Dissecting the metal ion dependence of DNA binding by PvuII endonuclease.
Divalent cations can provide an effective means of modulating the behavior of nucleic acid binding proteins. As a result, there is strong interest in understanding the role of metal ions in the function of both nucleic acid binding proteins and their enzymes. We have applied complementary fluorescence spectroscopic and nitrocellulose filter binding assays to quantitate the role of metal ions in mediating DNA binding and sequence specificity by the representative PvuII endonuclease. At pH 7.5 in the presence of the catalytically nonsupportive Ca(II), this enzyme binds the PvuII target sequence with a K(d) of 50 pM. Under strict metal-free conditions, the enzyme exhibits a K(d) of only 300 nM for the cognate sequence, an affinity which is weak relative to those measured for other systems in the absence of metal ions. This represents a 6000-fold increase in PvuII affinity for cognate DNA upon the addition of Ca(II). The pH dependences of both metal ion-dependent and metal ion-independent DNA binding are remarkably shallow throughout the physiological range; other characterized restriction enzymes exhibit more pronounced pH dependences of DNA binding even in the absence of metal ions. Similar measurements with noncognate sequences indicate that divalent metal ions are not important to nonspecific DNA binding; K(d) values are approximately equal to 200 nM throughout the physiological pH range, a behavior shared with other endonucleases. While some of these results extend somewhat the range of expected behavior for restriction enzymes, these results indicate that PvuII endonuclease shares with other characterized systems a mechanism by which cognate affinity and sequence discrimination are most effectively achieved in the presence of divalent metal ions.
Conlan
Lori H
LH
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
Dupureur
Cynthia M
CM
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D011485:Protein Binding; D013379:Substrate Specificity
D004247:DNA; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific