A two-site kinetic mechanism for ATP binding and hydrolysis by E-coli Rep helicase dimer bound to a single-stranded oligodeoxynucleotide

Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in reac tions that are coupled to ATP binding and hydrolysis. We have investigated the kinetic mechanism of ATP binding and hydrolysis by a proposed intermedi ate in Rep-catalyzed DNA unwinding, the Rep "P2S" dimer (formed with the si ngle-stranded (ss) oligodeoxynucleotide, (dT)(16)), in which only one subun it of a Rep homo-dimer is bound to ssDNA. Pre-steady-state quenched-flow st udies under both single turnover and multiple turnover conditions as well a s fluorescence stopped-flow studies were used (4 degrees C, pH 7.5, 6 mM Na Cl, 5 mM MgCl2, 10 % (v/v) glycerol). Although steady-state studies indicat e that a single ATPase site dominates the kinetics (k(cat)=17(+/-2)s(-1) K- M=3 mu M), pre-steady-state studies provide evidence for a two-ATP site mec hanism in which both sites of the dimer are catalytically active and commun icate allosterically. Single turnover ATPase studies indicate that Am hydro lysis does not require the simultaneous binding of two ATP molecules, and u nder these conditions release of product (ADP-P-i) is preceded by a slow ra te-limiting isomerization (similar to 0.2 s(-1)). However, product (ADP or P-i) release is not rate-limiting under multiple turnover conditions, indic ating the involvement of a second Am site under conditions of excess ATP. S topped-now fluorescence studies monitoring Am-induced changes in Rep's tryp tophan fluorescence displayed biphasic time courses. The binding of the fir st Am occurs by a two-step mechanism in which binding (k(+1)=1.5(+/-0.2)x 1 0(7) M-1 s(-1) k(-1)=29(+/-2)s(-1)) is followed by a protein conformational change (k(+2) = 23(+/-3) s(-1)), monitored by an enhancement of Trp fluore scence. The second Trp fluorescence quenching phase is associated with bind ing of a second ATP. The first ATP appears to bind to the DNA-free subunit and hydrolysis induces a global conformational change to form a high energy intermediate state with tightly bound (ADP-P-i). Binding of the second ATP then leads to the steady-state ATP cycle. As proposed previously, the role of steady-state ATP hydrolysis by the DNA-bound Rep subunit may be to main tain the DNA-free subunit in an activated state in preparation for binding a second fragment of DNA as needed for translocation and/or DNA unwinding. We propose that the roles of the two ATP sites may alternate upon binding D NA to the second subunit of the Rep dimer during unwinding and translocatio n using a subunit switching mechanism. (C) 1999 Academic Press.