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.

DNA polymerase is the critical enzyme maintaining genetic integrity during DNA replication. Individual steps in the replication process that contribute to DNA synthesis fidelity include nucleotide insertion, exonucleolytic proofreading, and binding to and elongation of matched and mismatched primer termini. Each process has been investigated using polyacrylamide gel electrophoresis (PAGE) to resolve 32P-labeled primer molecules extended by polymerase. We describe how integrated gel band intensities can be used to obtain site-specific velocities for addition of correct and incorrect nucleotides, extending mismatched compared to correctly matched primer termini and measuring polymerase dissociation rates and equilibrium DNA binding constants. The analysis is based on steady-state "single completed hit conditions", where polymerases encounter many DNA molecules but where each DNA encounters an enzyme at most once. Specific topics addressed include nucleotide misinsertion, mismatch extension, exonucleolytic proofreading, single nucleotide discrimination using PCR, promiscuous mismatch extension by HIV-1 and AMV reverse transcriptases, sequence context effects on fidelity and polymerase dissociation, structural and kinetic properties of mispairs relating to fidelity, error avoidance mechanisms, kinetics of copying template lesions, the "A-rule" for insertion at abasic template lesions, an interesting exception to the "A-rule", thermodynamic and kinetic determinants of base pair discrimination by polymerases.

The relation between DNA polymerase fidelity and base pairing stability is investigated by using DNA primer-template duplexes that contain a common 9-base template sequence but have either correct (APT) or incorrect (G-T, COT, T-T) base pairs at the primer 3' terminus. Thermal melting and enzyme kinetic measurements are compared for each kind of terminus. Analysis of melting temperatures finds that differences between the free energy changes upon disso- energies of dissociation of correct and incorrect base pairs account for nucleotide insertion fidelity? To address these questions, a thermodynamic analysis (3) is made of melting data for oligonucleotide duplexes containing matched and mismatched template-primer termini. The thermodynamic measurements are compared with enzyme kinetic data obtained with the same DNA sequences under the same conditions, for right and wrong nucleotide insertions (4), and for elongation from matched and mismatched template-primer termini. MATERIALS AND METHODSPurified Drosophila DNA polymerase a holoenzyme (5) was a generous gift of I. R. Lehman (Stanford University, Stanford, CA). Four versions of a 20-base DNA primer (5'-TGATATTCACAACGAATGGN-3'), where N = A, C, G, or T), complementary in sequence (except for terminal base N) to bases 2242-2222 in wild-type M13 DNA (6), were synthesized by conventional solid-phase methods. The template was single-stranded DNA isolated from wild-type M13 phage grown in Escherichia coli strain JM103. Each primer was labeled at the 5' end with 32P using [y-32P]ATP (4500 Ci/ mmol; 1 Ci = 37 GBq) purchased from ICN Radiochemicals and T4 polynucleotide kinase from United States Biochemicals, Cleveland, OH. Procedures for primer 5'-end-labeling and hybridizing to template were the same as described (4).Synthetic DNA duplexes used in melting experiments, representing the last 9 base pairs in the primer-template complexes and differing only in the terminal base pair (NOT), were prepared by annealing equimolar amounts of the component 9-base strands synthesized in the same way as primers.DNA Polymerase Reactions. To measure extension rates at primer 3' ends (N opposite T), with dTTP as substrate for addition of T opposite A, reactions were carried out in the same way with each of the four 5'-end-labeled primers hybridized to M13 template as illustrated in Fig. lb

Lengthy expansions of trinucleotide repeats are found in DNA of patients suffering severe neurodegenerative age-related diseases. Using a synthetic self-priming DNA, containing CAG and CTG repeats implicated in Huntington's disease and several other neurological disorders, we measure the equilibrium distribution of hairpin folding and generate triplet repeat expansions by polymerase-catalyzed extensions of the hairpin folds. Expansions occur by slippage in steps of two CAG triplets when the self-priming sequence (CTG) 16 (CAG) 4 is extended with proofreading-defective Klenow fragment (KF exo ؊ ) from Escherichia coli DNA polymerase I. Slippage by two triplets is 20 times more frequent than by one triplet, in accordance with our finding that hairpin loops with even numbers of triplets are 1-2 kcal/mol more stable than their odd-numbered counterparts. By measuring triplet repeat expansions as they evolve over time, individual rate constants for expansion from 4 to 18 CAG repeats are obtained. An empirical expression is derived from the data, enabling the prediction of slippage rates from the ratio of hairpin CTG/CTG interactions to CAG/CTG interactions. Slippage is initiated internally in the hairpin folds in preference to melting inward from the 3 terminus. The same triplet expansions are obtained using proofreading-proficient KF exo ؉

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