Modeling DNA shuffling.

In vitro evolution is a new, important laboratory method to evolve molecules with desired properties. It has been used in a variety of biological studies and drug development. In this paper, we study one important mutagenesis method used in in vitro evolution experiments called DNA shuffling. We construct a mathematical model for DNA shuffling and study the properties of molecules after DNA shuffling experiments based on this model. The model for DNA shuffling consists of two parts. First we apply the Lander-Waterman model for physical mapping by fingerprinting random clones to model the distribution of regions that can be reassembled through DNA shuffling. Then we present a model for recombination between different DNA species with different mutations. We compare our theoretical results with experimental data. Finally we propose novel applications of the theoretical results to the optimal design of DNA shuffling experiments and to physical mapping using DNA shuffling.     

On the fidelity of DNA replication. Isolation of high fidelity DNA polymerase-primase complexes by immunoaffinity chromatography

Error rates for conventionally purified DNA polymerase-alpha from calf thymus, chicken, and human sources have been reported to be one in 10,000 to one in 40,000 nucleotides incorporated. Isolation of polymerase-alpha by immunoaffinity chromatography yields a multiprotein high molecular weight replication complex that contains an associated DNA primase (Wong, S. W., Paborsky, L. R., Fisher, P. A., Wang, T. S-F., and Korn, D. (1986) J. Biol. Chem. 261, 7958-7968). We have isolated DNA polymerase-primase complexes from calf thymus, from a human lymphoblast cell line (TK-6), and from Chinese hamster lung cells (V-79) using two different methods of immunoaffinity chromatography. These enzyme complexes are 12- to 20-fold more accurate than conventionally purified calf thymus DNA polymerase-alpha when assayed using the phi X174am3 fidelity assay; estimated error rates are one in 460,000 to one in 830,000 nucleotides incorporated when the enzyme complex is freshly isolated. The polymerase-primase complex from calf thymus exhibited no detectable 3'----5' exonuclease activity using a heteroduplex substrate containing a single 3'-terminal mismatched nucleotide. Upon prolonged storage at -70 degrees C, the error rate of the immunoaffinity-purified calf thymus DNA polymerase-primase complex increases to about one in 50,000 nucleotides incorporated, an error rate similar to that exhibited by conventional isolates of DNA polymerase-alpha.     

Halogenated pentamethine cyanine dyes exhibiting high fidelity for G-quadruplex DNA

Design and optimization of quadruplex-specific small molecules is developing into an attractive strategy for anti-cancer therapeutics with some promising candidates in clinical trials. A number of therapeutically favorable features of cyanine molecules can be effectively exploited to develop them as promising quadruplex-targeting agents. Herein, the design, synthesis and evaluation of a series of dimethylindolenine cyanine dyes with varying halogen substitutions are reported. Their interactions with telomeric and c-myc quadruplexes as well as a reference duplex sequence have been evaluated using thermal melting, biosensor-surface plasmon resonance, circular dichroism, isothermal titration calorimetry and mass spectrometry. Thermal melting analysis indicates that these ligands exhibit significant quadruplex stabilization and a very low duplex binding, with the dimethyl incorporation of paramount importance for decreased duplex affinity. Circular dichroism studies showed that the interaction of cyanines with quadruplex structures are primarily through stacking at one or both ends of the terminal tetrads with the two (trimethylammonium)propyl groups interacting in the accessible quadruplex grooves. Surface plasmon resonance and mass spectral studies shows the formation of an initial strong 1:1 complex followed by a significantly weaker secondary binding. Isothermal calorimetry studies show that the interaction of cyanines is predominantly entropy driven. In line with the design principles, this work provides new insights for further developing potent, highly selective cyanines as promising quadruplex-specific agents.     

Degenerate oligonucleotide gene shuffling (DOGS): a method for enhancing the frequency of recombination with family shuffling.

Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Shuffling techniques can be used on a collection of mutants of the same gene, or related families of genes can be shuffled to produce mutants encoding chimeric gene products. One difficulty with current shuffling procedures is the predominance of unshuffled ("parental") molecules in the pool of mutants. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled and reduces the regeneration of unshuffled parental genes. This procedure has the advantage of avoiding the use of endonucleases for gene fragmentation prior to shuffling and allows the use of random mutagenesis of selected segments of the gene as part of the procedure. We illustrate the use of the technique with a diverse family of beta-xylanase genes that possess widely different G+C contents.     

Molecular mechanisms of exon shuffling: illegitimate recombination

Illegitimate recombination (IR) is a process that takes place far more often than homologous recombination and is characterized by the recombination between non-homologous or short homologous sequences. The consequences of IR frequently emerge after the introduction of DNA in cell lines because it more frequently integrates in non-homologous than in homologous regions of the host genome. As a result, unexpected truncated or elongated products may be found. By not discarding those products as transfection artifacts, but by studying how they are generated, it might elucidate a possible molecular mechanism of IR. Here we review the current literature describing different mechanisms by which non-homologous DNA recombination can be induced.     

DNA shuffling of subgenomic sequences of subtilisin.

DNA family shuffling of 26 protease genes was used to create a library of chimeric proteases that was screened for four distinct enzymatic properties. Multiple clones were identified that were significantly improved over any of the parental enzymes for each individual property. Family shuffling, also known as molecular breeding, efficiently created all of the combinations of parental properties, producing a great diversity of property combinations in the progeny enzymes. Thus, molecular breeding, like classical breeding, is a powerful tool for recombining existing diversity to tailor biological systems for multiple functional parameters.     

Saccharomyces cerevisiae DNA polymerase delta: high fidelity for base substitutions but lower fidelity for single- and multi-base deletions

Eukaryotic DNA polymerase delta (Pol delta) plays an essential role in replicating large nuclear genomes, a process that must be accurate to maintain stability over many generations. Based on kinetic studies of insertion of individual dNTPs opposite a template guanine, Pol delta is believed to have high selectivity for inserting correct nucleotides. This high selectivity, in conjunction with an intrinsic 3'-exonuclease activity, implies that Pol delta should have high base substitution fidelity. Here we demonstrate that the wild type Saccharomyces cerevisiae three-subunit Pol delta does indeed have high base substitution fidelity for the 12 possible base-base mismatches, producing on average less than 1.3 stable misincorporations/100,000 nucleotides polymerized. Measurements with exonuclease-deficient Pol delta confirm the high nucleotide selectivity of the polymerase and further indicate that proofreading enhances the base substitution fidelity of the wild type enzyme by at least 60-fold. However, Pol delta inefficiently proofreads single nucleotide deletion mismatches in homopolymeric runs, such that the error rate is 30 single nucleotide deletions/100,000 nucleotides polymerized. Moreover, wild type Pol delta frequently deletes larger numbers of nucleotides between distantly spaced direct repeats of three or more base pairs. Although wild type Pol delta and Pol epsilon both have high base substitution fidelity, Pol delta is much less accurate than Pol epsilon for deletions involving repetitive sequences. Thus, strand slippage during replication by wild type Pol delta may be a primary source of insertion and deletion mutagenesis in eukaryotic genomes.     

DNA structure and polymerase fidelity.

The accuracy of DNA replication results from both the intrinsic DNA polymerase fidelity and the DNA sequence. Although the recent structural studies on polymerases have brought new insights on polymerase fidelity, the role of DNA sequence and structure is less well understood. Here, the analysis of the crystal structures of hotspots for polymerase slippage including (CA)n and (A)n tracts in different intermolecular contexts reveals that, in the B-form, these sequences share common structural alterations which may explain the high rate of replication errors. In particular, a two-faced "Janus-like" structure with shifted base-pairs in the major groove but an apparent normal geometry in the minor groove constitutes a molecular decoy specifically suitable to mislead the polymerases. A model of the rat polymerase beta bound to this structure suggests that an altered conformation of the nascent template-primer duplex can interfere with correct nucleotide incorporation by affecting the geometry of the active site and breaking the rules of base-pairing, while at the same time escaping enzymatic mechanisms of error discrimination which scan for the correct geometry of the minor groove.In contrast, by showing that the A-form greatly attenuates the sequence-dependent structural alterations in hotspots, this study suggests that the A-conformation of the nascent template-primer duplex at the vicinity of the polymerase active site will contribute to fidelity. The A-form may play the role of a structural buffer which preserves the correct geometry of the active site for all sequences. The detailed comparison of the conformation of the nascent template-primer duplex in the available crystal structures of DNA polymerase-DNA complexes shows that polymerase beta, the least accurate enzyme, is unique in binding to a B-DNA duplex even close to its active site. This model leads to several predictions which are discussed in the light of published experimental data.     

透明颤菌血红蛋白的DNA改组研究

为提高透明颤菌血红蛋白在限氧条件下促进宿主细胞生长的能力,首先通过易错PCR向透明颤菌血红蛋白基因中引入突变,再结合DNA改组对其进行改造。将改组基因置于透明颤菌血红蛋白天然启动子下游,转化大肠杆菌DH5α,构建改组文库。以限氧培养条件下菌体沉淀的颜色为指标进行试管初筛,再以限氧和极端限氧条件下菌体湿重为指标进行摇瓶复筛,最终得到一个高活性突变蛋白VHb'042506。该蛋白使宿主的菌体湿重在限氧和极端限氧条件下较原基因转化子分别提高了31.25%和58.75%。经测序和比对,该基因与原基因相比发生了11处碱基点突变,致氨基酸4处错义突变。CO差光谱实验显示该蛋白具有更强的特征吸收。     

Evolution of a cytokine using DNA family shuffling.

DNA shuffling of a family of over 20 human interferon-alpha (Hu-IFN-alpha) genes was used to derive variants with increased antiviral and antiproliferation activities in murine cells. A clone with 135,000-fold improved specific activity over Hu-IFN-alpha2a was obtained in the first cycle of shuffling. After a second cycle of selective shuffling, the most active clone was improved 285,000-fold relative to Hu-IFN-alpha2a and 185-fold relative to Hu-IFN-alpha1. Remarkably, the three most active clones were more active than the native murine IFN-alphas. These chimeras are derived from up to five parental genes but contained no random point mutations. These results demonstrate that diverse cytokine gene families can be used as starting material to rapidly evolve cytokines that are more active, or have superior selectivity profiles, than native cytokine genes.     

Coordinating DNA polymerase traffic during high and low fidelity synthesis

With the discovery that organisms possess multiple DNA polymerases (Pols) displaying different fidelities, processivities, and activities came the realization that mechanisms must exist to manage the actions of these diverse enzymes to prevent gratuitous mutations. Although many of the Pols encoded by most organisms are largely accurate, and participate in DNA replication and DNA repair, a sizeable fraction display a reduced fidelity, and act to catalyze potentially error-prone translesion DNA synthesis (TLS) past lesions that persist in the DNA. Striking the proper balance between use of these different enzymes during DNA replication, DNA repair, and TLS is essential for ensuring accurate duplication of the cell's genome. This review highlights mechanisms that organisms utilize to manage the actions of their different Pols. A particular emphasis is placed on discussion of current models for how different Pols switch places with each other at the replication fork during high fidelity replication and potentially error-pone TLS.     

Anticipatory evolution and DNA shuffling

DNA shuffling has proven to be a powerful technique for the directed evolution of proteins. A mix of theoretical and applied research has now provided insights into how recombination can be guided to more efficiently generate proteins and even organisms with altered functions.     

Insights into the high fidelity of a DNA polymerase I mutant

Mutants of DNA polymerase I from Thermus aquaticus (Taq) with higher fidelity compared to the wild type enzyme were identified in an earlier study by Summerer et al. (Angew Chem Int Ed 44:4712–4715, 2005). Here, one of these mutants, PLQ (consensus residues 879–881), was analysed using molecular dynamics simulations. This was done by calculating the structures of the ternary complex comprising the enzyme, the DNA primer and template as well as the incoming nucleotide before the chemical reaction for the Watson-Crick and different mismatched base pairings. The results show that the high fidelity of the mutant can be explained partly by different specific interactions between the amino acids of the enzyme and the DNA primer end as well as, in some mismatches, a displacement of the primer relative to the incoming deoxyribonucleoside triphosphate and the catalytic magnesium ion. This displacement is facilitated by reduced steric interactions between the enzyme and the DNA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DNA replication fidelity

DNA replication fidelity plays fundamental role in faithful transmission of genetic material during cell division and during transfer of genetic material from parents to progeny. Replicative polymerases are the main guardian responsible for high replication fidelity of genomic DNA. DNA main replicative polymerases are also involved in many DNA repair processes. High fidelity of DNA replication is determined by correct nucleotide selectivity in polymerase active center, and exonucleolytic proofreading that removes mismatches from primer terminus. In this article we will focus on the mechanisms that are responsible for high fidelity of replications with the special emphasis on structural studies showing important conformational changes after substrate binding. We will also stress the importance of hydrogen bonding, base pair geometry, polymerase DNA interactions and the role of accessory proteins in replication fidelity.     

Ensuring the fidelity of recombination in mammalian chromosomes

Mammalian cells frequently depend on homologous recombination (HR) to repair DNA damage accurately and to help rescue stalled or collapsed replication forks. The essence of HR is an exchange of nucleotides between identical or nearly identical sequences. Although HR fulfills important biological roles, recombination between inappropriate sequence partners can lead to translocations or other deleterious rearrangements and such events must be avoided. For example, the recombination machinery must follow stringent rules to preclude recombination between the many repetitive elements in a mammalian genome that share significant but imperfect homology. This paper takes a conceptual approach in addressing the homology requirements for recombination in mammalian genomes as well as the general strategy used by cells to reject recombination between similar but imperfectly matched sequences. A mechanism of heteroduplex rejection that involves the unwinding of recombination intermediates that may form between mismatched sequences is discussed.     

Biochemical properties of a high fidelity DNA ligase from Thermus species AK16D.

NAD+-dependent DNA ligases from thermophilic bacteria Thermus species are highly homologous with amino acid sequence identities ranging from 85 to 98%. Thermus species AK16D ligase, the most divergent of the seven Thermus isolates collected worldwide, was cloned, expressed in Escherichia coli and purified to homogeneity. This Thermus ligase is similar to Thermus thermophilus HB8 ligase with respect to pH, salt, NAD+, divalent cation profiles and steady-state kinetics.However, the former is more discriminative toward T/G mismatches at the 3'-side of the ligation junction, as judged by the ratios of initial ligation rates of matched and mismatched substrates. The two wild-type Thermus ligases and a Tth ligase mutant (K294R) demonstrate 1-2 orders of magnitude higher fidelity than viral T4 DNA ligase. Both Thermus ligases are active with either the metal cofactor Mg2+, Mn2+or Ca2+but not with Co2+, Ni2+, Cu2+or Zn2+. While the nick closure step with Ca2+becomes rate-limiting which results in the accumulation of DNA-adenylate intermediate, Ni2+only supports intermediate formation to a limited extent. Both Thermus ligases exhibit enhanced mismatch ligation when Mn2+is substituted for Mg2+, but the Tsp. AK16D ligase remains more specific toward perfectly matched substrate.     

DNA polymerase insertion fidelity. Gel assay for site-specific kinetics

A quantitative assay based on gel electrophoresis is described to measure nucleotide insertion kinetics at an arbitrary DNA template site. The assay is used to investigate kinetic mechanisms governing the fidelity of DNA synthesis using highly purified Drosophila DNA polymerase alpha holoenzyme complex and M13 primer-template DNA. Km and Vmax values are reported for correct insertion of A and misinsertion of G, C, and T opposite a single template T site. The misinsertion frequencies are 2 X 10(-4) for G-T and 5 X 10(-5) for both C-T and T-T relative to normal A-T base pairs. The dissociation constant of the polymerase-DNA-dNTP complex, as measured by Km, plays a dominant role in determining the rates of forming right and wrong base pairs. Compared with Km for insertion of A opposite T (3.7 +/- 0.7 microM), the Km value is 1100-fold greater for misinsertion of G opposite T (4.2 +/- 0.4 mM), and 2600-fold greater for misinsertion of either C or T opposite T (9.8 +/- 4.2 mM). These Km differences indicate that in the enzyme binding site the stability of A-T base pairs is 4.3 kcal/mol greater than G-T pairs and 4.9 kcal/mol greater than C-T or T-T pairs. In contrast to the large differences in Km, differences in Vmax are relatively small. There is only a 4-fold reduction in Vmax for insertion of G opposite T and an 8-fold reduction for C or T opposite T, compared with the correct insertion of A. For the specific template T site investigated, the nucleotide insertion fidelity for Drosophila polymerase alpha seems to be governed primarily by a Km discrimination mechanism.