Lanthanide spectroscopic studies of the dinuclear and Mg(II)-dependent PvuII restriction endonuclease.

Type II restriction enzymes are homodimeric systems that bind four to eight base pair palindromic recognition sequences of DNA and catalyze metal ion-dependent phosphodiester cleavage. While Mg(II) is required for cleavage in these enzymes, in some systems Ca(II) promotes avid substrate binding and sequence discrimination. These properties make them useful model systems for understanding the roles of alkaline earth metal ions in nucleic acid processing. We have previously shown that two Ca(II) ions stimulate DNA binding by PvuII endonuclease and that the trivalent lanthanide ions Tb(III) and Eu(III) support subnanomolar DNA binding in this system. Here we capitalize on this behavior, employing a unique combination of luminescence spectroscopy and DNA binding assays to characterize Ln(III) binding behavior by this enzyme. Upon excitation of tyrosine residues, the emissions of both Tb(III) and Eu(III) are enhanced severalfold. This enhancement is reduced by the addition of a large excess of Ca(II), indicating that these ions bind in the active site. Poor enhancements and affinities in the presence of the active site variant E68A indicate that Glu68 is an important Ln(III) ligand, similar to that observed with Ca(II), Mg(II), and Mn(II). At low micromolar Eu(III) concentrations in the presence of enzyme (10-20 microM), Eu(III) excitation (7)F(0) --> (5)D(0) spectra yield one dominant peak at 579.2 nm. A second, smaller peak at 579.4 nm is apparent at high Eu(III) concentrations (150 microM). Titration data for both Tb(III) and Eu(III) fit well to a two-site model featuring a strong site (K(d) = 1-3 microM) and a much weaker site (K(d) approximately 100-200 microM). Experiments with the E68A variant indicate that the Glu68 side chain is not required for the binding of this second Ln(III) equivalent; however, the dramatic increase in DNA binding affinity around 100 microM Ln(III) for the wild-type enzyme and metal-enhanced substrate affinity for E68A are consistent with functional relevance for this weaker site. This discrimination of sites should make it possible to use lanthanide substitution and lanthanide spectroscopy to probe individual metal ion binding sites, thus adding an important tool to the study of restriction enzyme structure and function.

The UV response in Saccharomyces cerevisiae involves the mitogen-activated protein kinase Slt2p.

Exposure to UV causes a response in yeast and mammalian cells, which is distinct from the response to DNA damage. We report that the mitogen-activated protein kinase Slt2p is involved in this response in Saccharomyces cerevisiae. Thus, budding yeast and mammalian cells respond to UV by using very similar signal transduction pathways.

Binding and conformational analysis of phosphoramidate-restriction enzyme interactions.

Phosphoramidates are modified deoxyoligonucleotides that feature nitrogen in place of the 3'-oxygen of a phosphodiester linkage. Noted for stability against nuclease activity, these linkages are of both mechanistic and therapeutic interest. While a number of studies characterizing the properties of oligonucleotides composed entirely of phosphoramidate linkages have been published, little is known about how singly substituted phosphoramidate substitutions affect the thermodynamics and structure of protein-oligonucleotide interactions. We chose to investigate these interactions with PvuII endonuclease, the DNA binding behavior of which is well-characterized. Oligonucleotide duplexes containing a phosphoramidate substitution at the scissile phosphates were resistant to cleavage by the enzyme, even after extended incubations. However, the enzyme was able to cleave the native strand in a native:phosphoramidate heteroduplex at a rate comparable to that observed with the native substrate. Ca(II)-stimulated PvuII binding for a phosphoramidate-substituted oligonucleotide is comparable to that of the native duplex (K(d) approximately 200 pM). K(d) values obtained in the presence of Mg(II) are somewhat weaker (K(d) approximately 10 nM). Under metal-free conditions, the enzyme exhibited a remarkable approximately 50-fold greater affinity for the modified oligonucleotide relative to the native substrate (5 vs 240 nM). While (31)P NMR spectra indicate increased chemical shift dispersion in the free phosphoramidate duplex, the spectrum of the enzyme-bound duplex is similar to that of the native duplex. (1)H-(15)N HSQC analysis indicates that enzyme conformations in the presence of these oligonucleotides are also comparable. The tight binding of the phosphoramidate duplex under metal-free conditions and its resistance to cleavage are attributed to local conformational adjustments propagating from the O-->N substitution.

Investigation of restriction enzyme cofactor requirements: a relationship between metal ion properties and sequence specificity.

Restriction enzymes are important model systems for understanding the mechanistic contributions of metal ions to nuclease activity. These systems are unique in that they combine distinct functions which have been shown to depend on metal ions: high-affinity DNA binding, sequence-specific recognition of DNA, and Mg(II)-dependent phosphodiester cleavage. While Ca(II) and Mn(II) are commonly used to promote DNA binding and cleavage, respectively, the metal ion properties that are critical to the support of these functions are not clear. To address this question, we assessed the abilities of a series of metal ions to promote DNA binding, sequence specificity, and cleavage in the representative PvuII endonuclease. Among the metal ions tested [Ca(II), Sr(II), Ba(II), Eu(III), Tb(III), Cd(II), Mn(II), Co(II), and Zn(II)], only Mn(II) and Co(II) were similar enough to Mg(II) to support detectable cleavage activity. Interestingly, cofactor requirements for the support of DNA binding are much more permissive; the survey of DNA binding cofactors indicated that Cd(II) and the heavier and larger alkaline earth metal ions Sr(II) and Ba(II) were effective cofactors, stimulating DNA binding affinity 20-200-fold. Impressively, the trivalent lanthanides Tb(III) and Eu(III) promoted DNA binding as efficiently as Ca(II), corresponding to an increase in affinity over 1000-fold higher than that observed under metal-free conditions. The trend for DNA binding affinity supported by these ions suggests that ionic radius and charge are not critical to the promotion of DNA binding. To examine the role of metal ions in sequence discrimination, we determined specificity factors [K(a)(specific)/K(a)(nonspecific)] in the presence of Cd(II), Ba(II), and Tb(III). Most interestingly, all of these ions compromised sequence specificity to some degree compared to Ca(II), by either increased affinity for a noncognate sequence, decreased affinity for the cognate sequence, or both. These results suggest that while amino acid-base contacts are important for specificity, the properties of metal ion cofactors at the catalytic site are also critical for sequence discrimination. This insight is invaluable to our efforts to understand and subsequently design sequence-specific nucleases.