Terminal Mismatches Research Articles

Robust mapping of polyadenylated and non-polyadenylated RNA 3' ends at nucleotide resolution by 3'-end sequencing.

3'-end poly(A)+ sequencing is an efficient and economical method for global measurement of mRNA levels and alternative poly(A) site usage. A common method involves oligo(dT)19V reverse-transcription (RT)-based library preparation and high-throughput sequencing with a custom primer ending in (dT)19. While the majority of library products have the first sequenced nucleotide reflect the bona fide poly(A) site (pA), a substantial fraction of sequencing reads arise from various mis-priming events. These can result in incorrect pA site calls anywhere from several nucleotides downstream to several kilobases upstream from the bona fide pA site. While these mis-priming events can be mitigated by increasing annealing stringency (e.g. increasing temperature from 37 °C to 42 °C), they still persist at an appreciable level (∼10%) and computational methods must be used to prevent artifactual calls. Here we present a bioinformatics workflow for precise mapping of poly(A)+ 3' ends and handling of artifacts due to oligo(dT) mis-priming and sample polymorphisms. We test pA site calling with three different read mapping programs (STAR, BWA, and BBMap), and show that the way in which each handles terminal mismatches and soft clipping has a substantial impact on identifying correct pA sites, with BWA requiring the least post-processing to correct artifacts. We demonstrate the use of this pipeline for mapping pA sites in the model eukaryote S. cerevisiae, and further apply this technology to non-polyadenylated transcripts by employing in vitro polyadenylation prior to library prep (IVP-seq). As proof of principle, we show that a fraction of tRNAs harbor CCU 3' tails instead of the canonical CCA tail, and globally identify 3' ends of splicing intermediates arising from inefficiently spliced transcripts.

The 5′ Nuclease Domain of DNA Polymerase I Mediates a Novel DNA Transfer Pathway during Proofreading

The high fidelity of many DNA polymerases is due, in part, to their ability to remove erroneously incorporated nucleotides at the terminus of the growing primer strand. This “proofreading” activity is catalyzed by the 3′-5′ exonuclease (exo) domain in E. coli DNA polymerase I (Pol I). Structural studies have shown that this domain is spatially separated from the 5′-3′ polymerase domain (pol), which is responsible for incorporation of dNTPs by template-directed polymerization. Pol I also contains another spatially distinct domain, the 5′ nuclease (nuc) domain, which is connected to the enzyme core by a flexible amino acid linker. This domain is involved in cleavage of downstream flaps formed during Okazaki fragment maturation and base excision repair. Using a single-molecule FRET system, we resolved three distinct conformations of Pol I-DNA complexes. Experiments with Pol I mutants established that these states correspond to DNA bound at the pol, exo, or nuc domains. Notably, the DNA substrate can transfer among all three domains without dissociation from the enzyme. We determined rate constants for site switching of model DNA substrates containing a correctly paired primer terminus, a terminal mismatch or an internal mismatch. The presence of mismatches accelerated transfer of DNA from the pol domain to the exo domain, as expected during proofreading. Surprisingly, the mismatches also accelerated transfer of DNA along the pol to nuc to exo domain pathway. These observations reveal the existence of a second intramolecular proofreading pathway, in which mismatched DNA first transfers from the pol domain to the nuc domain and then to the exo domain. Our results suggest an unexpected role for the 5′ nuclease domain in the proofreading activity of Pol I.

Structures of DNA Polymerase Mispaired DNA Termini Transitioning to Pre-catalytic Complexes Support an Induced-Fit Fidelity Mechanism

High-fidelity DNA synthesis requires that polymerases display a strong preference for right nucleotide insertion. When the wrong nucleotide is inserted, thepolymerase deters extension from the mismatched DNA terminus. Twenty-three crystallographic structures of DNA polymerase β with terminal template-primer mismatches were determined as binary DNA and ternary pre-catalytic substrate complexes. These structures indicate that the mismatched termini adopt various distorted conformations that attempt to satisfy stacking and hydrogen-bonding interactions. The binary complex structures indicate an induced strain in the mismatched template nucleotide. Addition of a non-hydrolyzable incoming nucleotide stabilizes the templating nucleotide with concomitant strain in the primer terminus. Several dead-end ternary complex structures suggest that DNA synthesis might occur as the enzyme transitions from an open to a closed complex. The structures are consistent with an induced-fit mechanism where a mismatched terminus is misaligned relative to the correct incoming nucleotide to deter or delay further DNA synthesis.

Evaluating Hydrogen Bonds and Base Stacking of Single, Tandem and Terminal GU Mismatches in RNA with a Mesoscopic Model.

Guanine-Uracil (GU) mismatches are crucial to the stability of the RNA double helix and need to be considered in RNA folding algorithms for numerous biotechnological applications. Yet despite its importance, many aspects of GU base pairs are still poorly understood. There is also a lack of parametrization which prevents it to be considered in mesoscopic models. Here, we adapted the mesoscopic Peyrard-Bishop model to deal with context-dependent hydrogen bonds of GU mismatches and calculated the model parameters related to hydrogen bonding and base stacking from available experimental melting temperatures. The context-dependence causes a proliferation of parameters which made the problem computationally very demanding. We were able to overcome this problem by systematically regrouping the parameters during the minimization procedure. Our results not only provide the much needed parametrization but also answer several questions about the general properties of GU base pairs, as they can be associated straightforwardly to hydrogen bonding and base stacking. In particular, we found a very small Morse potential for tandem 5'-GU-3', which confirms a single hydrogen bond for this configuration, answering a long-standing question over conflicting experimental findings. Terminal GU base pairs are known to increase the duplex stability, but it is not clear why. Our results suggest that the increased terminal stability is mostly due to stronger hydrogen bonding.

Enhancing potency of siRNA targeting fusion genes by optimization outside of target sequence

Canonical siRNA design algorithms have become remarkably effective at predicting favorable binding regions within a target mRNA, but in some cases (e.g., a fusion junction site) region choice is restricted. In these instances, alternative approaches are necessary to obtain a highly potent silencing molecule. Here we focus on strategies for rational optimization of two siRNAs that target the junction sites of fusion oncogenes BCR-ABL and TMPRSS2-ERG. We demonstrate that modifying the termini of these siRNAs with a terminal G-U wobble pair or a carefully selected pair of terminal asymmetry-enhancing mismatches can result in an increase in potency at low doses. Importantly, we observed that improvements in silencing at the mRNA level do not necessarily translate to reductions in protein level and/or cell death. Decline in protein level is also heavily influenced by targeted protein half-life, and delivery vehicle toxicity can confound measures of cell death due to silencing. Therefore, for BCR-ABL, which has a long protein half-life that is difficult to overcome using siRNA, we also developed a nontoxic transfection vector: poly(lactic-coglycolic acid) nanoparticles that release siRNA over many days. We show that this system can achieve effective killing of leukemic cells. These findings provide insights into the implications of siRNA sequence for potency and suggest strategies for the design of more effective therapeutic siRNA molecules. Furthermore, this work points to the importance of integrating studies of siRNA design and delivery, while heeding and addressing potential limitations such as restricted targetable mRNA regions, long protein half-lives, and nonspecific toxicities.

Sequence and Temperature Influence on Kinetics of DNA Strand Displacement at Gold Electrode Surfaces.

Understanding complex contributions of surface environment to tethered nucleic acid sensing experiments has proven challenging, yet it is important because it is essential for interpretation and calibration of indispensable methods, such as microarrays. We investigate the effects of DNA sequence and solution temperature gradients on the kinetics of strand displacement at heated gold wire electrodes, and at gold disc electrodes in a heated solution. Addition of a terminal double mismatch (toehold) provides a reduction in strand displacement energy barriers sufficient to probe the secondary mechanisms involved in the hybridization process. In four different DNA capture probe sequences (relevant for the identification of genetically modified maize MON810), all but one revealed a high activation energy up to 200 kJ/mol during hybridization, that we attribute to displacement of protective strands by capture probes. Protective strands contain 4 to 5 mismatches to ease their displacement by the surface-confined probes at the gold electrodes. A low activation energy (30 kJ/mol) was observed for the sequence whose protective strand contained a toehold and one central mismatch, its kinetic curves displayed significantly different shapes, and we observed a reduced maximum signal intensity as compared to other sequences. These findings point to potential sequence-related contributions to oligonucleotide diffusion influencing kinetics. Additionally, for all sequences studied with heated wire electrodes, we observed a 23 K lower optimal hybridization temperature in comparison with disc electrodes in heated solution, and greatly reduced voltammetric signals after taking into account electrode surface area. We propose that thermodiffusion due to temperature gradients may influence both hybridization and strand displacement kinetics at heated microelectrodes, an explanation supported by computational fluid dynamics. DNA assays with surface-confined capture probes and temperature gradients should not neglect potential influences of thermodiffusion as well as sequence-related effects. Furthermore, studies attempting to characterize surface-tethered environments should consider thermodiffusion if temperature gradients are involved.

Mismatch extension of DNA polymerases and high-accuracy single nucleotide polymorphism diagnostics by gold nanoparticle-improved isothermal amplification.

Sequence mismatches may induce nonspecific extension reaction, causing false results for SNP diagnostics. Herein, we systematically investigated the impact of various 3'-terminal mismatches on isothermal amplification catalyzed by representative DNA polymerases. Despite their diverse efficiencies depending on types of mismatch and kinds of DNA polymerase, all 12 kinds of single 3'-terminal mismatches induced the extension reaction. Generally, only several mismatches (primer-template, C-C, G-A, A-G, and A-A) present an observable inhibitory effect on the amplification reaction, whereas other mismatches trigger amplified signals as high as those of Watson-Crick pairs. The related mechanism was deeply discussed, and a primer-design guideline for specific SNP analysis was summarized. Furthermore, we found that the addition of appropriate gold nanoparticles (AuNPs) can significantly inhibit mismatch extension and enhance the amplification specificity. Also the high-accuracy SNP analysis of human blood genomic DNA has been demonstrated by AuNPs-improved isothermal amplification, the result of which was verified by sequencing (the gold standard method for SNP assay). Collectively, this work provides mechanistic insight into mismatch behavior and achieves accurate SNP diagnostics, holding great potential for the application in molecular diagnostics and personalized medicine.

Modeling Alien-Line Impedance Mismatch in Wideband Vectored Wireline Systems

Sudden changes of channel coefficients in a wideband vectored wireline system (such as G.fast) due to changes in the terminating impedance of lines outside the vectored group can seriously degrade stability and throughput. This letter presents a model that predicts the impact of termination mismatch based exclusively on crosstalk data for the properly-terminated state. Experimental results confirm a tight fit between model and measurements. The model allows analysis of system performance and stability without dedicated crosstalk measurements for mismatch cases.

Mitochondrial tRNA 5′-Editing in Dictyostelium discoideum and Polysphondylium pallidum

Mitochondrial tRNA (mt-tRNA) 5'-editing was first described more than 20 years ago; however, the first candidates for 5'-editing enzymes were only recently identified in a eukaryotic microbe (protist), the slime mold Dictyostelium discoideum. In this organism, eight of 18 mt-tRNAs are predicted to be edited based on the presence of genomically encoded mismatched nucleotides in their aminoacyl-acceptor stem sequences. Here, we demonstrate that mt-tRNA 5'-editing occurs at all predicted sites in D. discoideum as evidenced by changes in the sequences of isolated mt-tRNAs compared with the expected sequences encoded by the mitochondrial genome. We also identify two previously unpredicted editing events in which G-U base pairs are edited in the absence of any other genomically encoded mismatches. A comparison of 5'-editing in D. discoideum with 5'-editing in another slime mold, Polysphondylium pallidum, suggests organism-specific idiosyncrasies in the treatment of U-G/G-U pairs. In vitro activities of putative D. discoideum editing enzymes are consistent with the observed editing reactions and suggest an overall lack of tRNA substrate specificity exhibited by the repair component of the editing enzyme. Although the presence of terminal mismatches in mt-tRNA sequences is highly predictive of the occurrence of mt-tRNA 5'-editing, the variability in treatment of U-G/G-U base pairs observed here indicates that direct experimental evidence of 5'-editing must be obtained to understand the complete spectrum of mt-tRNA editing events in any species.

Proper functioning of the GINS complex is important for the fidelity of DNA replication in yeast

The role of replicative DNA polymerases in ensuring genome stability is intensively studied, but the role of other components of the replisome is still not fully understood. One of such component is the GINS complex (comprising the Psf1, Psf2, Psf3 and Sld5 subunits), which participates in both initiation and elongation of DNA replication. Until now, the understanding of the physiological role of GINS mostly originated from biochemical studies. In this article, we present genetic evidence for an essential role of GINS in the maintenance of replication fidelity in Saccharomyces cerevisiae. In our studies we employed the psf1-1 allele (Takayama et al., 2003) and a novel psf1-100 allele isolated in our laboratory. Analysis of the levels and specificity of mutations in the psf1 strains indicates that the destabilization of the GINS complex or its impaired interaction with DNA polymerase epsilon increases the level of spontaneous mutagenesis and the participation of the error-prone DNA polymerase zeta. Additionally, a synergistic mutator effect was found for the defects in Psf1p and in the proofreading activity of Pol epsilon, suggesting that proper functioning of GINS is crucial for facilitating error-free processing of terminal mismatches created by Pol epsilon.

The Influence of Network Impedance on Conducted Disturbances Within the High-Voltage Traction Harness of Electric Vehicles

Currently, automotive traction systems are increasingly being revised from combustion engine drives to electric propulsion. The high power cables of electric vehicles are routed close to various communication, sensor, or control units. This circumstance results in a tightened challenge for the developing engineers, who need to ensure the electromagnetic compatibility of these miscellaneous and adjacent systems. A trial-and-error approach in the development process of electromagnetic interference filters can only be avoided if the influence of the network impedances within the high voltage traction harness on conducted RF disturbances is known. This contribution presents the current methodology of determining the conducted emissions of power inverters for electric driven cars. It highlights the differences between the traditional low-voltage cable wiring and novel high-voltage traction harnesses, and their impact on the network impedances. An adaption of the line impedance stabilization networks used recently to the characteristic impedance of the high-voltage cables applied shows the influence of termination mismatch on conducted emissions within the traction harness. High-voltage components employed in a car are usually supplied by a traction accumulator battery, which acts as transmission line termination. The influence of a battery's input impedances on disturbances within a component level EMC test setup is shown.

The Specificity and Flexibility of L1 Reverse Transcription Priming at Imperfect T-Tracts

L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP) first uses its endonuclease (EN) to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A) tail, a process known as target-primed reverse transcription (TPRT). Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate 5′-TTTT/A-3′ sites) and frequently preceded by imperfect T-tracts. However, it is currently unclear whether—and to which degree—the liberated 3′-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A) tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3′ end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3′ overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3′ end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.

Effects of Salt, Polyethylene Glycol, and Locked Nucleic Acids on the Thermodynamic Stabilities of Consecutive Terminal Adenosine Mismatches in RNA Duplexes

Consecutive terminal mismatches add thermodynamic stability to RNA duplexes and occur frequently in microRNA-mRNA interactions. Accurate thermodynamic stabilities of consecutive terminal mismatches contribute to the development of specific, high-affinity siRNA therapeutics. Consecutive terminal adenosine mismatches (TAMS) are studied at different salt concentrations, with polyethylene glycol cosolutes, and with locked nucleic acid (LNA) substitutions. These measurements provide benchmarks for the application of thermodynamic predictions to different physiological or therapeutic conditions. The salt dependence for RNA duplex stability is similar for TAMS, internal loops, and Watson-Crick duplexes. A unified model for predicting the free energy of an RNA duplex with or without loops and mismatches at lower sodium concentrations is presented. The destabilizing effects of PEG 200 are larger for TAMS than internal loops or Watson-Crick duplexes, which may result from different base stacking conformations, dynamics, and water hydration. In contrast, LNA substitutions stabilize internal loops much more than TAMS. Surprisingly, the average per adenosine increase in stability for LNA substitutions in internal loops is -1.82 kcal/mol and only -0.20 kcal/mol for TAMS. The stabilities of TAMS and internal loops with LNA substitutions have similar favorable free energies. Thus, the unfavorable free energy of adenosine internal loops is largely an entropic effect. The favorable stabilities of TAMS result mainly from base stacking. The ability of RNA duplexes to form extended terminal mismatches in the absence of proteins such as argonaute and identifying the enthalpic contributions to terminal mismatch stabilities provide insight into the physical basis of microRNA-mRNA molecular recognition and specificity.

New Prediction Model for Probe Specificity in an Allele-Specific Extension Reaction for Haplotype-Specific Extraction (HSE) of Y Chromosome Mixtures

Allele-specific extension reactions (ASERs) use 3′ terminus-specific primers for the selective extension of completely annealed matches by polymerase. The ability of the polymerase to extend non-specific 3′ terminal mismatches leads to a failure of the reaction, a process that is only partly understood and predictable, and often requires time-consuming assay design. In our studies we investigated haplotype-specific extraction (HSE) for the separation of male DNA mixtures. HSE is an ASER and provides the ability to distinguish between diploid chromosomes from one or more individuals. Here, we show that the success of HSE and allele-specific extension depend strongly on the concentration difference between complete match and 3′ terminal mismatch. Using the oligonucleotide-modeling platform Visual Omp, we demonstrated the dependency of the discrimination power of the polymerase on match- and mismatch-target hybridization between different probe lengths. Therefore, the probe specificity in HSE could be predicted by performing a relative comparison of different probe designs with their simulated differences between the duplex concentration of target-probe match and mismatches. We tested this new model for probe design in more than 300 HSE reactions with 137 different probes and obtained an accordance of 88%.

The Werner Syndrome Exonuclease Facilitates DNA Degradation and High Fidelity DNA Polymerization by Human DNA Polymerase δ

DNA Polymerase δ (Pol δ) and the Werner syndrome protein, WRN, are involved in maintaining cellular genomic stability. Pol δ synthesizes the lagging strand during replication of genomic DNA and also functions in the synthesis steps of DNA repair and recombination. WRN is a member of the RecQ helicase family, loss of which results in the premature aging and cancer-prone disorder, Werner syndrome. Both Pol δ and WRN encode 3' → 5' DNA exonuclease activities. Pol δ exonuclease removes 3'-terminal mismatched nucleotides incorporated during replication to ensure high fidelity DNA synthesis. WRN exonuclease degrades DNA containing alternate secondary structures to prevent formation and enable resolution of stalled replication forks. We now observe that similarly to WRN, Pol δ degrades alternate DNA structures including bubbles, four-way junctions, and D-loops. Moreover, WRN and Pol δ form a complex with enhanced ability to hydrolyze these structures. We also present evidence that WRN can proofread for Pol δ; WRN excises 3'-terminal mismatches to enable primer extension by Pol δ. Consistent with our in vitro observations, we show that WRN contributes to the maintenance of DNA synthesis fidelity in vivo. Cells expressing limiting amounts (∼10% of normal) of WRN have elevated mutation frequencies compared with wild-type cells. Together, our data highlight the importance of WRN exonuclease activity and its cooperativity with Pol δ in preserving genome stability, which is compromised by the loss of WRN in Werner syndrome.

A 5′‐Cap for DNA Probes Binding RNA Target Strands

Detecting short RNA strands with high fidelity at any of the bases of their sequence, including the termini, can be challenging, since fraying, wobbling, and refolding all compete with canonical base pairing. We performed a search for 5'-substituents of oligodeoxynucleotides that increase base pairing fidelity at the terminus of duplexes with RNA target strands. From a total of over 70 caps, differing in stacking moiety and linker, a phosphodiester-linked sequence of the residues of L-prolinol, glycine, and oxolinic acid, dubbed ogOA, was identified as a 5'-cap that stabilizes any of the four canonical base pairs, with ΔT(m) values of up to +13.1 °C for an octamer. At the same time, the cap increases discrimination against any of the 12 possible terminal mismatches, including mismatches that are more stable than their perfectly matched counterparts in the control duplex, such as A:A. A probe with the cap also showed increased selectivity in the detection of two closely related microRNAs, let7c and let7a, with a ΔT(m) value of 9.2 °C. Melting curves also yielded thermodynamic data that shed light on the uniformity of molecular recognition in the sequence space of DNA:DNA and DNA:RNA duplexes. Hybridization probes with fidelity-enhancing caps should find applications in the individual and parallel detection of biologically active RNA species.

Abstract 2118: Rare allele enrichment by ARMS combined with probe base genotyping techniques

Abstract Mutation detection plays a key role in several medical areas for diagnosis, treatment and prognosis. Additionally, the ability to detect low level mutations among mostly wild-type DNA is critical to areas such as cancer detection, prenatal testing and infectious diseases. For example, low-level (<10%) cancer mutations can not be sequenced by standard PCR or genotyping techniques. Many moderate- to high-selectivity PCR methods have been developed over the past two decades to enrich minority alleles for known mutations. One of the most widely used approaches is the amplification refractory mutation system (ARMS) which relies on the use of 3’ end terminal nucleotide mismatch to enhance the minor allele. The mutation detection sensitivity ranges from 1:10 to 1:1000. Probe based genotyping techniques such as fluorescent labeled probes, unlabeled probes and Snapback primers depend on short oligonucleotides that hybridize to the template for genotyping. We have combine allele enrichment by ARMS and probe based genotyping techniques to enhance allele discrimination. We designed a 3’ end blocked oligonucleotides to form a perfect match with the wild- type allele (mismatched to other alleles). Therefore, the probe blocks the primer from annealing to the wild-type template, while simultaneously permitting the primer to anneal to, and amplify, the mutant template. Wild-type amplification is completely blocked, and the mutation is enriched. The sensitivity of unlabeled probe-ARMS mutation enrichment detection is 1:30,000. Combining ARMS enrichment of minority alleles and snapback primers has the additional benefit that only two PCR primers are required, with the addition of a short tail of nucleotides on one primer that results in an intra-molecular hybridization probe. The sensitivity of snapback-ARMS mutation enrichment detection is 1:60,000. Fourty-four patients with paired thyroid biopsies and needle samples and 3 additional patients with only needle samples were tested for the BRAF mutation p.V600E (c.1799 T>A) by dual hybridization probes. A blinded test using unlabeled probe-ARMS and Snapback-ARMS enrichment techniques were compared to the dual hybridization probe method. All samples were concordant (47 positive and 42 negative) except 4 samples were positive using unlabeled probe-ARMS and Snapback-ARMS but were negative using hybridization probes. Minority allele enrichment using ARMS combined with probe base genotyping methods increases the mutation detection sensitivity 100 times or more. ARMS combined with an unlabeled probe or a snapback primer is an inexpensive and rapid enrichment technique. No separation, purification or addition steps that increase risk of contamination are necessary. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2118.

The interaction of p53 with 3'-terminal mismatched DNA

The diversity of p53 functions involves its interaction with sequence-specific, non-sequence-specific and various damaged sites in DNA. The preferential excision of misincorporated over correct nucleotides by the 3′→5′ exonuclease activity of p53 provides a molecular basis for p53 involvement in the correction of the DNA replication errors. However, p53 exhibits variations in its comparative efficiency to excise different 3'-terminal mismatched nucleotides. To determine the importance of the binding capacity of the protein to various 3'-terminal damaged sites, we have examined the interaction of p53 with linear dsDNAs containing various 3'-terminal mismatches, employing a gel retardation assay. The data demonstrate the intrinsic 3'-terminal mismatched DNA binding capacity of p53. Since p53 binds directly to various 3'-terminal purine:pyrimidine and purine:purine mispairs to an equal extent, p53 can be considered as a general 3'-mismatched DNA binding protein. Apparently, 3'-terminal mismatched bases are structural element to which p53 can bind, that extends the spectrum of damage sites to which p53 may respond. The formation of the p53-mismatched DNA complex is independent of the sequence context. Thus, the dissimilarities in mispair excision efficiency are probably due to an inherent property of the p53 in excision of 3'-mismatched nucleotides by a bound protein. The results establish a framework for understanding the mechanism of cooperative interaction between p53 and exonuclease-deficient DNA polymerase (e.g. HIV-1 RT). Within the context of error-correction events, p53 by recognition and excision of 3'-mismatched nucleotides from DNA, may be involved in DNA repair, thus increasing the accuracy of DNA synthesis by DNA polymerases.

Thermodynamic characterization of naturally occurring RNA tetraloops

Although tetraloops are one of the most frequently occurring secondary structure motifs in RNA, less than one-third of the 30 most frequently occurring RNA tetraloops have been thermodynamically characterized. Therefore, 24 stem-loop sequences containing common tetraloops were optically melted, and the thermodynamic parameters DeltaH degrees , DeltaS degrees , DeltaG degrees (37,) and T(M) for each stem-loop were determined. These new experimental values, on average, are 0.7 kcal/mol different from the values predicted for these tetraloops using the model proposed by Vecenie CJ, Morrow CV, Zyra A, Serra MJ. 2006. Biochemistry 45: 1400-1407. The data for the 24 tetraloops reported here were then combined with the data for 28 tetraloops that were published previously. A new model, independent of terminal mismatch data, was derived to predict the free energy contribution of previously unmeasured tetraloops. The average absolute difference between the measured values and the values predicted using this proposed model is 0.4 kcal/mol. This new experimental data and updated predictive model allow for more accurate calculations of the free energy of RNA stem-loops containing tetraloops and, furthermore, should allow for improved prediction of secondary structure from sequence. It was also shown that tetraloops within the sequence 5'-GCCNNNNGGC-3' are, on average, 0.6 kcal/mol more stable than the same tetraloop within the sequence 5'-GGCNNNNGCC-3'. More systemic studies are required to determine the full extent of non-nearest-neighbor effects on tetraloop stability.

First-Principles Prediction of the Sequence-Dependent Stability of RNA Hairpin Loop

We develop a statistical mechanical model to predict the sequence-dependent folding stability from the sequence for simple RNA hairpin loops. We use a recently developed 3-vector virtual bond-based RNA folding model which enables rigorous computation of RNA chain entropy. Enumeration of all the possible arrangements of base pairs and the corresponding conformational entropies, through exhaustive self-avoiding random walks of the virtual bonds in a diamond lattice, gives the partition function and free energy for a given RNA sequence. The new model developed here has two unique advantages. First, the model is based on the complete conformational ensemble, including loop conformations that contain all the possible intra-loop base pairs and/or terminal mismatches. Therefore, intra-loop base pairs and mismatches are the results predicted from the model instead of known input information for the model. Second, the model gives accurate estimation for the dramatic entropic changes caused by the formation of the intra-loop base pairs and mismatches. Our first principles calculations for the chain entropy for each given set of base pairs, in combination with the empirical energy function provided from Turner rules, provide ab initio predictions for the sequence-dependent loop stability from RNA sequence. Tests against experimental data indicate that the theory can give improved predictions for the sequence-dependent RNA hairpin loop stability than the nearest-neighbor model.