Fig. 4.

The DNA end resection activities of drNurA-HerA and mutants. A–C The DNA end resection activities of different mutants have been compared on the same gel. Briefly, 400 nM substrate DNA was incubated with drNurA (4 μM protomer) and drHerA (12 μM protomer) in the presence of 2 mM MgCl₂ and 8 mM MnCl₂. Whenever needed, 1 mM ligand (ADP, ADPNP, or ATP) was added to the reaction system. Products were analyzed on 15% denaturing TBE-PAGE. The same gels were imaged at FAM fluorescent mode (upper) and Cy5 fluorescent mode (lower) separately. The types of the generated products before denaturing have been annotated at the right side of the gel to aid the interpretation of the bands on the gels. Markers were created by mixing different lengths of 3′ FAM or 5′ Cy5 labeled DNA oligos together. dPin, NurA^dPin; 487/8, HerA^E487A/E488A; m, HerA^m; 13, NurA^F13A; 8, NurA^W8A; dN7, drNurAdN7; 53, NurA^D53A; 201, NurA^K201A; 309, NurA^R309A; 96, HerA^R96A; 495, HerA^R495A; 552, HerA^R552A. D The predicted drNurA-HerA-dsDNA complex model. dsDNA was docked into the DNA binding channel of drHerA. The carbon atoms on the Cα backbone of the residues which were predicted important for translocation and catalysis are shown as spheres and are colored blue and red, respectively. The pin-like motif is shown as spheres and colored cyan. E The cutting pattern analysis of the hairpin substrate O1. Putative major cutting sites are marked by arrows. Red arrows indicate that the cleavage requires drNurA-HerA complex in a translocation mode (during translocation), the blue arrows indicate that the cleavage requires drNurA-HerA complex in a steady state mode (translocation stopped or initiated), and the green arrows indicate that the cleavage are drHerA independent and ATP independent. The size of the arrow indicates the digestion efficiency of each site, that the bigger the arrow, the more efficient the cutting