Microarray-based DNA resequencing using 3' blocked primers

To exceed the throughput and accuracy of conventional sequencing technologies, we tested a method (pyrophosphorolysis-activated polymerization [PAP]) of nucleic acid amplification that uses 3' blocked primers (P*s). As proof-of-principle, we resequenced a 20-bp region of the factor IX gene with a microarray of P*s. P*s discriminate 3' end mismatches with ultra-high specificity as well as mismatches along their lengths with high specificity. We correctly identified two wild-type samples as well as all mismatches, including three single-base substitutions, one microdeletion, one microinsertion, and one heterozygous mutation. Despite limitations in the primer purity, the signal/noise ratio between the matched and mismatched P*s sometimes exceeded 1000. Thus, PAP resequencing shows great potential for accurate and high-throughput microarray-based resequencing.     

Single-base discrimination mediated by proofreading 3' phosphorothioate-modified primers

It has been well known for decades that deoxyribonucleic acid (DNA) polymerases with proofreading function have a higher fidelity in primer extension as compared to those without 3' exonuclease activities. However, polymerases with proofreading function have not been used in single nucleotide polymorphism (SNP) assays. Here, we describe a new method for single-base discrimination by proofreading the 3' phosphorothioate-modified primers using a polymerase with proofreading function. Our data show that the combination of a polymerase with 3' exonuclease activity and the 3' phosphorothioate-modified primers work efficiently as a single-base mismatch-operated on/off switch. DNA polymerization only occurred from matched primers, whereas mismatched primers were not extended at the broad range of annealing temperature tested in our study. This novel single-base discrimination method has potential in SNP assays.     

Human exonuclease I is required for 5' and 3' mismatch repair.

We have partially purified a human activity that restores mismatch-dependent, bi-directional excision to a human nuclear extract fraction depleted for one or more mismatch repair excision activities. Human EXOI co-purifies with the excision activity, and the purified activity can be replaced by near homogeneous recombinant hEXOI. Despite the reported 5' to 3' hydrolytic polarity of this activity, hEXOI participates in mismatch-provoked excision directed by a strand break located either 5' or 3' to the mispair. When the strand break that directs repair is located 3' to the mispair, hEXOI- and mismatch-dependent gap formation in excision-depleted extracts requires both hMutSalpha and hMutLalpha. However, excision directed by a 5' strand break requires hMutSalpha but can occur in absence of hMutLalpha. In systems comprised of pure components, the 5' to 3' hydrolytic activity of hEXOI is activated by hMutSalpha in a mismatch-dependent manner. These observations indicate a hydrolytic function for hEXOI in 5'-heteroduplex correction. The involvement of hEXOI in 3'-heteroduplex repair suggests that it has a regulatory/structural role in assembly of the 3'-excision complex or that the protein possesses a cryptic 3' to 5' hydrolytic activity.     

Nucleotide 2',3'-cyclic monophosphokinase from actinomycetes

Streptomyces nucleotide 3'-pyrophosphokinase does not only transfer the 5'-beta, gamma-pyrophosphoryl group of ATP, ATP 3'-pyrophosphate or dATP to a variety of nucleotides at the 3'-OH site, but also adds 2',3'-cyclic terminal monophosphate to some suitable nucleotides with the use of diadenosine 5',5'-polyphosphates (n = 3-5). Examples are pA greater than p, ppA greater than p, pG greater than p, CpG greater than p, etc.     

Nucleotide excision repair 3' endonuclease XPG stimulates the activity of base excision repairenzyme thymine glycol DNA glycosylase.

An ionizing radiation-induced DNA lesion, thymine glycol, is removed from DNA by a thymine glycol DNA glycosylase with an apurinic/apyrimidinic (AP) lyase activity encoded by the Escherichia coli endonuclease III ( nth ) gene and its homolog in humans. Cells from Cockayne syndrome patients with mutations in the XPG gene show approximately 2-fold reduced global repair of thymine glycol. Hence, I decided to investigate the molecular mechanism of the effect of XPG protein observed in vivo on thymine glycol removal by studying the interactions of XPG protein and human endonuclease III (HsNTH) protein in vitro and the effect of XPG protein on the activity of HsNTH protein on a substrate containing thymine glycol. The XPG protein stimulates the binding of HsNTH protein to its substrate and increases its glycosylase/AP lyase activity by a factor of approximately 2 through direct interaction between the two proteins. These results provide in vitro evidence for a second function of XPG protein in DNA repair and a mechanistic basis for its stimulatory activity on HsNTH protein.     

Induction of multiple replacement mutations by oligonucleotide-directed mutagenesis with extended mismatch primers

We have developed a method called oligo-scanning mutagenesis that uses oligonucleotides to mutate up to 12 contiguous bases in a single step. Some advantages of this procedure are that the position and sequence of the replacement mutations are completely specified by the investigator, and combinations of mutations can easily be generated. The technique uses a gapped substrate and the Escherichia coli dam methylation error-correcting mechanism to increase the yield of mutants.     

The conversion of 3' UTRs into coding regions

A possible origin of novel coding sequences is the removal of stop codons, leading to the inclusion of 3' untranslated regions (3' UTRs) within genes. We classified changes in the position of stop codons in closely related Saccharomyces species and in a mouse/rat comparison as either additions to or subtractions from coding regions. In both cases, the position of stop codons is highly labile, with more subtractions than additions found. The subtraction bias may be balanced by the input of new coding regions through gene duplication. Saccharomyces shows less stop codon lability than rodents, probably due to greater selective constraint. A higher proportion of 3' UTR incorporation events preserve frame in Saccharomyces. This higher proportion is consistent with the action of the [PSI(+)] prion as an evolutionary capacitor to facilitate 3' UTR incorporation in yeast.     

Thermodynamic analysis of 5' and 3' single- and 3' double-nucleotide overhangs neighboring wobble terminal base pairs

Thermodynamic parameters are reported for duplex formation of 40 self-complementary RNA duplexes containing wobble terminal base pairs with all possible 3′ single and double-nucleotide overhangs, mimicking the structures of short interfering RNAs (siRNA) and microRNAs (miRNA). Based on nearest neighbor analysis, the addition of a single 3′ dangling nucleotide increases the stability of duplex formation up to 1 kcal/mol in a sequence-dependent manner. The addition of a second dangling nucleotide increases the stability of duplexes closed with wobble base pairs in an idiosyncratic manner. The results allow for the development of a nearest neighbor model, which improves the predication of free energy and melting temperature for duplexes closed by wobble base pairs with 3′ single or double-nucleotide overhangs. Phylogenetic analysis of naturally occurring miRNAs was performed. Selection of the effector miR strand of the mature miRNA duplex appears to be dependent on the orientation of the GU closing base pair rather than the identity of the 3′ double-nucleotide overhang. Thermodynamic parameters for the 5′ single terminal overhangs adjacent to wobble closing base pairs are also presented.     

The Activity of 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase in Rat Tissues

Abstract: The activity of the myelin-associated enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) was measured in 14 rat tissues and in subcellular fractions of rat liver by a sensitive fluorometric method, using cyclic NADP as substrate. CNP activity in brain (339 μmol/h/mg protein) was fourfold that of the sciatic nerve. The activities in tissues outside the nervous system ranged from a low of 0.42 μmol/h/mg protein in the unwashed red blood cell to a high of 9.96 in the spleen. The activity was highest in tissues containing cells with membranes capable of undergoing transformation and elaboration (spleen and thymus) and low in those in which the cell membranes are morphologically stable (muscle and red cell). The enzyme was found in all major liver subtractions, with the highest activities in the microsomal and nuclear fractions. Despite the large difference in the maximal velocities of CNP in brain and liver, the affinity of the liver enzyme for the substrate ( k _m) was similar to that of brain enzyme. Brain CNP was stable over a 48-h postmortem period.     

Ability of base analogs to induce the SOS response: effect of a dam mutation and mismatch repair system

Summary 2-Aminopurine, 2-amino-N⁶-hydroxyadenine, 2-amino-N⁶-methoxyadenine and 2-amino-N⁶-methyl-N⁶-hydroxyadenine (but not N⁴-hydroxycytidine), strong mutagens of base analog type, may induce the SOS response in E. coli cells. This ability is greatly enhanced in dam3 mutants and abolished in dam3mutS, dam3mutH, and dam3mutL strains, thereby suggesting that the mismatch repair system is involved in the mechanism of induction.Abbreviations n²Pur 2-aminopurine - n²oh⁶Ade 2-amino-N⁶-hydroxyadenine - n²om⁶Ade 2-amino-N⁶-methyoxyadenine - n²-m⁶oh⁶Ade 2-amino-N⁶-methyl-N⁶-hydroxyadenine - oh⁴Cyd N⁴-hydroxycytidine - MC mitomycin C     

Demonstration of 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase in Cultured Human Schwann Cells

Abstract: Schwann cell cultures were established from adult human sural nerve biopsies. 2'3'-Cyclic nucleotide 3'-phosphohydrolase (CNPase) activity was estimated in the homogenates of those cells by a sensitive isotope assay using [³H]2',3'-cyclic AMP as substrate. A high level of CNPase activity was observed in cultured Schwann cells, whereas cultured human muscle and skin fibroblasts contained negligible levels of CNPase activity. CNPase of human Schwann cells followed typical enzyme-substrate kinetics, with an apparent K _m of 1.6 m M for 2',3'-cyclic AMP, and the enzyme was stimulated by detergents such as Triton X-100 and deoxycholate. It was inhibited by p -chloromercuricbenzoate and 2'-AMP. These properties are typical of CNPase isolated from adult brain and spinal cord. CNPase can serve as a new biochemical marker of normal cultured human Schwann cells and can be useful in analyzing the properties of cultured Schwann cells from patients with dysschwannian neuropathies.     

Differential requirement for proliferating cell nuclear antigen in 5' and 3' nick-directed excision in human mismatch repair

Proliferating cell nuclear antigen (PCNA) is involved in mammalian mismatch repair at a step prior to or at mismatch excision, but the molecular mechanism of this process is not fully understood. To examine the role of PCNA in mismatch-provoked and nick-directed excision, orientation-specific mismatch removal of heteroduplexes with a pre-existing nick was monitored in human nuclear extracts supplemented with the PCNA inhibitor protein p21. We show here that, whereas 3' nick-directed mismatch excision was completely inhibited by low concentrations of p21 or a p21 C-terminal fusion protein, 5' nick-directed excision was only partially blocked under the same conditions. No further reduction of the 5' excision was detected when a much higher concentration of p21 C-terminal protein was used. These results suggest the following. (i) There is a differential requirement for PCNA in 3' and 5' nick-directed excision; and (ii) 5' nick-directed excision is conducted by a manner either dependent on or independent of PCNA. Our in vitro reconstitution experiments indeed identified a 5' nick-directed excision pathway that is dependent on PCNA, hMutSalpha, and a partially purified fraction from a HeLa nuclear extract.     

Detection of single DNA base mutations with mismatch repair enzymes.

A novel method for identifying DNA point mutations has been developed by using mismatch repair enzymes. The high specificity of the Escherichia coli MutY protein has permitted the development of a reliable and sensitive method for the detection and characterization of point mutations in the human genome. The MutY protein is involved in a repair pathway that can convert A/G or A/C mismatches to C/G or G/C basepairs, respectively. A/G or A/C mismatches formed by hybridization between two amplified genomic DNA samples or between specific DNA probes and target DNA are nicked at the mispaired adenine strand by MutY protein. As little as 1% of the mutant sequence can be detected by the mismatch repair enzyme cleavage (MREC) method in a mixture of normal and mutated DNAs (e.g., mutant cells are only present in 1% of the normal cell background). By using different probes, the assay also can determine the nucleotide sequence of the mutation. We have applied this method to detect single-base substitutions in human oncogenes.     

Nucleotide sequence of the 3' end of MCF 247 murine leukemia virus

We isolated DNA clones of MCF 247, a leukemogenic, recombinant type C virus obtained from the thymus of an AKR mouse. We determined the nucleotide sequence of the viral long terminal repeat (LTR) and the 3' end of env, and we compared the sequences to corresponding sequences of the genome of Akv virus, the putative ecotropic parent of MCF 247. By analogy with Moloney leukemia virus, we identified the amino terminus of Prp15E, the C-terminal proteolytic cleavage product of env and precursor to mature virion p15E. In MCF 247 the presumptive Prp15E is encoded by a 603-nucleotide open reading frame. The majority of this sequence is identical to that of Akv. However, a recombination event near the 3' end of the Prp15E-coding region introduces nonecotropic sequences into MCF 247, and these extend to the 3' end through the U3 portion of the LTR. The U3 regions of Akv and MCF 247 are about 83% homologous. The R and U5 regions of the LTR of MCF 247 and Akv are identical. Large RNase T1-resistant oligonucleotides analyzed previously in numerous ecotropic and MCF viral genomes were located within the Akv and MCF 247 DNA sequences. The resulting precise T1 oligonucleotide maps of the 3' ends of MCF viral genomes reveal that the biologically defined, leukemogenic class I MCFs isolated from thymic neoplasms of inbred mice all share the sequence pattern seen in MCF 247, a representative of this group; they possess recombinant Prp15E genes and derive U3 from their nonecotropic parents.     

Isothermal amplification of DNA using quadruplex primers with fluorescent pteridine base analogue 3‐methyl isoxanthopterin

We previously developed a method, known as quadruplex priming amplification (QPA), which greatly simplifies DNA amplification and quantification assays. QPA employs specific primers based on GGGTGGGTGGGTGGG (G3T) sequence, which upon polymerase elongation spontaneously dissociates from the target and folds into a stable quadruplex. Fluorescent nucleotide analogs, when incorporated into these primers, emit light upon quadruplex formation and permit simple, specific, and sensitive quantification without the attachment of probe molecules. Here, we studied optical [fluorescence and circular dichroism (CD)] and thermodynamic properties of the G3T sequence and variants incorporating 3‐methylisoxanthopterin (3MI), a highly fluorescent nucleotide analog suitable for QPA. CD studies demonstrate that the incorporation of 3MI does not change the overall tertiary structure of G3T; however, thermal unfolding experiments revealed that it significantly destabilizes the quadruplex. Enzymatic studies revealed that Taq and Bst are practically unable to incorporate any nucleotides opposite to template 3MI. Based on this knowledge, we designed QPA assays with truncated targets that demonstrate efficient amplification around 55°C. Overall, these studies suggest that 3MI‐based QPA is a useful assay for DNA amplification and detection. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 583–590, 2014.