John Petruska

Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation

Activation-induced cytidine deaminase (AID) is a protein required for B cells to undergo class switch recombination and somatic hypermutation (SHM)—two processes essential for producing high-affinity antibodies. Purified AID catalyses the deamination of C to U on single-stranded (ss)DNA. Here, we show in vitro that AID-catalysed C deaminations occur preferentially on 5′ WRC sequences in accord with SHM spectra observed in vivo. Although about 98% of DNA clones suffer no mutations, most of the remaining mutated clones have 10–70 C to T transitions per clone. Therefore, AID carries out multiple C deaminations on individual DNA strands, rather than jumping from one strand to another. The avid binding of AID to ssDNA could result from its large net positive charge (+11) at pH 7.0, owing to a basic amino-terminal domain enriched in arginine and lysine. Furthermore, AID exhibits a 15-fold preference for C deamination on the non-transcribed DNA strand exposed by RNA polymerase than the transcribed strand protected as a RNA–DNA hybrid. These deamination results on ssDNA bear relevance to three characteristic features of SHM: preferential mutation at C sites within WRC hotspot sequences, the broad clonal mutagenic heterogeneity of antibody variable regions targeted for mutation, and the requirement for active transcription to obtain mutagenesis.

Gene Targeting in Rat Embryo Fibroblasts Promoted by the Polyomavirus Large T Antigen Associated With Neurological Diseases

Expansions of trinucleotide repeats in DNA, a novel source of mutations associated with human disease, may arise by DNA replication slippage initiated by hairpin folding of primer or template strands containing such repeats. To evaluate the stability of single-strand folding by repeating triplets of DNA bases, thermal melting profiles of (CAG)10, (CTG)10, (GAC)10 and (GTC)10 strands are determined at low and physiological salt concentrations, and measurements of melting temperature and enthalpy change are made in each case. Comparisons are made to strands with three times as many repeats, (CAG)30 and (CTG)30. Evidence is presented for stable intrastrand folding by the CAG/CTG class of triplet repeats. Relative to the GAC/GTC class not associated with disease, the order of folding stability is found to be CTG > GAC approximately = CAG > GTC for 10 repeats. Surprisingly, the folds formed by 30 repeats of CTG or CAG have no higher melting temperature and are only 40% more stable in free energy than those formed by 10 repeats. This finding suggests that triplet expansions with higher repeat number may result from the formation of more folded structures with similar stability rather than fewer but longer folds of greater stability.

Weak Strand Displacement Activity Enables Human DNA Polymerase β to Expand CAG/CTG Triplet Repeats at Strand Breaks

Using synthetic DNA constructs in vitro, we find that human DNA polymerase β effectively catalyzes CAG/CTG triplet repeat expansions by slippage initiated at nicks or 1-base gaps within short (14 triplet) repeat tracts in DNA duplexes under physiological conditions. In the same constructs, Escherichia coli DNA polymerase I Klenow Fragment exo− is much less effective in expanding repeats, because its much stronger strand displacement activity inhibits slippage by enabling rapid extension through two downstream repeats into flanking non-repeat sequence. Polymerase β expansions of CAG/CTG repeats, observed over a 32-min period at rates of ∼1 triplet added per min, reveal significant effects of break type (nick versus gap), strand composition (CTG versus CAG), and dNTP substrate concentration, on repeat expansions at strand breaks. At physiological substrate concentrations (1–10 μm of each dNTP), polymerase β expands triplet repeats with the help of weak strand displacement limited to the two downstream triplet repeats in our constructs. Such weak strand displacement activity in DNA repair at strand breaks may enable short tracts of repeats to be converted into longer, increasingly mutable ones associated with neurological diseases.

Biochemical Basis of Immunological and Retroviral Responses to DNA-targeted Cytosine Deamination by Activation-induced Cytidine Deaminase and APOBEC3G

Activation-induced cytidine deaminase (AID) and APOBEC3G catalyze deamination of cytosine to uracil on single-stranded DNA, thereby setting in motion a regulated hypermutagenic process essential for human well-being. However, if regulation fails, havoc ensues. AID plays a central role in the synthesis of high affinity antibodies, and APOBEC3G inactivates human immunodeficiency virus-1. This minireview highlights biochemical and structural properties of AID and APOBEC3G, showing how studies using the purified enzymes provide valuable insight into the considerably more complex biology governing antibody generation and human immunodeficiency virus inactivation.

Bromodeoxyuridine substitution in mammalian DNA can both stimulate and inhibit restriction cleavage

Abstract Total replacement of thymidine by 5-bromodeoxyuridine in mammalian DNA causes a 5-fold stimulation in the rate of DNA cleavage by Mbo I. This is the first report of the stimulation of restriction endonuclease activity by 5-bromodeoxyuridine and is in contrast to the inhibition found with other restriction enzymes. We propose a hypothesis to rationalize these results.

Kinetic selection vs. free energy of DNA base pairing in control of polymerase fidelity.

Significance We address a fundamental biological issue: the source of free energy enabling high-fidelity DNA replication. DNA polymerase errors occur at about 1 per 1,000–10,000 bp, indicating “right” vs. “wrong” free energy differences ΔΔGinc = 3–5 kcal/mol. A recent paper using high inorganic pyrophosphate concentrations to “equilibrate” right and wrong forward and reverse incorporation reactions concluded that base pairing in DNA alone is sufficient to account for polymerase fidelity. By performing an explicit analysis of forward and reverse reactions, we show that steady-state pol incorporation levels are far from equilibrium for wrong incorporations, so that polymerase fidelity cannot depend solely on intrinsic DNA properties. DNA polymerases must operate under kinetic control to achieve high fidelity. What is the free energy source enabling high-fidelity DNA polymerases (pols) to favor incorporation of correct over incorrect base pairs by 103- to 104-fold, corresponding to free energy differences of ΔΔGinc ∼ 5.5–7 kcal/mol? Standard ΔΔG° values (∼0.3 kcal/mol) calculated from melting temperature measurements comparing matched vs. mismatched base pairs at duplex DNA termini are far too low to explain pol accuracy. Earlier analyses suggested that pol active-site steric constraints can amplify DNA free energy differences at the transition state (kinetic selection). A recent paper [Olson et al. (2013) J Am Chem Soc 135:1205–1208] used Vent pol to catalyze incorporations in the presence of inorganic pyrophosphate intended to equilibrate forward (polymerization) and backward (pyrophosphorolysis) reactions. A steady-state leveling off of incorporation profiles at long reaction times was interpreted as reaching equilibrium between polymerization and pyrophosphorolysis, yielding apparent ΔG° = −RT ln Keq, indicating ΔΔG° of 3.5–7 kcal/mol, sufficient to account for pol accuracy without need of kinetic selection. Here we perform experiments to measure and account for pyrophosphorolysis explicitly. We show that forward and reverse reactions attain steady states far from equilibrium for wrong incorporations such as G opposite T. Therefore, ΔΔGinc° values obtained from such steady-state evaluations of Keq are not dependent on DNA properties alone, but depend largely on constraints imposed on right and wrong substrates in the polymerase active site.

Relating DNA base-pairing in aqueous media to DNA polymerase fidelity

Controversy surrounds the perceived absence of a relationship between DNA polymerase fidelity (kinetic discrimination) and free energy changes determined from DNA melting studies (thermodynamic discrimination). Thermodynamic discrimination together with aqueous solvent effects can account for kinetic fidelities on the order of those observed experimentally.

Protein synthesis in homologous and heterologous cell-free systems from chick embryo connective tissues.

Two homologous systems for cell-free protein synthesis from chick embryo connective tissues are described. Both the skin polysomes and the wing-leg polysomes are active in collagen synthesis, but they have different requirements for optimum protein synthesis. Protein synthesis was not dependent on tissue-specific factors, since heterologous preparations of supernatant enzymes or initiation factors were able to stimulate maximum protein synthesis with each fraction of polysomes.