[New derivatives of azobenzene for the directed modification of proteins].

Derivatives of azobenzene which contained a maleimide group in one of the benzene rings (for binding to a protein cysteine residue) and maleimide, hydroxyl, or carboxyl substitutes in another benzene ring were synthesized. The reactivity of these compounds towards a cysteine residue of a protein and their optical properties in a free state and after their attachment to the mutant forms of the SsoII restriction endonuclease were studied.

Sau3AI, a monomeric type II restriction endonuclease that dimerizes on the DNA and thereby induces DNA loops.

Here, we report that Sau3AI, an unusually large type II restriction enzyme with sequence homology to the mismatch repair protein MutH, is a monomeric enzyme as shown by gel filtration and ultracentrifugation. Structural similarities in the N- and C-terminal halves of the protein suggest that Sau3AI is a pseudo-dimer, i.e. a polypeptide with two similar domains. Since Sau3AI displays a nonlinear dependence of cleavage activity on enzyme concentration and a strong preference for substrates with two recognition sites over those with only one, it is likely that the functionally active form of Sau3AI is a dimer of a pseudo-dimer. Indeed, electron microscopy studies demonstrate that two distant recognition sites are brought together through DNA looping induced by the simultaneous binding of two Sau3AI molecules to the DNA. We suggest that the dimeric form of Sau3AI supplies two DNA-binding sites, one that is associated with the catalytic center and one that serves as an effector site.

Immobilization of sugar-non-specific nucleases by utilizing the streptavidin--biotin interaction.

Due to their high enzymatic activity, the sugar-non-specific endonucleases from Serratia marcescens and Anabaena can be used for a number of applications, such as the removal of contaminating genetic material from biological preparations, footprinting studies, and the determination of nucleic acids in biochemical samples. These methods would benefit from immobilized nucleases. For this purpose, a single cysteine residue was added at the N-terminus of the Serratia and Anabaena nucleases and subsequently modified with a maleimide-biotin conjugate. Alternatively, a biotin acceptor domain was fused to the Anabaena nuclease, allowing biotinylation during expression in E. coli without a further chemical step. The attachment of biotin-modified nucleases to streptavidin-coated paramagnetic beads and to streptavidin-coated surface plasmon resonance sensor chips (to study interactions with substrate and inhibitor) worked well when aggregates present in the protein preparations were removed by ultrafiltration. These methods should be of general use for similar enzyme systems.

Identification of Asp218 and Asp326 as the principal Mg2+ binding ligands of the homing endonuclease PI-SceI.

The monomeric homing endonuclease PI-SceI harbors two catalytic centers which cooperate in the cleavage of the two strands of its extended recognition sequence. Structural and biochemical data suggest that catalytic center I contains Asp218, Asp229, and Lys403, while catalytic center II contains Asp326, Thr341, and Lys301. The analogy with I-CreI, for which the cocrystal structure with the DNA substrate has been determined, suggests that Asp218 and Asp229 in catalytic center I and Asp326 and Thr341 in catalytic center II serve as ligands for Mg(2+), the essential divalent metal ion cofactor which can be replaced by Mn(2+) in vitro. We have carried out a mutational analysis of these presumptive Mg(2+) ligands. The variants carrying an alanine or asparagine substitution bind DNA, but (with the exception of the D229N variant) are inactive in DNA cleavage in the presence of Mg(2+), demonstrating that these residues are important for cleavage. Our finding that the PI-SceI variants carrying single cysteine substitutions at these positions are inactive in the presence of the oxophilic Mg(2+) but active in the presence of the thiophilic Mn(2+) suggests that the amino acid residues at these positions are involved in cofactor binding. From the fact that in the presence of Mn(2+) the D218C and D326C variants are even more active than the wild-type enzyme, it is concluded that Asp218 and Asp326 are the principal Mg(2+) ligands of PI-SceI. On the basis of these findings and the available structural information, a model for the composition of the two Mg(2+) binding sites of PI-SceI is proposed.

Influence of DNA target melting behavior on real-time PCR quantification.

BACKGROUND: Quantitative real-time PCR is increasingly used to quantify copy numbers of nucleic acids for clinical applications. We observed that the measurements of allele imbalances of the tumor suppressor gene p16 and the oncogene ErbB-2 yielded results with variable precision under certain experimental conditions. METHODS: We used the LightCycler(TM) real-time PCR system to quantify different genomic target sequences using hybridization probes or SYBR Green for detection. RESULTS: With two primer/template systems (p16 and ErbB-2), we observed sinusoidal scattering of the threshold cycle values depending on the capillary position in the thermostated reaction chamber. This scattering depended on the denaturation temperature only when complete genomic DNA was used as template and did not occur when PCR product or restricted or boiled genomic DNA was used or the denaturation temperature in the first cycles was increased (and other targets, such as p53, HBB, IGF-1, GAPDH, and PBGD, did not show this behavior). CONCLUSIONS: Before a primer system is used for precise quantitative real-time PCR, the dependence of the quantification results on the positions of reaction tubes in the thermocycler should be tested. Our data indicate that amplification efficiencies, especially in the first cycles, depend not only on the priming efficiencies of the primers and the melting temperature of the amplicon, but also on the melting behavior of the amplicon's genomic vicinity. Complete denaturation of genomic DNA is necessary to maximize precision of quantitative PCR. Higher denaturation temperatures in the initial cycles or boiling of DNA before the PCR can improve the accuracy of quantification in some cases.

A model for the PI-SceIxDNA complex based on multiple base and phosphate backbone-specific photocross-links.

We have synthesized different oligodeoxynucleotides carrying, in single positions of the >36 bp recognition site of PI-SceI, photoreactive base analogues (5-iododeoxypyrimidines) or phosphate modifications (p-azidophenacylphosphorothioates) and used them in photocross-linking experiments with PI-SceI to probe the protein-DNA interface of the specific complex between the homing endonuclease PI-SceI and its DNA substrate. One base-specific and several backbone-specific cross-links were analyzed in detail: the cross-linking positions were identified by Edman degradation of isolated cross-linked peptidexoligodeoxynucleotide adducts and confirmed by site-directed mutagenesis. Based on these results and the crystal structure of PI-SceI, a model for the structure of the PI-SceIxDNA complex is proposed.

Mechanism of DNA cleavage by the DNA/RNA-non-specific Anabaena sp. PCC 7120 endonuclease NucA and its inhibition by NuiA.

A structural model of the DNA/RNA non-specific endonuclease NucA from Anabaena sp. PCC7120 that has been obtained on the basis of the three-dimensional structure of the related Serratia nuclease, suggests that the overall architecture of the active site including amino acid residues H124, N155 and E163 (corresponding to H89, N119 and E127 in Serratia nuclease) is similar in both nucleases. Substitution of these residues by alanine leads to a large reduction in activity (<0.1 %), similarly as observed for Serratia nuclease demonstrating that both enzymes share a similar mechanism of catalysis with differences only in detail. NucA is inhibited by its specific polypeptide inhibitor with a K(i) value in the subpicomolar range, while the related Serratia nuclease at nanomolar concentrations is only inhibited at an approximately 1000-fold molar excess of NuiA. The artificial chromophoric substrate deoxythymidine 3',5'-bis-(p-nitrophenyl phosphate) is cleaved by NucA as well as by Serratia nuclease. Cleavage of this analogue by NucA, however, is not inhibited by NuiA, suggesting that small molecules gain access to the active site of NucA in the enzyme-inhibitor complex under conditions where cleavage of DNA substrates is completely inhibited. The active site residue E163 seems to be the main target amino acid for inhibition of NucA by NuiA, but R93, R122 and R167 (corresponding to K55, R87, R131 in Serratia nuclease) are also involved in the NucA/NuiA interaction. NuiA deletion mutants show that the structural integrity of the N and C-terminal region of the inhibitor is important for complex formation with NucA and inhibition of nuclease activity. Based on these results a mechanism of DNA cleavage by NucA and its inhibition by NuiA is proposed.

The monomeric homing endonuclease PI-SceI has two catalytic centres for cleavage of the two strands of its DNA substrate.

The monomeric homing endonuclease PI-SceI cleaves the two strands of its DNA substrate in a concerted manner, which raises the question of whether this enzyme harbours one or two catalytic centres. If PI-SceI has only one catalytic centre, one would expect that cross-linking enzyme and substrate should prevent reorientation of the enzyme required to perform the second cut after having made the first cut: PI-SceI, however, when cross-linked to its substrate, is able to cleave both DNA strands. If PI-SceI has two catalytic centres, one would expect that it should be possible to inactivate one catalytic centre by mutation and obtain a variant with preference for a substrate nicked in one strand; such variants have been found. The structural homology between the catalytic domain of PI-SceI having a pseudo 2-fold symmetry, and I-CreI, a homodimeric homing endonuclease, suggests that in PI-SceI active site I, which attacks the top strand, comprises Asp218, Asp229 and Lys403, while Asp326, Thr341 and Lys301 make up active site II, which cleaves the bottom strand. Cleavage experiments with modified oligodeoxynucleotides and metal ion mapping experiments demonstrate that PI-SceI interacts differently with the two strands at the cleavage position, supporting a model of two catalytic centres.

Photocross-linking of the homing endonuclease PI-SceI to its recognition sequence.

PI-SceI is an intein-encoded protein that belongs to the LAGLIDADG family of homing endonucleases. According to the crystal structure and mutational studies, this endonuclease consists of two domains, one responsible for protein splicing, the other for DNA cleavage, and both presumably for DNA binding. To define the DNA binding site of PI-SceI, photocross-linking was used to identify amino acid residues in contact with DNA. Sixty-three double-stranded oligodeoxynucleotides comprising the minimal recognition sequence and containing single 5-iodopyrimidine substitutions in almost all positions of the recognition sequence were synthesized and irradiated in the presence of PI-SceI with a helium/cadmium laser (325 nm). The best cross-linking yield (approximately 30%) was obtained with an oligodeoxynucleotide with a 5-iododeoxyuridine at position +9 in the bottom strand. The subsequent analysis showed that cross-linking had occurred with amino acid His-333, 6 amino acids after the second LAGLIDADG motif. With the H333A variant of PI-SceI or in the presence of excess unmodified oligodeoxynucleotide, no cross-linking was observed, indicating the specificity of the cross-linking reaction. Chemical modification of His residues in PI-SceI by diethylpyrocarbonate leads to a substantial reduction in the binding and cleavage activity of PI-SceI. This inactivation can be suppressed by substrate binding. This result further supports the finding that at least one His residue is in close contact to the DNA. Based on these and published results, conclusions are drawn regarding the DNA binding site of PI-SceI.

Synthesis and biochemical characterization of obligatory dimers of the sugar non-specific nuclease from Serratia marcescens using specifically designed bismaleimidoalkanes as SH-specific crosslinking reagents.

The genetically engineered S140C variant of the homodimeric nuclease from Serratia marcescens was crosslinked across the dimer interface at the Cys 140 residues using bifunctional SH-specific 1,1'-alkanediyl-bis-pyrrole-2,5-diones of different lengths. These bismaleimidoalkanes were synthesized by the condensation of n-alkyldiamines with maleic anhydride and subsequent cyclization with acetic anhydride and sodium acetate. Bismaleimidohexane (BMH) which gave the best crosslinking yield was used to produce in preparative amounts crosslinked Serratia nuclease. The crosslinked protein has the same secondary structure and exhibits the same guanidinium chloride unfolding behavior as the wild type enzyme or the non-covalently linked S 140C variant. In contrast, in thermal unfolding experiments the crosslinked dimer behaves differently from the wild type enzyme or the non-covalently linked S140C variant. CD-spectra recorded during temperature rise showed only minor changes of the secondary structure composition for the wild type enzyme and the non-covalently linked S140C variant, whereas in the case of the crosslinked S140C dimer a distinct increase of the CD effect was observed corresponding to an increase in alpha-helix. Our results demonstrate that bismaleimidoalkanes are very well suited to covalently link subunits of proteins, provided suitably located cysteine residues are present.

Quantitative PCR with Internal Standardization and OLA-ELISA Product Analysis for the p53 Tumor Suppressor Gene.

Over the last nine years, several quantitative polymerase chain reaction (QPCR) techniques have been developed, and these are now frequently used for the quantification of DNA template copy numbers. However, only few of these PCR techniques are suitable for the precise and absolute quantification of the template copy number (1,2). For this purpose, we describe here a quantitative PCR strategy that uses a known amount of an internal standard DNA that is amplified in competition with the sample template, using one common PCR primer pair and identical primer binding sites for both templates (2-4). In the literature, several variants of internal control sequences were used for the purpose of standardization, e.g., (i) homologous gene sequences of closely related species; differing in few bp, slightly in length and/or absence or presence of restriction sites (5,6), (ii) sample DNA derived sequences that are shortened by a deletion (7) or (iii) lengthened by an insertion (8); (iv) competitor fragments that contain more (9); or (v) less (10) extended heterologous sequence strings; or (vi) differ only by one or two bp, thereby replacing a sample specific restriction site by unique one specific to the internal control DNA (1,11,12). But only the last type of the internal control templates that differ in a negligible manner from the sample DNA sequenc is suitable for precise quantifications, as could be shown by theoretical considerations (2) as well as experimentally (1). Even in the case of a homologous internal control sequence of identical length as the sample sequence and differing by less than 5% in sequence, the two templates are not amplified with the same efficiency and therefore do not fulfill the criteria of ideal competition (2), as could be shown (6).

The protein splicing domain of the homing endonuclease PI-sceI is responsible for specific DNA binding.

The homing endonuclease PI- Sce I consists of a protein splicing domain (I) and an endonucleolytic domain (II). To characterize the two domains with respect to their contribution to DNA recognition we cloned, purified and characterized the isolated domains. Both domains have no detectable endonucleolytic activity. Domain I binds specifically to the PI- Sce I recognition sequence, whereas domain II displays only weak non-specific DNA binding. In the specific complex with domain I the DNA is bent to a similar extent as observed with the initial complex formed between PI- Sce I and DNA. Our results indicate that protein splicing domain I is also involved in recognition of the DNA substrate.

Structural and functional analysis of the homing endonuclease PI-sceI by limited proteolytic cleavage and molecular cloning of partial digestion products.

PI-SceI is a member of an unusual class of rare cutting homing endonucleases produced by an autocatalytic protein splicing from a precursor. To analyze the structural and functional domain organization of the endonuclease PI-SceI and to examine whether the DNA binding activity can be structurally separated from the catalytic activity, we performed limited proteolytic digestion experiments with various proteases. Two protease-resistant fragments spanning the N- and C-terminal halves of the nuclease were identified using different proteases which cleave the protein in the same region. Each fragment contains one of the two conserved LAGLIDADG motifs. The products of the limited proteolytic digests were shown to remain associated and to exhibit specific DNA binding but to be inactive in DNA cleavage. Different from what is observed with native PI-SceI, only one complex is formed as shown in an electrophoretic mobility shift assay. Expression clones for the N- and C-terminal protein fragments obtained by tryptic digestion were constructed, and the proteins PI-SceI-N and PI-SceI-C were purified. Only PI-SceI-N exhibits DNA binding activity. Bending experiments with PI-SceI-N, a mixture of PI-SceI-N and PI-SceI-C, as well as the products of the limited tryptic digest show that a DNA substrate with the full length recognition sequence is bent by 45;. This degree of bending is also observed with a DNA containing only the right side of the recognition sequence, corresponding to one of the DNA cleavage products of PI-SceI. Our results demonstrate that the N-terminal half of PI-SceI which lacks one of the two LAGLDADG motifs is able to bind to DNA specifically and to induce one of the distortions observed to occur in the process of DNA binding by PI-SceI. These results are discussed in light of the recently solved crystal structure of PI-SceI and used to refine a model for the mechanism of DNA binding and cleavage by PI-SceI.

Characterization of the interaction between the restriction endonuclease McrBC from E. coli and its cofactor GTP.

McrBC, a GTP-dependent restriction enzyme from E. coli K-12, cleaves DNA containing methylated cytosine residues 40 to 80 residues apart and 3'-adjacent to a purine residue (PumCN40-80PumC). The presence of the three consensus sequences characteristic for guanine nucleotide binding proteins in one of the two subunits of McrBC suggests that this subunit is responsible for GTP binding and hydrolysis. We show here that (i) McrB binds GTP with an affinity of 10(6) M-1 and that GTP binding stabilizes McrB against thermal denaturation. (ii) McrB binds GDP about 50-fold and ATP at least three orders of magnitude more weakly than GTP. (iii) McrB hydrolyzes GTP in the presence of Mg2+ with a steady-state rate of approximately 0.5 min-1. (iv) McrC stimulates GTP hydrolysis 30-fold, but substrate DNA has no detectable effect on the GTPase activity of McrB, neither by itself nor in the presence of McrC. (v) Substitution of N339 and N376 with alanine allowed us to identify NTAD (339 to 342) rather than NKKA (376 to 379) as the equivalent of the third consensus sequence motif characteristic for guanine nucleotide binding proteins, NKXD.

Binding, bending and cleavage of DNA substrates by the homing endonuclease Pl-SceI.

To characterize the interaction between the homing endonuclease PI-SceI and DNA, we prepared different DNA substrates containing the natural recognition sequence or parts thereof. Depending on the nature of the substrates, efficient cleavage is observed with a DNA containing approximatel 30 bp of the natural recognition sequence using supercoiled plasmids, approximately 40-50 bp using linearized plasmids and > 50 bp using synthetic double-stranded oligodeoxynucleotides. Cleavage of supercoiled plasmids occurs without accumulation of the nicked intermediate. In the presence of Mn2+, DNA cleavage by PI-SceI is more efficient than with Mg2+ and already occurs with substrates containing a shorter part of the recognition sequence. The requirements for strong binding are less stringent: a 35 bp oligodeoxynucleotide which is not cleaved is bound as firmly as other longer oligodeoxynucleotides. PI-SceI binds with high affinity to one of its cleavage products, a finding which may explain why PI-SceI hardly shows enzymatic turnover in vitro. Upon binding, two complexes are formed, which differ in the degree of bending (45 degrees versus 75 degrees). According to a phasing analysis bending is directed into the major groove. Strong binding, not, however, cleavage is also observed with the genetically engineered enzymatically inactive variant comprising amino acids 1-277. Models for binding and cleavage of DNA by PI-SceI are discussed based on these results.

Semiautomated quantitative detection of loss of heterozygosity in the tumor suppressor gene p53.

The most frequently altered gene in diverse tumor types is the tumor suppressor gene p53. Typically, normal function is inactivated by point mutation of one allele and deletion of the other. Therefore, loss of heterozygosity (LOH) of intragenic polymorphic markers is a strong indication for p53 involvement in a cancerous lesion. This study shows that a highly polymorphic short tandem repeat (STR) within intron 1 of p53 is an excellent marker for quantitative evaluation of LOH in tumor samples, whose multicolor, fluorescently tagged PCR products are analyzed and quantitated on an automated DNA sequencer. The range of error was analyzed in detail. Discrete allelic profiles were obtained following amplification of DNA from microdissected cell samples of patients with urogenital tumors. By calculating qLOH, the relative allele ratio of a tumor compared with healthy tissue, a quantitative expression for the LOH is obtained. PCR-based tumor DNA typing using fluorescent STR primers and automated analysis provides an enhanced level of accuracy and sensitivity required for routine analysis in clinical practice, where large numbers of tumor samples have to be processed.

Polymorphism of the pentanucleotide repeat d(AAAAT) within intron 1 of the human tumor suppressor gene p53 (17p13.1).

DyeDeoxy terminator cycle sequencing of allele-specific polymerase chain reaction products has shown that there is a highly polymorphic d(AAAAT) pentanucleotide repeat within the first intron of the human p53 gene. This provides a genetic marker for tumor suppressor p53 gene alterations.