The expression of activation-induced cytidine deaminase (AID) is prerequisite to a “trifecta” of key molecular events in B cells: class-switch recombination and somatic hypermutation in humans and mice and gene conversion in chickens. Although this critically important enzyme shares common sequence motifs with apolipoprotein B mRNA-editing enzyme, and exhibits deaminase activity on free deoxycytidine in solution, it has not been shown to act on either RNA or DNA. Recent mutagenesis data in Escherichia coli suggest that AID may deaminate dC on DNA, but its putative biochemical activities on either DNA or RNA remained a mystery. Here, we show that AID catalyzes deamination of dC residues on single-stranded DNA in vitro but not on double-stranded DNA, RNA–DNA hybrids, or RNA. Remarkably, it has no measurable deaminase activity on single-stranded DNA unless pretreated with RNase to remove inhibitory RNA bound to AID. AID catalyzes dC → dU deamination activity most avidly on double-stranded DNA substrates containing a small “transcription-like” single-stranded DNA bubble, suggesting a targeting mechanism for this enigmatic enzyme during somatic hypermutation.

In Escherichia coli, cell survival and genomic stability after UV radiation depends on repair mechanisms induced as part of the SOS response to DNA damage. The early phase of the SOS response is mostly dominated by accurate DNA repair, while the later phase is characterized with elevated mutation levels caused by error-prone DNA replication. SOS mutagenesis is largely the result of the action of DNA polymerase V (pol V), which has the ability to insert nucleotides opposite various DNA lesions in a process termed translesion DNA synthesis (TLS). Pol V is a low-fidelity polymerase that is composed of UmuD′₂C and is encoded by the umuDC operon. Pol V is strictly regulated in the cell so as to avoid genomic mutation overload. RecA nucleoprotein filaments (RecA*), formed by RecA binding to single-stranded DNA with ATP, are essential for pol V-catalyzed TLS both in vivo and in vitro. This review focuses on recent studies addressing the protein composition of active DNA polymerase V, and the role of RecA protein in activating this enzyme. Based on unforeseen properties of RecA*, we describe a new model for pol V-catalyzed SOS-induced mutagenesis.

APOBEC3G (Apo3G) is a single-stranded DNA-dependent deoxycytidine deaminase, which, in the absence of the human immunodeficiency virus (HIV) viral infectivity factor, is encapsulated into HIV virions. Subsequently, Apo3G triggers viral inactivation by processively deaminating C-->U, with 3'-->5' polarity, on nascent minus-strand cDNA. Apo3G has a catalytically inactive N-terminal CD1 domain and an active C-terminal CD2 domain. Apo3G exists as monomers, dimers, tetramers, and higher order oligomers whose distributions depend on DNA substrate and salt. Here we use multiangle light scattering and atomic force microscopy to identify oligomerization states of Apo3G. A double mutant (F126A/W127A), designed to disrupt dimerization at the predicted CD1-CD1 dimer interface, predominantly converts Apo3G to a monomer that binds single-stranded DNA, Alu RNA, and catalyzes processive C-->U deaminations with 3'-->5' deamination polarity, similar to native Apo3G. The CD1 domain is essential for both processivity and polarity. We propose a structure-based model to explain the scanning and catalytic behavior of Apo3G.

APOBEC3G (A3G) restricts HIV-1 infection by catalyzing processive C → U deaminations on single-stranded DNA (ssDNA) with marked 3′ → 5′ deamination polarity. Here we show that A3G exists in oligomeric states whose composition is dictated primarily by interactions with DNA, with salt playing an important, yet secondary, role. Directional deaminations correlate with the presence of dimers, tetramers, and larger oligomers observed by atomic force microscopy, and random deaminations appear to correlate mainly with monomers. The presence of a 30-nt weakly deaminated “dead” zone located at the 3′-ssDNA end implies the presence of a preferred asymmetric direction for A3G catalysis. Single turnover reaction rates reveal a salt-dependent inhibition of C deamination toward the 3′-ssDNA region, offering a molecular basis underlying A3G deamination polarity. Presteady state analysis demonstrates rapid diffusion-limited A3G-ssDNA binding, a slower salt-dependent conformational change, possibly indicative of DNA wrapping, and long (5–15 min) protein-DNA complex lifetimes. We suggest that diverse A3G oligomerization modes contribute to the human immunodeficiency virus, type 1, proviral DNA mutational bias.