Metal ion dependence of DNA cleavage by SepMI and EhoI restriction endonucleases.

Most of type II restriction endonucleases show an absolute requirement for divalent metal ions as cofactors for DNA cleavage. While Mg(2+) is the natural cofactor other metal ions can substitute it and mediate the catalysis, however Ca(2+) (alone) only supports DNA binding. To investigate the role of Mg(2+) in DNA cleavage by restriction endonucleases, we have studied the Mg(2+) and Mn(2+) concentration dependence of DNA cleavage by SepMI and EhoI. Digestion reactions were carried out at different Mg(2+) and Mn(2+) concentrations at constant ionic strength. These enzymes showed different behavior regarding the ions requirement, SepMI reached near maximal level of activity between 10 and 20mM while no activity was detected in the presence of Mn(2+) and in the presence of Ca(2+) cleavage activity was significantly decreased. However, EhoI was more highly active in the presence of Mn(2+) than in the presence of Mg(2+) and can be activated by Ca(2+). Our results propose the two-metal ion mechanism for EhoI and the one-metal ion mechanism for SepMI restriction endonuclease. The analysis of the kinetic parameters under steady state conditions showed that SepMI had a K(m) value for pTrcHisB DNA of 6.15 nM and a V(max) of 1.79×10(-2)nM min(-1), while EhoI had a K(m) for pUC19 plasmid of 8.66 nM and a V(max) of 2×10(-2)nM min(-1).

Ca(2+) binding to the ExDxD motif regulates the DNA cleavage specificity of a promiscuous endonuclease.

Most of the restriction endonucleases (REases) are dependent on Mg(2+) for DNA cleavage, and in general, Ca(2+) inhibits their activity. R.KpnI, an HNH active site containing ßßα-Me finger nuclease, is an exception. In presence of Ca(2+), the enzyme exhibits high-fidelity DNA cleavage and complete suppression of Mg(2+)-induced promiscuous activity. To elucidate the mechanism of unusual Ca(2+)-mediated activity, we generated alanine variants in the putative Ca(2+) binding motif, E(132)xD(134)xD(136), of the enzyme. Mutants showed decreased levels of DNA cleavage in the presence of Ca(2+). We demonstrate that ExDxD residues are involved in Ca(2+) coordination; however, the invariant His of the catalytic HNH motif acts as a general base for nucleophile activation, and the other two active site residues, D148 and Q175, also participate in Ca(2+)-mediated cleavage. Insertion of a 10-amino acid linker to disrupt the spatial organization of the ExDxD and HNH motifs impairs Ca(2+) binding and affects DNA cleavage by the enzyme. Although ExDxD mutant enzymes retained efficient cleavage at the canonical sites in the presence of Mg(2+), the promiscuous activity was greatly reduced, indicating that the carboxyl residues of the acidic triad play an important role in sequence recognition by the enzyme. Thus, the distinct Ca(2+) binding motif that confers site specific cleavage upon Ca(2+) binding is also critical for the promiscuous activity of the Mg(2+)-bound enzyme, revealing its role in metal ion-mediated modulation of DNA cleavage.

Characterizing the residue level folding of the intrinsically unstructured IA3.

Residue level analysis of the folding of simple proteins may hold the key to understanding folding pathways and aid in structure prediction. IA(3), the endogenous inhibitor of yeast aspartic proteinase A (YPrA), is an unstructured protein in solution. Comparison of the 2D (15)N-HSQC spectra of IA(3) in water and in 23% 2,2,2-trifluoroethanol (TFE) shows that the individual residue cross peaks of IA(3) become more dispersed in the presence of TFE, indicating that the protein undergoes an unstructured to structured transition in the presence of TFE. This transition can be monitored by the movements of the cross peaks. Following the individual cross peaks, however, is complicated and does not establish whether a single transition occurs globally in the sequence. In this equilibrium study, we apply singular value decomposition (SVD) to elucidate both the main features of the TFE-driven transition and the residue-level deviations from the average behavior. This analysis has yielded a two-state folding description as well as specifics of NMR frequency shifts of individual residues, indicating that the N-terminus of IA(3) has a higher helical propensity than the C-terminus. Additionally, we discuss possible mechanisms for observed deviations from a two-state folding transition. When combined with a traditional biochemical understanding of interactions between individual residues, this approach leads to a better understanding of protein folding.

PfoI, a unique type II restriction endonuclease that recognises the sequence 5'-T downward arrow CCNGGA-3'.

A new type II restriction endonuclease designated PfoI has been partially purified from Pseudomonas fluorescens biovar 126. PfoI recognises the interrupted hexanucleotide palindromic sequence 5'-T downward arrow CCNGGA-3' and cleaves DNA to produce protruding pentanucleotide 5'-ends.

Induction of multiple double-strand breaks within an hsr by meganucleaseI-SceI expression or fragile site activation leads to formation of double minutes and other chromosomal rearrangements.

Gene amplification is frequently associated with tumor progression, hence, understanding the underlying mechanisms is important. The study of in vitro model systems indicated that different initial mechanisms accumulate amplified copies within the chromosomes (hsr) or on extra-chromosomal elements (dmin). It has long been suggested that formation of dmin could also occur following hsr breakdown. In order to check this hypothesis, we developed an approach based on the properties of the I-SceI meganuclease, which induces targeted DNA double-strand breaks. A clone containing an I-SceI site, integrated by chance close to an endogenous dhfr gene locus, was used to select for methotrexate resistant mutants. We recovered clones in which the I-SceI site was passively co-amplified with the dhfr gene within the same hsr. We show that I-SceI-induced hsr breakdown leads to the formation of dmin and creates different types of chromosomal rearrangements, including inversions. This demonstrates, for the first time, a direct relationship between double-strand breaks and inversions. Finally, we show that activation of fragile sites by aphidicolin or hypoxia in hsr-containing cells also generates dmin and a variety of chromosomal rearrangements. This may constitute a valuable model to study the consequences of breaks induced in hsr of cancer cells in vivo.

Restriction fragment analysis as a source of error in detection of heteroplasmic mtDNA mutations.

The transition from A to G at nt 5656 (5656A-->G) in mitochondrial DNA has been suggested to be a pathogenic mutation and, furthermore, a heteroplasmic one. We found that the mutation was present in 14 out of 83 healthy controls from northern Finland and that 5656A-->G was exclusively associated with mtDNA haplogroup U. Interestingly, 5656A-->G appeared to be heteroplasmic in NheI digestion of PCR fragments that were amplified by using a mismatched oligonucleotide primer creating a digestion site in the presence of the mutant variant. However, we did not detect the wild type genome in clones from such a sample and subsequent experiments revealed that the apparent heteroplasmy was due to inhibition of NheI by NaCl. Our results suggest that 5656A-->G is a polymorphism and it may be highly characteristic for Finns. Furthermore, new heteroplasmic mutations identified by restriction fragment analysis should be adequately controlled for any false positive results that may be due to incomplete digestion.

Specific binding by EcoRV endonuclease to its DNA recognition site GATATC.

Restriction endonuclease EcoRV has been reported to be unable to distinguish its specific DNA site, GATATC, from non-specific DNA sites in the absence of the catalytic cofactor Mg2+, and thus to exercise sequence specificity solely in the catalytic step. In contrast, we show here that under appropriate conditions of pH and salt concentration, specific complexes with oligonucleotides containing the GATATC site can be detected by either filter-binding or gel-retardation. Equilibrium binding constants (K(A)) are easily measured by both direct equilibrium and equilibrium-competition methods. The preference for "specific" over "non-specific" binding at pH 7 in the absence of divalent cations is about 1000-fold (per mole of oligonucleotide) or 12,000-fold (per mole of binding sites). Ethylation-interference footprinting shows that the "specific" complex includes strong contacts to the phosphate groups GpApTpApTC. Specific DNA binding is strongly pH-dependent, decreasing about 15-fold for each increase of one pH unit above pH 6, but non-specific binding is not; thus, binding specificity decreases with increasing pH. Gel retardation and filter-binding at pH < or = 7 yield essentially identical values of K(A) for specific-site binding, but at pH > 7 gel retardation significantly underestimates K(A). Specific-site binding is stimulated about 700-fold by Ca2+ (not a cofactor for cleavage), but with non-cleavable 3'-phosphorothiolate and 4'-thiodeoxyribose derivatives whose response to Ca2+ is similar to that of the parent oligonucleotide, Mg2+ stimulates binding only fourfold and twofold, respectively. Thus, binding specificity is not dramatically enhanced by Mg2+. Taking into account discrimination in binding and in the first-order rate constant for phosphodiester bond scission, the overall discrimination exercised against the incorrect site GTTATC is about 10(7)-fold. EcoRV endonuclease is thus not a "new paradigm" for site-specific interaction without binding specificity, but like other type II restriction endonucleases achieves sequence specificity by discriminating both in DNA binding and in catalysis.

Formation and repair of O6-methylguanine in recombination hot spots of plant chromosomes.

Mutagen-induced chromatid aberrations are not randomly distributed along the metaphase chromosomes. In the field bean (Vicia faba), defined late-replicating and transcriptionally inactive heterochromatic regions are preferentially involved. After exposure to the alkylating agent N-methyl-N-nitrosourea (MNU) (10(-3) M, 1 hour), 70% of all aberrations are clustered within 6 segments containing tandemly repeated FokI elements of 59 bp, which comprise approximately 10% of the genome. Using immuno-slot-blot analyses, we have studied the frequency of O6-methylguanine (O6-MeG), a mutagenic lesion important for aberration induction, in total genomic DNA as well as in FokI sequences of the field bean after exposure to MNU. In either case, similar numbers of adducts per nucleotide were found immediately after treatment as well as after 18 hours of recovery, when most adducts were removed and significant amounts of chromatid aberrations were detectable. Peculiarities of long FokI element arrays (e.g., formation of specific tertiary structures), resulting in error-prone recombination repair, rather than preferential formation or delayed repair of O6-MeG are apparently responsible for aberration clustering in these hot spot regions.

Properties of triple helix formation with oligodeoxyribonucleotides containing 8-oxo-2'-deoxyadenosine and 2'-modified nucleoside derivatives.

The ability of homopyrimidine oligonucleotides containing 8-oxo-2'-deoxyadenosine (dAOH), 2'-methoxyuridine (Um), 2'-fluorouridine (Uf), 2'-methoxycytidine (Cm), and 2'-fluorocytidine (Cf) to form stable, triple-helical structures with sequences containing the recognition site for the class II-S restriction enzyme, Ksp632-I, was studied as a function of pH. The 8-oxo-2'-deoxyadenosine substituted oligomers were shown to bind within the physiological pH range in a pH-independent fashion, without a compromise in specificity. In particular, the substitutions of three deoxycytidine residues with 8-oxo-2'-deoxyadenosine showed higher endonuclease inhibition than the substitution of either one or two deoxycytidine residues with 8-oxo-2'-deoxyadenosine. In contrast, the oligonucleotides containing 2'-modified nucleosides (Uf, Um, Uf-Cf, Um-Cm, dAOH-Uf, and dAOH-Um) bind in a pH-dependent manner to the target duplex.

Photoaffinity cross-linking of TaqI restriction endonuclease using an aryl azide linked to the phosphate backbone.

In an effort to identify amino acid (aa) residues near the active site of TaqI restriction endonuclease (ENase), a sequence-specific photoaffinity reagent was designed. This reagent exploits the finding that modification of the Rp oxygen of the scissile phosphate does not interfere with substrate binding. The TpCGA phosphate was substituted with an Rp phosphorothioate group to direct the placement of the heterobifunctional reagent p-azidophenacyl bromide. TaqI bound the photoaffinity reagent specifically and formed a covalent adduct with the ENase in the presence of UV light. The modified aa was identified as Tyr161. This aa was changed to Phe by site-directed mutagenesis, and the resulting Y161F mutant was characterized. Removal of the Tyr161 hydroxyl group lowered both the kcat and the Km fivefold, indicating that, while this aa may be near the scissile phosphate, it is not critically required for catalysis.

Purification, properties, and sequence specificity of SslI, a new type II restriction endonuclease from Streptococcus salivarius subsp. thermophilus.

SslI, a type II restriction endonuclease, was purified from Streptococcus salivarius subsp. thermophilus strain BSN 45. SslI is an isoschizomer of BstNI. SslI activity was maximum at pH 8.8, 0 to 50 mM NaCl, 2 to 8 mM Mg2+, and 42 degrees C. Activity against phage DNA in vitro was demonstrated.