Mutant DNAs Research Articles

Distribution of mutation frequencies among Salmonella enterica isolates from animal and human sources and genetic characterization of a Salmonella Heidelberg hypermutator

Hypermutation is an important mechanism used by different Salmonella enterica subspecies enterica to regulate genetic stability in adaptation to changing environments, including antimicrobial treatments and industrial processes. Strong hypermutator strains generally contain a mutation in genes of the methyl mismatch repair (MMR) system and have mutation frequencies up to 1000-fold higher than wild type strains. The objectives of this study were to determine the distribution of mutation frequencies from a collection of 209 Salmonella strains, to genetically characterize a strong mutator, and to study MMR mutated protein–DNA binding interactions. Only one strain of S. Heidelberg was determined to have a hypermutator phenotype by virtue of its high mutation rate. Sequencing of genes of the MMR system showed a 12 bp deletion in the mutS gene was present. The MMR mutated protein-DNA binding interactions were studied by bioanalysis, using the available crystal structure of a similar MutS protein from Escherichia coli. This analysis showed the small deletion in the Salmonella MutS was localized within the core domain. A retardation assay with MutS from hypermutable and wild type strains showed this mutation has no effect on MutS DNA binding. A better understanding of the genetic mechanisms of hypermutation will help to anticipate the behavior of hypermutator strains in various conditions.

The selection and characterization of the mutants in bioflocculant bacteria C8 induced by 3 MeV helium ion irradiation

A 3 MeV He 2+ beam was used to irradiate C8 (a flocculant-producing bacteria) with a fluence ranging from 10 11 to 10 13 ions/cm 2. The effects on the survival ratio, TTC-dehydrogenase activity, flocculating activity and RAPD analysis are reported. The survival ratio curve caused by irradiation is proved to be “saddle-shaped”. Eleven mutants were obtained, all of which had a significant change in dehydrogenase activity and most showed a positive change in flocculating activity. RAPD measurements were used to analyse the DNA of mutants with a flocculating activity over 80%, which indicated that all their DNA had been changed by irradiation.

Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone

The biogenesis of rat thyrotropin releasing hormone (TRH) involves the processing of its precursor (proTRH) into five biologically active TRH peptides and several non-TRH peptides where two of them had been attributed potential biological functions. This process implicates 1) proper folding of proTRH in the endoplasmic reticulum after its biosynthesis and exit to the Golgi apparatus and beyond, 2) initial processing of proTRH in the trans Golgi network and, 3) sorting of proTRH-derived peptides to the regulated secretory pathway. Previous studies have focused on elucidating the processing and sorting determinants of proTRH. However, the role of protein folding in the sorting of proTRH remains unexplored. Here we have investigated the role in the secretion of proTRH of a sequence comprising 22 amino acid residues, located at the N-terminal region of proTRH, residues 31-52. Complete deletion of these 22 amino acids dramatically compromised the biosynthesis of proTRH, manifested as a severe reduction in the steady state level of proTRH in the endoplasmic reticulum. This effect was largely reproduced by the deletion of only three amino acid residues, 40PGL42, within the proTRH31-52 sequence. The decreased steady state level of the mutant DeltaPGL was due to enhanced endoplasmic reticulum-associated protein degradation. However, the remnant of DeltaPGL that escaped degradation was properly processed and sorted to secretory granules. Thus, these results suggest that the N-terminal domain within the prohormone sequence does not act as "sorting signal" in late secretion; instead, it seems to play a key role determining the proper folding pathway of the precursor and, thus, its stability.

Successful treatment with lamivudine may correlate with reduction of serum ferritin levels in the patients with chronic hepatitis and liver cirrhosis type B

PurposeTo study the changes in serum ferritin levels in lamivudine (LAM)-treated patients with chronic hepatitis and liver cirrhosis type B and determine whether successful treatment with LAM results in a reduction of serum ferritin levels.MethodsThirty patients with chronic hepatitis B virus (HBV) infection were followed prospectively during their treatment with LAM for 12 months. Serum HBV DNA, ferritin levels, and emergence of YMDD mutants were monitored. A case of severe liver cirrhosis with hepatic hemosiderosis that was treated successfully with LAM also is shown as a representative case.ResultsSerum alanine aminotransferase and ferritin levels decreased significantly more in the patients treated with LAM without YMDD mutants (n = 23) than those with mutants (n = 7). Hepatic hemosiderosis along with serum iron markers improved greatly in the representative patient.ConclusionSuccessful treatment with LAM may reduce serum ferritin levels and improve hepatic siderosis in a subset of patients with chronic HBV infection.

Depletion of RNase HI activity in Escherichia coli lacking DNA topoisomerase I leads to defects in DNA supercoiling and segregation

Gyrase-mediated hypernegative supercoiling is one manifestation of R-loop formation, a phenomenon that is normally suppressed by topoisomerase I (topA) in Escherichia coli. Overproduction of RNase HI (rnhA), an enzyme that removes the RNA moiety of R-loops, prevents hypernegative supercoiling and allows growth of topA null mutants. We previously showed that topA and rnhA null mutations are incompatible. We now report that such mutants were viable when RNase HI or topoisomerase III was expressed from a plasmid-borne gene. Surprisingly, DNA of topA null mutants became relaxed rather than hypernegatively supercoiled following depletion of RNase HI activity. This result failed to correlate with the cellular concentration of gyrase or topoisomerase IV (the other relaxing enzyme in the cell) or with transcription-induced supercoiling. Rather, intracellular DNA relaxation in the absence of RNase HI was related to inhibition of gyrase activity both in vivo and in extracts. Cells lacking topA and rnhA also exhibited properties consistent with segregation defects. Overproduction of topoisomerase III, an enzyme that can carry out DNA decatenation, corrected the segregation defects without restoring supercoiling activity. Collectively these data reveal (i) the existence of a cellular response to loss of RNase HI that counters the supercoiling activity of gyrase, and (ii) supercoiling-independent segregation defects due to loss of RNase HI from topA null mutants. Thus RNase HI plays a more central role in DNA topology than previously thought.

Minisequencing mitochondrial DNA pathogenic mutations.

BackgroundThere are a number of well-known mutations responsible of common mitochondrial DNA (mtDNA) diseases. In order to overcome technical problems related to the analysis of complete mtDNA genomes, a variety of different techniques have been proposed that allow the screening of coding region pathogenic mutations.MethodsWe here propose a minisequencing assay for the analysis of mtDNA mutations. In a single reaction, we interrogate a total of 25 pathogenic mutations distributed all around the whole mtDNA genome in a sample of patients suspected for mtDNA disease.ResultsWe have detected 11 causal homoplasmic mutations in patients suspected for Leber disease, which were further confirmed by standard automatic sequencing. Mutations m.11778G>A and m.14484T>C occur at higher frequency than expected by change in the Galician (northwest Spain) patients carrying haplogroup J lineages (Fisher's Exact test, P-value < 0.01). The assay performs well in mixture experiments of wild:mutant DNAs that emulate heteroplasmic conditions in mtDNA diseases.ConclusionWe here developed a minisequencing genotyping method for the screening of the most common pathogenic mtDNA mutations which is simple, fast, and low-cost. The technique is robust and reproducible and can easily be implemented in standard clinical laboratories.

A Common Mechanism for the ATP-DnaA-dependent Formation of Open Complexes at the Replication Origin

Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. Escherichia coli DnaA protein forms a homomultimeric complex with the replication origin (oriC). ATP-DnaA multimers unwind the duplex within the oriC unwinding element (DUE). In this study, structural analyses suggested that several residues exposed in the central pore of the putative structure of DnaA multimers could be important for unwinding. Using mutation analyses, we found that, of these candidate residues, DnaA Val-211 and Arg-245 are prerequisites for initiation in vivo and in vitro. Whereas DnaA V211A and R245A proteins retained normal affinities for ATP/ADP and DNA and activity for the ATP-specific conformational change of the initiation complex in vitro, oriC complexes of these mutant proteins were inactive in DUE unwinding and in binding to the single-stranded DUE. Unlike oriC complexes including ADP-DnaA or the mutant DnaA, ATP-DnaA-oriC complexes specifically bound the upper strand of single-stranded DUE. Specific T-rich sequences within the strand were required for binding. The corresponding conserved residues of the DnaA ortholog in Thermotoga maritima, an ancient eubacterium, were also required for DUE unwinding, consistent with the idea that the mechanism and regulation for DUE unwinding can be evolutionarily conserved. These findings provide novel insights into mechanisms for pore-mediated origin unwinding, ATP/ADP-dependent regulation, and helicase loading of the initiation complex.

X-ray Structure of the Complete ABC Enzyme ABCE1 from Pyrococcus abyssi

The ATP binding cassette enzyme ABCE1 (also known as RNase-L (ribonuclease L) inhibitor, Pixie, and HP68), one of the evolutionary most sequence-conserved enzymes, functions in translation initiation, ribosome biogenesis, and human immunodeficiency virus capsid assembly. However, its structural mechanism and biochemical role in these processes have not been revealed. We determined the crystal structure of Pyrococcus abyssi ABCE1 in complex with Mg(2+) and ADP to 2.8A resolution. ABCE1 consists of four structural domains. Two nucleotide binding domains are arranged in a head-to-tail orientation by a hinge domain, suggesting that these domains undergo the characteristic tweezers-like powerstroke of ABC enzymes. In contrast to all other known ABC enzymes, ABCE1 has a N-terminal iron-sulfur-cluster (FeS) domain. The FeS domain contains two [4Fe-4S] clusters and is structurally highly related to bacterial-type ferredoxins. However, one cluster is coordinated by an unusual CX(4)CX(3/4)C triad. Surprisingly, intimate interactions of the FeS domain with the adenine and ribose binding Y-loop on nucleotide binding domain 1 suggest a linkage between FeS domain function and ATP-induced conformational control of the ABC tandem cassette. The structure substantially expands the functional architecture of ABC enzymes and raises the possibility that ABCE1 is a chemomechanical engine linked to a redox process.

Development of SYBR Green I-based real-time PCR assay for detection of drug resistance mutations in cytomegalovirus

Ganciclovir (GCV) is an antiviral drug that is used to treat cytomegalovirus (CMV) infection. However, long-term monotherapy does not commonly result in complete suppression of viral replication and is associated with the emergence of resistant mutants. In this study, a method for detecting CMV resistance mutations was carried out by real-time amplification refractory mutation system PCR (real-time ARMS PCR) using SYBR Green I fluorescent dye. Three recombinant plasmids were constructed by overlapping extension PCR to be used as standard mutation or wild-type models. Four pairs of primers were used to amplify the approximately 150 bp of the UL97 gene spanning codon 460, where mutations associated with resistance to GCV invariably occur. As little as 20% mutants DNA in 10 7 copies/ml mixture DNA were detected. Though this approach was not more sensitive than PCR-restriction fragment length polymorphism (RFLP) for the detection of the presence of mixtures, it was a high-throughput and automation method, and the specific mutation type can be deduced by the real-time ARMS PCR data. Overall, this study has demonstrated an approach that could be a sensitive and rapid method for the detection of GCV resistance-associated mutation in CMV.

The functional analysis of YabA, which interacts with DnaA and regulates initiation of chromosome replication in Bacillus subtils

The initiation of bacterial chromosome DNA replication and its regulation are critical events. DnaA is essential for initiation of DNA replication and is conserved throughout bacteria. In Escherichia coli, hydrolysis of ATP-DnaA is promoted by Hda through formation of a ternary complex with DnaA and DnaN, ensuring the timely inactivation of DnaA during the replication cycle. In Bacillus subtilis, YabA also forms a ternary complex with DnaA and DnaN, and negatively regulates the initiation step of DNA replication. However, YabA shares no structural homology with Hda and the regulatory mechanism itself has not been clarified. Here, in contrast to Hda, we observed that dnaA transcription was stable during under- and overexpression of YabA. ChAP-chip assays showed that the depletion of YabA did not affect DNA binding by DnaA. On the other hand, yeast two-hybrid analysis indicated that the DnaA ATP-binding domain interacts with YabA. Moreover, mutations in YabA interaction-deficient mutants, isolated by yeast two-hybrid analysis, are located at the back of the ATP-binding domain, whereas Hda is thought to interact with the ATP-binding pocket itself. The introduction into B. subtilis of a dnaA(Y144C) mutation, which disabled the interaction with YabA but did not affect interactions either with DnaA itself or with DnaD, resulted in over-initiation and asynchronous initiation of replication and disabled the formation of YabA foci, further demonstrating that the amino acid on the opposite side to the ATP-binding pocket is important for YabA binding. These results indicate that YabA indeed regulates the initiation of DNA replication by a different mechanism from that used by Hda in the E. coli RIDA system. Interestingly, all DnaA mutants deficient in YabA binding also displayed reduced DnaD binding in yeast two-hybrid assays, suggesting that YabA can inhibit replication initiation through competitive inhibition of DnaD binding to DnaA.

Polymer Microfluidic Chips with Integrated Waveguides for Reading Microarrays

A microfluidic chip with an integrated planar waveguide was fabricated in poly(methyl methacrylate), PMMA, using a single-step, double-sided hot-embossing approach. The waveguide was embedded in air on three sides, the solution being interrogated on the fourth. DNA probes were covalently attached to the waveguide surface by plasma activating the PMMA and the use of carbodiimide coupling chemistry. Successful hybridization events were read using evanescent excitation monitored by an imaging microscope, which offered high spatial resolution (2 microm) and a large field-of-view (20 mm diameter field-of-view), providing imaging of the entire array without scanning. The application of the microfluidic/waveguide assembly was demonstrated by detecting low abundant point mutations; insertion C mutations in BRCA1 genes associated with breast cancer were analyzed using a universal array coupled to an allele-specific ligation assay. DNA probes consisting of amine-terminated oligonucleotides were printed inside the microfluidic channel using a noncontact microspotter. Mutant and wild-type genomic DNAs of BRCA1 were PCR (polymerase chain reaction) amplified, with the amplicons subjected to ligation detection reactions (LDRs). LDR solutions were allowed to flow over the microarray positioned on the polymer waveguide with successful ligation events discerned through fluorescence signatures present at certain locations of the array. The microfluidic/waveguide assembly could detect polymorphisms present at <1% of the total DNA content.

Ralstonia solanacearum strains from Martinique (French West Indies) exhibiting a new pathogenic potential.

We investigated a destructive pathogenic variant of the plant pathogen Ralstonia solanacearum that was consistently isolated in Martinique (French West Indies). Since the 1960s, bacterial wilt of solanaceous crops in Martinique has been caused primarily by strains of R. solanacearum that belong to either phylotype I or phylotype II. Since 1999, anthurium shade houses have been dramatically affected by uncharacterized phylotype II strains that also affected a wide range of species, such as Heliconia caribea, cucurbitaceous crops, and weeds. From 1989 to 2003, a total of 224 R. solanacearum isolates were collected and compared to 6 strains isolated in Martinique in the 1980s. The genetic diversity and phylogenetic position of selected strains from Martinique were assessed (multiplex PCRs, mutS and egl DNA sequence analysis) and compared to the genetic diversity and phylogenetic position of 32 reference strains covering the known diversity within the R. solanacearum species complex. Twenty-four representative isolates were tested for pathogenicity to Musa species (banana) and tomato, eggplant, and sweet pepper. Based upon both PCR and sequence analysis, 119 Martinique isolates from anthurium, members of the family Cucurbitaceae, Heliconia, and tomato, were determined to belong to a group termed phylotype II/sequevar 4 (II/4). While these strains cluster with the Moko disease-causing strains, they were not pathogenic to banana (NPB). The strains belonging to phylotype II/4NPB were highly pathogenic to tomato, eggplant, and pepper, were able to wilt the resistant tomato variety Hawaii7996, and may latently infect cooking banana. Phylotype II/4NPB constitutes a new pathogenic variant of R. solanacearum that has recently appeared in Martinique and may be latently prevalent throughout Caribbean and Central/South America.

The reactivation of DnaA(L366K) requires less acidic phospholipids supporting their role in the initiation of chromosome replication in Escherichia coli

DnaA(L366K), in concert with a wild-type DnaA (wtDnaA) protein, restores the growth of Escherichia coli cells arrested in the absence of adequate levels of cellular acidic phospholipids. In vitro and in vivo studies showed that DnaA(L366K) alone does not induce the initiation of replication, and wtDnaA must also be present. Hitherto the different behavior of wt and mutant DnaA were not understood. We now demonstrate that this mutant may be activated at significantly lower concentrations of acidic phospholipids than the wild-type protein, and this may explain the observed growth restoration in vivo.

Modulation of λ plasmid and phage DNA replication by Escherichia coli SeqA protein

SeqA protein, a main negative regulator of the replication initiation of the Escherichia coli chromosome, also has several other functions which are still poorly understood. It was demonstrated previously that in seqA mutants the copy number of another replicon, the lambda plasmid, is decreased, and that the activity of the lambda p(R) promoter (whose function is required for stimulation of ori lambda) is lower than that in the wild-type host. Here, SeqA-mediated regulation of lambda phage and plasmid replicons was investigated in more detail. No significant influence of SeqA on ori lambda-dependent DNA replication in vitro was observed, indicating that a direct regulation of lambda DNA replication by this protein is unlikely. On the other hand, density-shift experiments, in which the fate of labelled lambda DNA was monitored after phage infection of host cells, strongly suggested the early appearance of sigma replication intermediates and preferential rolling-circle replication of phage DNA in seqA mutants. The directionality of lambda plasmid replication in such mutants was, however, only slightly affected. The stability of the heritable lambda replication complex was decreased in the seqA mutant relative to the wild-type host, but a stable fraction of the lambda O protein was easily detectable, indicating that such a heritable complex can function in the mutant. To investigate the influence of seqA gene function on heritable complex- and transcription-dependent lambda DNA replication, the efficiency of lambda plasmid replication in amino acid-starved relA seqA mutants was measured. Under these conditions, seqA dysfunction resulted in impairment of lambda plasmid replication. These results indicate that unlike oriC, SeqA modulates lambda DNA replication indirectly, most probably by influencing the stability of the lambda replication complex and the transcriptional activation of ori lambda.

Regulation of p53 Nuclear Export through Sequential Changes in Conformation and Ubiquitination

Wild-type p53 is a conformationally labile protein that undergoes nuclear-cytoplasmic shuttling. MDM2-mediated ubiquitination promotes p53 nuclear export by exposing or activating a nuclear export signal (NES) in the C terminus of p53. We observed that cancer-derived p53s with a mutant (primary antibody 1620-/pAb240+) conformation localized in the cytoplasm to a greater extent and displayed increased susceptibility to ubiquitination than p53s with a more wild-type (primary antibody 1620+/pAb240-) conformation. The cytoplasmic localization of mutant p53s required the C-terminal NES and an intact ubiquitination pathway. Mutant p53 ubiquitination occurred at lysines in both the DNA-binding domain (DBD) and C terminus. Interestingly, Lys to Arg mutations that inhibited ubiquitination restored nuclear localization to mutant p53 but had no apparent effect on p53 conformation. Further studies revealed that wild-type p53, like mutant p53, is ubiquitinated by MDM2 in both the DBD and C terminus and that ubiquitination in both regions contributes to its nuclear export. MDM2 binding can induce a conformational change in wild-type p53, but this conformational change is insufficient to promote p53 nuclear export in the absence of MDM2 ubiquitination activity. Taken together, these results support a stepwise model for mutant and wild-type p53 nuclear export. In this model, the conformational change induced by either the cancer-derived mutation or MDM2 binding precedes p53 ubiquitination. The addition of ubiquitin to DBD and C-terminal lysines then promotes nuclear export via the C-terminal NES.

A novel class of mutations that affect DNA replication in E. coli

Over-initiation of DNA replication in cells containing the cold-sensitive dnaA(cos) allele has been shown to lead to extensive DNA damage, potentially due to head-to-tail replication fork collisions that ultimately lead to replication fork collapse, growth stasis and/or cell death. Based on the assumption that suppressors of the cold-sensitive phenotype of the cos mutant should include mutations that affect the efficiency and/or regulation of DNA replication, we subjected a dnaA(cos) mutant strain to transposon mutagenesis and selected mutant derivatives that could form colonies at 30 degrees C. Four suppressors of the dnaA(cos)-mediated cold sensitivity were identified and further characterized. Based on origin to terminus ratios, chromosome content per cell, measured by flow cytometry, and sensitivity to the replication fork inhibitor hydroxyurea, the suppressors fell into two distinct categories: those that directly inhibit over-initiation of DNA replication and those that act independently of initiation. Mutations that decrease the cellular level of HolC, the chi subunit of DNA polymerase, or loss of ndk (nucleoside diphosphate kinase) function fall into the latter category. We propose that these novel suppressor mutations function by decreasing the efficiency of replication fork movement in vivo, either by decreasing the dynamic exchange of DNA polymerase subunits in the case of HolC, or by altering the balance between DNA replication and deoxynucleoside triphosphate synthesis in the case of ndk. Additionally, our results indicate a direct correlation between over-initiation and sensitivity to replication fork inhibition by hydroxyurea, supporting a model of increased head-to-tail replication fork collisions due to over-initiation.

Minor Capsid Proteins of Simian Virus 40 Are Dispensable for Nucleocapsid Assembly and Cell Entry but Are Required for Nuclear Entry of the Viral Genome

We investigated the roles of simian virus 40 capsid proteins in the viral life cycle by analyzing point mutants in Vp1 and Vp2/3, as well as a deletion mutant lacking the Vp2/3 coding sequence. The Vp1 mutants (V243E and L245E) and the Vp2/3 mutants (F157E-I158E and P164R-G165E-G166R) were previously shown to be defective in Vp1-Vp2/3 interaction and to be noninfectious or poorly infectious, respectively. Here, we show that all these point mutants form stable particles following DNA transfection into cells. The Vp2/3-mutant particles contained very low levels of Vp2/3, whereas the Vp1 mutant particles contained no detectable Vp2/3. As expected, the deletion mutant also formed particles that were noninfectious. We further characterized the two Vp1 point mutants and the deletion mutant. All three mutant particles comprised Vp1 and histone-associated viral DNA, and all were able to enter cells. However, the mutant complexes failed to associate with host importins (owing to the loss of the Vp2/3 nuclear localization signal), and the mutant viral DNAs prematurely dissociated from the Vp1s, suggesting that the nucleocapsids did not enter the nucleus. Consistently, all three mutant particles failed to express large T antigen. Together, our results demonstrate unequivocally that Vp2/3 is dispensable for the formation of nucleocapsids. Further, the nucleocapsids' ability to enter cells implies that Vp1 contains the major determinants for cell attachment and entry. We propose that the major role of Vp2/3 in infectivity is to mediate the nuclear entry of viral DNA.

Simple sequence repeats in mycobacterial genomes

Simple sequence repeats (SSRs) or microsatellites are the repetitive nucleotide sequences of motifs of length 1-6 bp. They are scattered throughout the genomes of all the known organisms ranging from viruses to eukaryotes. Microsatellites undergo mutations in the form of insertions and deletions (INDELS) of their repeat units with some bias towards insertions that lead to microsatellite tract expansion. Although prokaryotic genomes derive some plasticity due to microsatellite mutations they have in-built mechanisms to arrest undue expansions of microsatellites and one such mechanism is constituted by post-replicative DNA repair enzymes MutL, MutH and MutS. The mycobacterial genomes lack these enzymes and as a null hypothesis one could expect these genomes to harbour many long tracts. It is therefore interesting to analyse the mycobacterial genomes for distribution and abundance of microsatellites tracts and to look for potentially polymorphic microsatellites. Available mycobacterial genomes, Mycobacterium avium, M. leprae, M. bovis and the two strains of M. tuberculosis (CDC1551 and H37Rv) were analysed for frequencies and abundance of SSRs. Our analysis revealed that the SSRs are distributed throughout the mycobacterial genomes at an average of 220-230 SSR tracts per kb. All the mycobacterial genomes contain few regions that are conspicuously denser or poorer in microsatellites compared to their expected genome averages. The genomes distinctly show scarcity of long microsatellites despite the absence of a post-replicative DNA repair system. Such severe scarcity of long microsatellites could arise as a result of strong selection pressures operating against long and unstable sequences although influence of GC-content and role of point mutations in arresting microsatellite expansions can not be ruled out. Nonetheless, the long tracts occasionally found in coding as well as non-coding regions may account for limited genome plasticity in these genomes.

Single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe for the detection of rare mutations

The detection of rare mutant DNA from a background of wild-type alleles usually requires laborious manipulations, such as restriction enzyme digestion and gel electrophoresis. Here, we describe a protocol for homogeneous detection of rare mutant DNA in a single tube. The protocol uses a peptide nucleic acid (PNA) as both PCR clamp and sensor probe. The PNA probe binds tightly to perfectly matched wild-type DNA template but not to mismatched mutant DNA sequences, which specifically inhibits the PCR amplification of wild-type alleles without interfering with the amplification of mutant DNA. A fluorescein tag (which undergoes fluorescence resonance energy transfer with the adjacent fluorophore of an anchor probe when both are annealed to the template DNA) also allows the PNA probe to generate unambiguous melting curves to detect mutant DNA during real-time fluorescent monitoring. The whole assay takes about only 1 h. This protocol has been used for detecting mutant K-ras DNA and could be applied to the detection of other rare mutant DNAs.

The NH2-terminal php Domain of the α Subunit of the Escherichia coli Replicase Binds the ϵ Proofreading Subunit

The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.