P1 nuclease cleavage is dependent on length of the mismatches in DNA

P1 nuclease is one of the most extensively used single-strand DNA specific nucleases in molecular biology. In modern biology, it is used as an enzymatic probe to detect altered DNA conformations. It is well documented that P1 cleaves single-stranded nucleic acids and single-stranded DNA regions. The fact that P1 can act under a wide range of conditions, including physiological pH and temperature make it the most commonly used enzymatic probe in DNA structural studies. Surprisingly, to this date, there is no study to characterize the influence of length of mismatches on P1 sensitivity. Using a series of radioactively labeled oligomeric DNA substrates-containing mismatches, we find that P1 nuclease cleavage is dependent on the length of mismatches. P1 does not cleave DNA when there is a single-base mismatch. P1 cleavage efficiency is optimum when mismatch length is 3 nt or more. Changing the position of the mismatches also does not make any difference in cleavage efficacy. These novel findings on P1 properties have implications for its use in DNA structure and DNA repair studies.     

A versatile mismatch recognition agent: specific cleavage of a plasmid DNA at a single base mispair

[Rh(bpy)2(chrysi)]3+ is a novel, sterically bulky DNA intercalator that has been designed to bind specifically in the destabilized regions near DNA base mismatches and, upon photoactivation, to cleave the DNA backbone. Here the molecule is shown to be both a general and remarkably specific mismatch recognition agent. Specific DNA cleavage is observed at over 80% of mismatch sites in all the possible single base pair sequence contexts around the mispaired bases. Moreover, the complex is highly site-specific; it is shown to recognize and photocleave at a single base mismatch in a 2725 base pair linearized plasmid heteroduplex. Sterically demanding intercalators such as [Rh(bpy)2(chrysi)]3+ may have application both in mutation detection systems and as mismatch-specific chemotherapeutic agents.     

Requirement for Phe36 for DNA binding and mismatch repair by Escherichia coli MutS protein

The MutS family of DNA repair proteins recognizes base pair mismatches and insertion/deletion mismatches and targets them for repair in a strand-specific manner. Photocrosslinking and mutational studies previously identified a highly conserved Phe residue at the N-terminus of Thermus aquaticus MutS protein that is critical for mismatch recognition in vitro. Here, a mutant Escherichia coli MutS protein harboring a substitution of Ala for the corresponding Phe36 residue is assessed for proficiency in mismatch repair in vivo and DNA binding and ATP hydrolysis in vitro. The F36A protein is unable to restore mismatch repair proficiency to a mutS strain as judged by mutation to rifampicin or reversion of a specific point mutation in lacZ. The F36A protein is also severely deficient for binding to heteroduplexes containing an unpaired thymidine or a G:T mismatch although its intrinsic ATPase activity and subunit oligomerization are very similar to that of the wild-type MutS protein. Thus, the F36A mutation appears to confer a defect specific for recognition of insertion/deletion and base pair mismatches.     

Recognition of DNA insertion/deletion mismatches by an activity in Saccharomyces cerevisiae.

An activity in nuclear extracts of S.cerevisiae binds specifically to heteroduplexes containing four to nine extra bases in one strand. The specificity of this activity (IMR, for insertion mismatch recognition) in band shift assays was confirmed by competition experiments. IMR is biochemically and genetically distinct from the MSH2 dependent, single base mismatch binding activity. The two activities migrate differently during electrophoresis, they are differentially competable and their spectra of mispair binding are distinct. Furthermore, IMR activity is observed in extracts from an msh2- msh3- msh4- strain. IMR exhibits specificity for insertion mispairs in two different sequence contexts. Binding is influenced by the structure of the mismatch since an insertion with a hairpin configuration is not recognized by this activity. IMR does not result from single-strand binding because single-stranded probes to not yield IMR complex and single-stranded competitors are unable to displace insertion heteroduplexes from the complex. Similar results with intrinsically bent duplexes make it unlikely that recognition is conferred by a bend alone. Heteroduplexes bound by IMR do not contain any obvious damage. These findings are consistent with the idea that yeast contains a distinct recognition factor, IMR that is specific for insertion/deletion mismatches.     

Repair of specific base pair mismatches formed during meiotic recombination in the yeast Saccharomyces cerevisiae.

Heteroduplexes formed between DNA strands derived from different homologous chromosomes are an intermediate in meiotic crossing over in the yeast Saccharomyces cerevisiae and other eucaryotes. A heteroduplex formed between wild-type and mutant genes will contain a base pair mismatch; failure to repair this mismatch will lead to postmeiotic segregation (PMS). By analyzing the frequency of PMS for various mutant alleles in the yeast HIS4 gene, we showed that C/C mismatches were inefficiently repaired relative to all other point mismatches. These other mismatches (G/G, G/A, T/T, A/A, T/C, C/A, A/A, and T/G) were repaired with approximately the same efficiency. We found that in spores with unrepaired mismatches in heteroduplexes, the nontranscribed strand of the HIS4 gene was more frequently donated than the transcribed strand. In addition, the direction of repair for certain mismatches was nonrandom.     

Production and characterization of the celery mismatch endonuclease CEL II using baculovirus/silkworm expression system

Mutation and polymorphism detection by nucleases has become a more important tool in clinical and biological researches. There are several kinds of single-stranded nucleases for detecting mismatched DNAs. One of them, CEL II, was isolated from Apium graveolens and cleaves DNA with high specificity at sites of mismatch. High-throughput mutation scanning requires large quantity of CEL II endonuclease. Here, we demonstrate high-level expression of CEL II using silkworm-baculovirus system. The recombinant CEL II secreted in silkworm hemolymph was glycosylated and susceptible to N-glycosidase F. Additionally, larger metal ions such as Ca²⁺ and Sr²⁺ were able to replace Mg²⁺ and enhanced mismatch cleavage activity of CEL II. These results indicate that the silkworm-baculovirus platform is a good alternative system to obtain the functional CEL II.     

Action of single-strand specific nucleases on model DNA heteroduplexes of defined size and sequence.

The sensitivity of the model DNAs containing dA-dG and dtg-dG heteroduplex regions of defined length to S1 and mung bean single-strand specific nucleases was tested by polyacrylamide gel electrophoretic analysis of the distribution of product oligonucleotides. Single-base mismatch heteroduplexes were extremely resistant to these nucleases, although low levels of cleavage at the heteroduplex nucleotide were observed at high nuclease concentrations. The nuclease sensitivity of dA-dtg heteroduplex regions increased gradually as the length of the heteroduplex region increased frome one to six nucleotides. The sensitivity of dG-dG heteroduplexes three to five nucleotides long was considerably greater than that of the single dtg-dG mismatch.     

Position and degree of mismatches and the mobility of DNA heteroduplexes

Heteroduplex mobility assay (HMA) is a fast and inexpensive method for determining relatedness between DNA sequences. Rapidly evolving viruses such as HIV-1 develop marked sequence differences in their genomes over the course of the epidemic and infection in a single individual. HMA can be used to monitor both processes. Here, we systematically evaluated the influence of single base mismatches on heteroduplex mobility. The impact of mismatches at nine different positions in 559 bp double-stranded DNA molecules, within a background of overall sequence divergence ranging from 1.97 to 9.65%, was evaluated in both non-denaturing and partially-denaturing acrylamide gels. We found that the electrophoretic mobility of heteroduplexes was proportional to the level of mismatch when that level exceeded 4.5%. Overall, mismatches near the center of the fragment and clustered mismatches tended to have an exaggerated influence on the mobility of heteroduplexes. Thus, the use of HMA for quantitative inference of genetic distances under the conditions we describe is of greatest utility at levels of mismatch >5%.     

Microarray-based method to evaluate the accuracy of restriction endonucleases HpaII and MspI

A double-strand DNA (ds DNA) microarray was fabricated to analyze the structural perturbations caused by methylation and the different base mismatches in the interaction of the restriction endonucleases HpaII and MspI with DNA. First, a series of synthesized oligonucleotides were arrayed on the aldehyde-coated glass slides. Second, these oligonucleotides were hybridized with target sequences to obtain ds DNA microarray, which includes several types of double strands with the fully methylated, semi-methylated, and unmethylated canonical recognition sequences, semi-methylated and unmethylated base mismatches within the recognition sequences. The cleavage experiments were carried out under normal buffer conditions. The results indicated that MspI could partially cleave methylated and semi-methylated canonical recognition sequences. In contrast, HpaII could not cleave methylated and semi-methylated canonical recognition sequences. HpaII and MspI could both cleave the unmethylated canonical recognition sequence. However, HpaII could partially cleave the sequence containing one GG mismatch and not cleave other base mismatches in the corresponding recognition site. In contrast, MspI could not recognize the base mismatches within the recognition sequence. A good reproducibility was observed in several parallel experiments. The experiment indicates that the microarray technology has great potentials in high-throughput identifying important interactions between protein and DNA.     

Mutation detection using a novel plant endonuclease.

We have discovered a useful new reagent for mutation detection, a novel nuclease CEL I from celery. It is specific for DNA distortions and mismatches from pH 6 to 9. Incision is on the 3'-side of the mismatch site in one of the two DNA strands in a heteroduplex. CEL I-like nucleases are found in many plants. We report here that a simple method of enzyme mutation detection using CEL I can efficiently identify mutations and polymorphisms. To illustrate the efficacy of this approach, the exons of the BRCA1 gene were amplified by PCR using primers 5'-labeled with fluorescent dyes of two colors. The PCR products were annealed to form heteroduplexes and subjected to CEL I incision. In GeneScan analyses with a PE Applied Biosystems automated DNA sequencer, two independent incision events, one in each strand, produce truncated fragments of two colors that complement each other to confirm the position of the mismatch. CEL I can detect 100% of the sequence variants present, including deletions, insertions and missense alterations. Our results indicate that CEL I mutation detection is a highly sensitive method for detecting both polymorphisms and disease-causing mutations in DNA fragments as long as 1120 bp in length.     

One tube mutation detection using sensitive fluorescent dyeing of MutS protected DNA

A novel, universal method for mutation detection utilising the ability of MutS protein to recognise DNA incomplementarities is proposed. The examined and reference DNA fragments are PCR amplified. The PCR products are purified, mixed, heated and cooled to form heteroduplexes. In the case of mutation the heteroduplex DNA containing mismatch is protected against exonuclease digestion by MutS, while the DNA without mismatches is degraded. The protection effect is visualised by the direct addition of a highly sensitive fluorescent dye (SYBR-Gold) selectively binding DNA. The Thermus thermophilus recombined His-tagged MutS and 3′–5′ exonuclease activity of T4 DNA polymerase were used in the assay.     

Potassium permanganate and tetraethylammonium chloride are a safe and effective substitute for osmium tetroxide in solid-phase fluorescent chemical cleavage of mismatch.

Whilst chemical cleavage of mismatch (CCM) detects all point mutations in DNA, its widespread use has been hampered by the complex multistage methodology and the need for toxic chemicals, in particular osmium tetroxide. Here we show that osmium tetroxide can be replaced by potassium permanganate, giving the same spectrum of mutation detection, but with greater sensitivity. The use of potassium permanganate is compatible with solid phase capture and fluorescent detection, giving a safer method of mutation detection. We present here a comparison of CCM with osmium tetroxide and with potassium permanganate, tested on a complete set of single base pair mismatches and a number of small insertion/deletions.     

Comparative reactivity of mismatched and unpaired bases in relation to their type and surroundings. Chemical cleavage of DNA mismatches in mutation detection analysis

Systematic study of chemical reactivity of non-Watson–Crick base pairs depending on their type and microenvironment was performed on a model system that represents two sets of synthetic DNA duplexes with all types of mismatched and unmatched bases flanked by T·A or G·C pairs. Using comparative cleavage pattern analysis, we identified the main and additional target bases and performed quantitative study of the time course and efficacy of DNA modification caused by potassium permanganate or hydroxylamine. Potassium permanganate in combination with tetraethylammonium chloride was shown to induce DNA cleavage at all mismatched or bulged T residues, as well as at thymines of neighboring canonical pairs. Other mispaired (bulged) bases and thymine residues located on the second position from the mismatch site were not the targets for KMnO₄ attack. In contrast, hydroxylamine cleaved only heteroduplexes containing mismatched or unmatched C residues, and did not modify adjacent cytosines. However when G·C pairs flank bulged C residue, neighboring cytosines are also attacked by hydroxylamine due to defect migration. Chemical reactivity of target bases was shown to correlate strongly with the local disturbance of DNA double helix at mismatch or bulge site. With our model system, we were able to prove the absence of false-negative and false-positive results. Portion of heteroduplex reliably revealed in a mixture with corresponding homoduplex consists of 5% for bulge bases and “open” non-canonical pairs, and 10% for wobble base pairs giving minimal violations in DNA structure. This study provides a complete understanding of the principles of mutation detection methodology based on chemical cleavage of mismatches and clarifies the advantages and limitations of this approach in various biological and conformational studies of DNA.     

Structures of mismatched base pairs in DNA and their recognition by the Escherichia coli mismatch repair system.

The Escherichia coli mismatch repair system does not recognize and/or repair all mismatched base pairs with equal efficiency: whereas transition mismatches (G X T and A X C) are well repaired, the repair of some transversion mismatches (e.g. A X G or C X T) appears to depend on their position in heteroduplex DNA of phage lambda. Undecamers were synthesized and annealed to form heteroduplexes with a single base-pair mismatch in the centre and with the five base pairs flanking each side corresponding to either repaired or unrepaired heteroduplexes of lambda DNA. Nuclear magnetic resonance (n.m.r.) studies show that a G X A mismatch gives rise to an equilibrium between fully helical and a looped-out structure. In the unrepaired G X A mismatch duplex the latter predominates, while the helical structure is predominant in the case of repaired G X A and G X T mismatches. It appears that the E. coli mismatch repair enzymes recognize and repair intrahelical mismatched bases, but not the extrahelical bases in the looped-out structures.     

Mispair specificity of methyl-directed DNA mismatch correction in vitro

To evaluate the substrate specificity of methyl-directed mismatch repair in Escherichia coli extracts, we have constructed a set of DNA heteroduplexes, each of which contains one of the eight possible single base pair mismatches and a single hemimethylated d(GATC) site. Although all eight mismatches were located at the same position within heteroduplex molecules and were embedded within the same sequence environment, they were not corrected with equal efficiencies in vitro. G-T was corrected most efficiently, with A-C, C-T, A-A, T-T, and G-G being repaired at rates 40-80% of that of the G-T mispair. Correction of each of these six mispairs occurred in a methyl-directed manner in a reaction requiring mutH, mutL, and mutS gene products. C-C and A-G mismatches showed different behavior. C-C was an extremely poor substrate for correction while repair of A-G was anomalous. Although A-G was corrected to A-T by the mutHLS-dependent, methyl-directed pathway, repair of A-G to C-G occurred largely by a pathway that is independent of the methylation state of the heteroduplex and which does not require mutH, mutL, or mutS gene products. Similar results were obtained with a second A-G mismatch in a different sequence environment suggesting that a novel pathway may exist for processing A-G mispairs to C-G base pairs. As judged by DNase I footprint analysis, MutS protein is capable of recognizing each of the eight possible base-base mismatches. Use of this method to estimate the apparent affinity of MutS protein for each of the mispairs revealed a rough correlation between MutS affinity and efficiency of correction by the methyl-directed pathway. However, the A-C mismatch was an exception in this respect indicating that interactions other than mismatch recognition may contribute to the efficiency of repair.     

Chemical cleavage of DNA with single base mismatches for detection of mutations of unknown localization

The most promising approach for detection of random point mutations relies upon the DNA chemical cleavage near associated mismatching base pairs. In our study, the series of heteroduplexes with all types of mismatches and extrahelical nucleotide residues surrounded by both A x T and G x C pairs were performed via hybridization of 50-mer synthetic oligonucleotides differing in only one nucleotide at the central position. The chemical cleavage of DNA duplexes immobilized on magnetic beads by means of biotin-streptavidin interaction was carried out with chemicals, which able to attack only nucleobases flipped out of the base stack: potassium permanganate and hydroxylamine reacting to T and C respectively. The chemical reactivity of different mismatches was shown to correlate clearly with the target local structure in a particular sequence context. This work makes up for a deficiency in systematic study of DNA cleavage near mismatches in dependence on their type, orientation and flanking nucleotides. The model system elaborated may be applied to estimate the sensitivity of the methodology and to control the possibility of false-positive and false-negative result appearance, when different protocols for detection of DNA mutations are used. The modification of heteroduplex mixtures by potassium permanganate and hydroxylamine allows to reveal any non-canonical base pair and suggest its type and neighboring nucleotides from the nature of chemical as well as its localization from the length of cleavage products.     

Incision at nucleotide insertions/deletions and base pair mismatches by the SP nuclease of spinach

Spinach leaves contain a highly active nuclease called SP. The purified enzyme incises single-stranded DNA, RNA, and double-stranded DNA that has been destabilized by A-T-rich regions and DNA lesions [Strickland et al. (1991) Biochemistry 30, 9749-9756]. This broad range of activity has suggested that SP may be similar to a family of nucleases represented by S1, P1, and the mung bean nuclease. However, unlike these single-stranded nucleases that require acidic pH and low ionic strength conditions, SP has a neutral pH optimum and is active over a wide range of salt concentrations. We have extended these findings and showed that an outstanding substrate for SP is a mismatched DNA duplex. For base-substitution mismatches, SP incises at all mismatches except those containing a guanine residue. SP also cuts at insertion/deletions of one or more nucleotides. Where the extrahelical DNA loop contains one nucleotide, the preference of extrahelical nucleotide is A > T approximately C but undetectable at G. The inability of SP to cut at guanine residues and the favoring of A-T-rich regions distinguish SP from the CEL I family of neutral pH mismatch endonucleases recently discovered in celery and other plants [Oleykowski et al. (1998) Nucleic Acids Res. 26, 4597-4602]. SP, like CEL I, does not turn over after incision at a mismatched site in vitro. Similar to CEL I, the presence of a DNA polymerase or a DNA ligase allows SP to turn over and stimulate its activity in vitro by about 20-fold. The possibility that the SP nuclease may be a natural variant of the CEL I family of mismatch endonucleases is discussed.     

The structural basis of damaged DNA recognition and endonucleolytic cleavage for very short patch repair endonuclease

Endonucleases in DNA repair must be able to recognize damaged DNA as well as cleave the phosphodiester backbone. These functional prerequisites are manifested in very short patch repair (Vsr) endonuclease through a common endonuclease topology that has been tailored for recognition of TG mismatches. Structural and biochemical comparison with type II restriction enzymes illustrates how Vsr resembles these endonucleases in overall topology but also how Vsr diverges in terms of the detailed catalytic mechanism. A histidine and two metal–water clusters catalyze the phosphodiester cleavage. The mode of DNA damage recognition is also unique to Vsr. All other structurally characterized DNA damage-binding enzymes employ a nucleotide flipping mechanism for substrate recognition and for catalysis. Vsr, on the other hand, recognizes the TG mismatch as a wobble base pair and penetrates the DNA with three aromatic residues on one side of the mismatch. Thus, Vsr endonuclease provides important counterpoints in our understanding of endonucleolytic mechanisms and of damaged DNA recognition.     

Anomalous RNA substrates for mammalian tRNA 3' processing endoribonuclease

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) is an enzyme responsible for the removal of a 3' trailer from pre-tRNA. The enzyme can also recognize and cleave any target RNA that forms a pre-tRNA-like complex with another RNA. To investigate the interaction between 3' tRNase and substrates, we tested various anomalous pre-tRNA-like complexes for cleavage by pig 3' tRNase. We examined how base mismatches in the acceptor stem affect 3' tRNase cleavage of RNA complexes, and found that even one base mismatch in the acceptor stem drastically reduces the cleavage efficiency. Mammalian 3' tRNase was able to recognize complexes between target RNAs and 5'-half tDNAs, and cleave the target RNAs, although inefficiently, whereas the enzyme had no activity to cleave phosphodiester bonds of DNA. A relatively long RNA target, the Escherichia coli chloramphenicol acetyltransferase (CAT) mRNA, was cleaved by 3' tRNase in the presence of appropriate 5'-half tRNAs. We also demonstrated that an RNA complex of lin-4 and lin-14 from Caenorhabditis elegans can be recognized and cleaved by pig 3' tRNase.     

Detection of 100% of mutations in 124 individuals using a standard UV/Vis microplate reader: a novel concept for mutation scanning

We report the development of a simple and inexpensive assay for the detection of DNA polymorphisms and mutations that is based on the modification of mismatched bases by potassium permanganate. Unlike the chemical cleavage of mismatch assay, which also exploits the reactivity of potassium permanganate to detect genomic variants, the assay we describe here does not require a cleavage manipulation and therefore does not require expensive or toxic chemicals or a separation step, as mismatches are detected using direct optical methods in a microplate format. Studies with individual deoxynucleotides demonstrated that the reactivity with potassium permanganate resulted in a specific colour change. Furthermore, studies with synthetic oligonucleotide heteroduplexes demonstrated that this colour change phenomenon could be applied to detect mismatched bases spectrophotometrically. A collection of plasmids carrying single point mutations in the mouse β-globin promoter region was used as a model system to develop a functional mutation detection assay. Finally, the assay was validated as 100% effective in detecting mismatches in a blinded manner using DNA from patients previously screened for mutations using established techniques, such as sequencing, SSCP and denaturing high-performance liquid chromatography (DHPLC) analysis in DNA fragments up to 300 bp in length.