Background: RecBCD helicase is involved in repair of double-stranded DNA breaks. Results: The 5Ј to 3Ј ssDNA translocation rate of RecBCD is faster than the 3Ј to 5Ј rate in the absence of a CHI site, and the rates are coupled asymmetrically. Conclusion: RecBC controls 3Ј to 5Ј and 5Ј to 3Ј translocation, but RecD controls only 5Ј to 3Ј translocation. Significance: Asymmetric regulation may explain how RecBCD is regulated after CHI recognition.

Ester hydrolysis is one of the most ubiquitous reactions in biochemistry. Many of these reactions rely on metal ions for various mechanistic steps. A large number of metal dependent nucleases have been crystallized with two metal ions in their active sites. In spite of an ongoing discussion about the roles of these metal ions in nucleic acid hydrolysis, there are very few studies which examine this issue using the native cofactor Mg(II) and global fitting of reaction progress curves. As part of a comprehensive study of the representative homodimeric PvuII endonuclease, we have collected single turnover DNA cleavage data as a function of Mg(II) concentration and globally fit these data to a number of models which test various aspects of metallonuclease mechanism. DNA association rate constants are about 100-fold higher in the presence of the catalytically nonsupportive Ca(II) vs. the native cofactor Mg(II), highlighting an interesting cofactor difference. A pathway in which metal ions bind prior to DNA is kinetically favored. The data fit well to a model in which both one and two metal ions per active site (EM2S and EM4S) support cleavage. Interestingly, the cleavage rate for EM2S is about 100-fold slower than that displayed by EM4S. Collectively, these data indicate that for the PvuII system, catalysis involving one metal ion per active site can indeed occur, but that a more efficient two metal ion mechanism can be operative under saturating metal ion (in vitro) conditions.

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Most nucleases rely on divalent cations as cofactors to catalyze the hydrolysis of nucleic acid phosphodiester bonds. Here both equilibrium and kinetic experiments are used to test recently proposed models regarding the metal ion dependence of product release and the degree of cooperativity between metal ions bound in the active sites of the homodimeric PvuII endonuclease. Equilibrium fluorescence anisotropy studies indicate that product binding is dramatically weakened in the presence of metal ions. Pre steady state kinetics indicate that product release is at least partially rate limiting. Steady state and pre steady state data fit best to models in which metals remain bound to the enzyme after the release of product. Finally, analysis of cooperative and independent binding models for metal ions indicates that single turnover kinetic data are consistent with little to no positive cooperativity between the two metal ions binding each active site. Keywordsmetallonuclease; mechanism; kinetics; product release; metal ions; cooperativity Nucleases are unique among hydrolytic enzymes in their challenge: To catalyze the cleavage of a phosphodiester bond, one of the most stable bonds in biochemistry [1], in a charge repulsive, polyanionic context. Due the high negative charge density and relatively high natural abundance [2], divalent cations like Mg(II) and Ca(II) are natural cofactors for this process. Since the early 1990's many metallonucleases have been crystallized, and a large fraction of these enzyme active sites feature more than one metal ion [3]. These structures have served as the inspiration for mechanisms involving two metal ions. Indeed, given the steps necessary to achieve the hydrolysis of phosphodiester bonds, it makes mechanistic sense to distribute the various steps between metal ions. In most proposed mechanisms, one metal ion is principally involved in nucleophile activation (metal A in Fig. 1), while the other (metal B) is often depicted as contributing to transition state stabilization but also involved in facilitating product release by coordinating a water molecule which donates a proton to the leaving group.With regard to the roles of the metal ions in various mechanistic steps, most of the attention has been devoted to the early steps of substrate binding [4], nucleophile activation [5], and other aspects of the chemical step. Indeed, these have been analyzed in some detail for the restriction enzyme PvuII endonuclease [3,6]. Like many of its relatives, this metallonuclease ; E-mail: cdup@umsl.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal perta...

Metallonucleases conduct metal dependent nucleic acid hydrolysis. While metal ions serve in multiple mechanistic capacities in these enzymes, precisely how the attacking water is activated remains unclear for those lacking an obvious general base. All arguments hinge on appropriate pK a 's for active site moieties very close to this species, and measurement of the pK a of a specific water molecule is difficult to access experimentally. Here we describe a computational approach for exploring the local electrostatic influences on the water-derived nucleophile in metallonucleases featuring the common PD…(D/E)xK motif. We utilized UHBD to predict the pK a 's of active site groups, including that of a water molecule positioned to act as a nucleophile. The pK a of a Mg(II)-ligated water molecule hydrogen bonded to the conserved Lys70 in a Mg(II)-PvuII enzyme complex was calculated to be 6.5. The metal and the charge on the Lys group were removed in separate experiments; both resulted in the elevation of the pK a of this water molecule, consistent with contributions from both moieties to lowering this pK a . This behavior is preserved among other PD…(D/E)xK metallonucleases. pK a 's extracted from the pH dependence of the single turnover rate constant are compared to previous experimental data and the above predicted pK a 's.