PrgJ (left) and PrgJ.K471E (right) binding to the DIG-labeled ssDNA 50-mers listed. The lanes are the same as in panel A, right; in all cases, competitor DNA was the unlabeled form of the test substrate at a 100-fold-higher concentration.