Mechanism of template-independent nucleotide incorporation catalyzed by a template-dependent DNA polymerase

Numerous template-dependent DNA polymerases are capable of catalyzing template-independent nucleotide additions onto blunt-end DNA. Such non-canonical activity has been hypothesized to increase the genomic hypermutability of retroviruses including human immunodeficiency viruses. Here, we employed pre-steady state kinetics and X-ray crystallography to establish a mechanism for blunt-end additions catalyzed by Sulfolobus solfataricus Dpo4. Our kinetic studies indicated that the first blunt-end dATP incorporation was 80-fold more efficient than the second, and among natural deoxynucleotides, dATP was the preferred substrate due to its stronger intrahelical base-stacking ability. Such base-stacking contributions are supported by the 41-fold higher ground-state binding affinity of a nucleotide analog, pyrene nucleoside 5'-triphosphate, which lacks hydrogen bonding ability but possesses four conjugated aromatic rings. A 2.05 A resolution structure of Dpo4*(blunt-end DNA)*ddATP revealed that the base and sugar of the incoming ddATP, respectively, stack against the 5'-base of the opposite strand and the 3'-base of the elongating strand. This unprecedented base-stacking pattern can be applied to subsequent blunt-end additions only if all incorporated dAMPs are extrahelical, leading to predominantly single non-templated dATP incorporation.     

Critical evaluation of random mutagenesis by error-prone polymerase chain reaction protocols, Escherichia coli mutator strain, and hydroxylamine treatment

Random mutagenesis methods constitute a valuable protein modification toolbox with applications ranging from protein engineering to directed protein evolution studies. Although a variety of techniques are currently available, the field is lacking studies that would directly compare the performance parameters and operational range of different methods. In this study, we have scrutinized several of the most commonly used random mutagenesis techniques by critically evaluating popular error-prone polymerase chain reaction (PCR) protocols as well as hydroxylamine and a mutator Escherichia coli strain mutagenesis methods. Relative mutation frequencies were analyzed using a reporter plasmid that allowed direct comparison of the methods. Error-prone PCR methods yielded the highest mutation rates and the widest operational ranges, whereas the chemical and biological methods generated a low level of mutations and exhibited a narrow range of operation. The repertoire of transitions versus transversions varied among the methods, suggesting the use of a combination of methods for high-diversity full-scale mutagenesis. Using the parameters defined in this study, the evaluated mutagenesis methods can be used for controlled mutagenesis, where the intended average frequency of induced mutations can be adjusted to a desirable level.     

Identification of Critical Residues for the Tight Binding of Both Correct and Incorrect Nucleotides to Human DNA Polymerase λ

DNA polymerase λ (Pol λ) is a novel X-family DNA polymerase that shares 34% sequence identity with DNA polymerase β. Pre-steady-state kinetic studies have shown that the Pol λ-DNA complex binds both correct and incorrect nucleotides 130-fold tighter, on average, than the DNA polymerase β-DNA complex, although the base substitution fidelity of both polymerases is 10^− 4 to 10^− 5. To better understand Pol λ's tight nucleotide binding affinity, we created single-substitution and double-substitution mutants of Pol λ to disrupt the interactions between active-site residues and an incoming nucleotide or a template base. Single-turnover kinetic assays showed that Pol λ binds to an incoming nucleotide via cooperative interactions with active-site residues (R386, R420, K422, Y505, F506, A510, and R514). Disrupting protein interactions with an incoming correct or incorrect nucleotide impacted binding to each of the common structural moieties in the following order: triphosphate ? base > ribose. In addition, the loss of Watson-Crick hydrogen bonding between the nucleotide and the template base led to a moderate increase in K_d. The fidelity of Pol λ was maintained predominantly by a single residue, R517, which has minor groove interactions with the DNA template.     

Intense neutral drifts yield robust and evolvable consensus proteins

What changes occur when a natural protein that had been under low mutation rates is subjected to a neutral drift at high mutational loads, thus generating genetically diverse (polymorphic) gene ensembles that all maintain the protein's original function and structure? To address this question we subjected large populations of TEM-1 beta-lactamase to a prolonged neutral drift, applying high mutation rates and purifying selection to maintain TEM-1's existing penicillinase activity. Purging of deleterious mutations and enrichment of beneficial ones maintained the sequence of these ensembles closer to TEM-1's family consensus and inferred ancestor. In particular, back-to-consensus/ancestor mutations that increase TEM-1's kinetic and thermodynamic stability were enriched. These acted as global suppressors and enabled the tolerance of a broad range of deleterious mutations, thus further increasing the genetic diversity of the drifting populations. The probability of a new function emerging (cefotaxime degradation) was also substantially increased in these ensembles owing to the presence of many gene variants carrying the global suppressors. Our findings indicate the unique features of large, polymorphic neutral ensembles generated under high mutational loads and prompt the speculation that the progenitors of today's proteins may have evolved under high mutational loads. The results also suggest that predictable back-to-consensus/ancestor changes can be used in the laboratory to generate highly diverse and evolvable gene libraries.     

Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease affecting cattle that is transmissible to humans, manifesting as a variant of Creutzfeldt-Jakob disease (vCJD) likely following the consumption of meat contaminated with BSE prions. High-affinity antibodies are a prerequisite for the development of simple, highly sensitive and non-invasive diagnostic tests that are able to detect even small amounts of the disease-associated PrP conformer (PrP(Sc)). We describe here the affinity maturation of a single-chain Fv antibody fragment with a binding affinity of 1 pM to a peptide derived from the unstructured region of bovine PrP (BoPrP (90-105)). This is the tightest peptide-binding antibody reported to date and may find useful application in diagnostics, especially when PrP(Sc) is pretreated by denaturation and/or proteolysis for peptide-like presentation. Several rounds of directed evolution and off-rate selection with ribosome display were performed using an antibody library generated from a single PrP binder with error-prone PCR and DNA-shuffling. As the correct determinations of affinities in this range are not straightforward, competition biosensor techniques and KinExA methods were both applied and compared. Structural interpretation of the affinity improvement was performed based on the crystal structure of the original prion binder in complex with the BoPrP (95-104) peptide by modeling the corresponding mutations.