Binding of the Single-Stranded DNA Binding Protein (gp32) of T4 Bacteriophage Induces Position-Specific Local Conformational Changes in DNA Lattices that can be Monitored by Fluorescent Probes

Abstract

Reverse gyrase, found in hyperthermophiles, is the only enzyme known to overwind (introduce positive supercoils into) DNA. The ATP-dependent activity, detected at >70 C, has so far been studied solely by gel electrophoresis, and thus the reaction dynamics remain obscure. Here we image the overwinding reaction at 71 C under a microscope, using DNA containing consecutive 30 mismatched base pairs that serve as a well-defined substrate site. A single reverse gyrase molecule processively winds the DNA for >100 turns. Bound enzyme shows moderate temperature dependence, retaining significant activity down to 50 C. Unloaded reaction rate at 71 C exceeds 5 turns s À1 , which is >10 2 -fold higher than hitherto indicated but lower than the ATPase rate measured in bulk of 20 s À1 , indicating loose coupling. The overwinding reaction sharply slows down as the torsional stress accumulates in DNA, and ceases at stress of mere~5 pN,nm, where one more turn would cost only six times the thermal energy. The enzyme would thus keep DNA in a slightly overwound state to protect, but not overprotect, the genome of hyperthermophiles against thermal melting. Overwinding activity is also highly sensitive to DNA tension, with an effective interaction length exceeding the size of reverse gyrase, implying requirement for slack DNA. All results point to the mechanism where strand passage relying on thermal motions, as in topoisomerase IA, is actively, but loosely, biased toward overwinding.