./. NoE. Riol. (1979) 135, 391-411 Base-pair Opening and Closing Reactions in the Double Helix A Stopped-flow Hydrogen Exchange Study in Poly(rA)‘Poly(rU) CHHABINATH MANDAL~, NEVILLE R. KALLENBACH~ AND S. WALTER ENGLANDER~ ‘Department of Biochemistry and Biophysics and ‘Departmeni of Biology University of Pennsylvania Philadelphia, PA 19104. U.S.A. (Received 10 April 1979) Tile llydrogen~deuterium exchange of AMP, uridinc, poly(rA), and poly(rA) . poly(rU) was investigated by a spectral difference met,hod using stopped-flow spectroptlotolnetry. Proton exchange rat,es were measured as a frmction of pH, added catalysts, temperature and salt, concentration. The results confirm and extend previous conclusions on t,ho H-exchange chemist,ry of the bases, on the large equilibrium opening of the double Ilelix, and on its slow opening and closing rat,es, but an n.ltcmativo conformation for the major open st,ate is considered. Two H-exctlange rate classes are found in poly(rA)-poty(rU). The slower class represents t.hc* two exocyclic amino protons of A which exchange t,hrouph a pre- equilibrium opening mechanism, thereforr re\-ealing the fraction of time the helix is open. Base-pairs arc open So& of the time at 25°C. Thr faster class is assigned to tire U-N-3 H proton, tile rat? of wtlicll is limited by helix opening. Both opening and reclosing of the duplex are slow. 2 s-l and 40 s-l, respectively, at 25’C. Thermodynamic parameters for tllr equilibrium helix opening and for t,he rate of opening were determilled. These propertics may be consistent with a sirllplr openirlg involving swinging ollt. of the U base while retaining A more or 1~s stacked within the duplex. The rc*sults demonnt~rate t,hat 110 faster or morr populated tlelix-open stat,e OCCIII’S (w-hell st,ructklre is stable). It appears that,, unlike opening-closing reactions at a llelix end or a helix --coil boundary, internal base opening and closing are innately slow. One implication of this is t,tiat any rtiemical or biological process requiring access to sequences in the interior of a closed stable DNA duplex may be constrained to proceed only on a time scale of seconds. and not in milliseconds or microseconds. 1. Introduction Because nucleic acid hydrogen exchange depends on helix opening-closing behavior. hpdrogen exchange measurements can yield direct information on this interesting and potentially important aspect of struct’ure. Beyond this H-exchange provides a, probe for amount of double-helical structure, helix stability, and changes in helix structure. Hydrogen exchange studies seem especially promising for these measure- ments. since this approach does not perturb the structural parameters to be studied. Most previous hydrogen exchange work on both proteins and nucleic acids has used thr~ t’ritium-Sephadex method (Englander Ri Englander. 1978). It, now appears :I!)I NE:! ~zri:lli!‘i’3!:)411.;!)121 $02.00/0 i? 1070 ;2cii(lcmir I’rcSs Inc. (I, rl,tlon) T,tll.