Memory B cells formed in response to microbial antigens provide immunity to later infections; however, the inability to detect rare endogenous antigen-specific cells limits current understanding of this process. Using an antigen-based technique to enrich these cells, we found that immunization with a model protein generated B memory cells that expressed isotype-switched immunoglobulins (swIg) or retained IgM. The more numerous IgM+ cells were longer lived than the swIg+ cells. However, swIg+ memory cells dominated the secondary response due to the capacity to become activated in the presence of neutralizing serum Ig. Thus, we propose that memory relies on swIg+ cells until they disappear and serum Ig falls to a low level, in which case memory resides with durable IgM+ reserves.

In response to an assault by foreign organisms, peripheral B cells can change their antibody affinity and isotype by somatically mutating their genomic DNA. The ability of a cell to modify its DNA is exceptional in light of the potential consequences of genetic alterations to cause human disease and cancer. Thus, as expected, this mechanism of antibody diversity is tightly regulated and coordinated through one protein, activation induced deaminase (AID). AID produces diversity by converting cytosine to uracil within the immunoglobulin loci. The deoxyuracil residue is mutagenic when paired with deoxyguanosine, since it mimics thymidine during DNA replication. Additionally, B cells can manipulate the DNA repair pathways so that deoxyuracils are not faithfully repaired. Therefore, an intricate balance exists which is regulated at multiple stages to promote mutation of immunoglobulin genes, while retaining integrity of the rest of the genome. Here we discuss and summarize the current understanding of how AID functions to cause somatic hypermutation.

Enzymes of the AID/APOBEC family, characterized by the targeted deamination of cytosine to generate uracil within DNA, mediate numerous critical immune responses. One family member, activation-induced cytidine deaminase (AID), selectively introduces uracil into antibody variable and switch regions, promoting antibody diversity through somatic hypermutation or class switching. Other family members, including APOBEC3F and APOBEC3G, play an important role in retroviral defense by acting on viral reverse transcripts. These enzymes are distinguished from one another by targeting cytosine within different DNA sequence contexts; however, the reason for these differences is not known. Here, we report the identification of a recognition loop of 9 -11 amino acids that contributes significantly to the distinct sequence motifs of individual family members. When this recognition loop is grafted from the donor APOBEC3F or 3G proteins into the acceptor scaffold of AID, the mutational signature of AID changes toward that of the donor proteins. These loop-graft mutants of AID provide useful tools for dissecting the biological impact of DNA sequence preferences upon generation of antibody diversity, and the results have implications for the evolution and specialization of the AID/APOBEC family.The polynucleotide cytosine deaminases have been identified as key contributors to both the adaptive and innate immune responses to pathogens. This enzyme family includes activation-induced cytidine deaminase (AID), 2 which initiates antibody diversification, and the APOBEC3 enzymes, which inhibit retroviral infection (1, 2). Although the enzymes share a common chemical mechanism for catalyzing the deamination of cytosine to generate mutagenic uracil within DNA, they have distinct biological functions based on differences in their expression, localization, and DNA sequence specificity (2, 3).AID is a B-cell-specific enzyme whose catalytic action on mammalian antibody genes initiates somatic hypermutation and class switch recombination (4, 5). The uracil deamination product is processed by familiar DNA repair proteins, including uracil DNA N-glycosylase (UNG) and mismatch repair enzymes. However, the cellular environment within activated B-cells promotes mutagenic processing by these pathways, resulting in point mutations that ultimately enhance antibody affinity in somatic hypermutation or promote clustered double-strand breaks that alter antibody isotype in class switch recombination (6, 7).The activity of AID is restricted to relatively small 3-kb regions within the immunoglobulin locus around rearranged variable genes and heavy chain switch regions. Within these regions, the targeting of AID is partially dictated by its DNA sequence preferences. For example, it has been noted that the mutable complementarity determining regions from variable gene segments have an abundance of AGC codons as compared with adjacent framework regions that function in stabilizing antibody structure (8). In vivo, somatic hypermutation is focused on WRCW (W ϭ A...

The actions of Escherichia coli DNA Polymerase IV (Pol IV) in mutagenesis are managed by its interaction with the ␤ sliding clamp. In the structure reported by Bunting et al. [EMBO J (2003) 22:5883-5892], the C-tail of Pol IV contacts a hydrophobic cleft on the clamp, while residues V303-P305 reach over the dimer interface to contact the rim of the adjacent clamp protomer. Using mutant forms of these proteins impaired for either the rim or the cleft contacts, we determined that the rim contact was dispensable for Pol IV replication in vitro, while the cleft contact was absolutely required. Using an in vitro assay to monitor Pol III*-Pol IV switching, we determined that a single cleft on the clamp was sufficient to support the switch, and that both the rim and cleft contacts were required. Results from genetic experiments support a role for the cleft and rim contacts in Pol IV function in vivo. Taken together, our findings challenge the toolbelt model and suggest instead that Pol IV contacts the rim of the clamp adjacent to the cleft that is bound by Pol III* before gaining control of the same cleft that is bound by Pol III*.mutagenesis ͉ toolbelt ͉ translesion DNA synthesis ͉ Pol IV ͉ DinB E ndogenous and exogenous agents continuously damage cellular DNA. As a result, faithful duplication of an organism's genetic information requires both high fidelity DNA polymerases (Pols), as well as a multitude of DNA repair and DNA damage tolerance mechanisms (reviewed in ref.

Activation-induced deaminase (AID) initiates diversity of immunoglobulin genes through deamination of cytosine to uracil. Two opposing models have been proposed for the deamination of DNA or RNA by AID. Although most data support DNA deamination, there is no physical evidence of uracil residues in immunoglobulin genes. Here we demonstrate their presence by determining the sensitivity of DNA to digestion with uracil DNA glycosylase (UNG) and abasic endonuclease. Using several methods of detection, we identified uracil residues in the variable and switch regions. Uracil residues were generated within 24 h of B cell stimulation, were present on both DNA strands and were found to replace mainly cytosine bases. Our data provide direct evidence for the model that AID functions by deaminating cytosine residues in DNA.

Repetitive DNA sequences in the immunoglobulin switch μ region form RNA-containing secondary structures and undergo hypermutation by activation-induced deaminase (AID). To examine how DNA structure affects transcription and hypermutation, we mapped the position of RNA polymerase II molecules and mutations across a 5-kb region spanning the intronic enhancer to the constant μ gene. For RNA polymerase II, the distribution was determined by nuclear run-on and chromatin immunoprecipitation assays in B cells from uracil-DNA glycosylase (UNG)–deficient mice stimulated ex vivo. RNA polymerases were found at a high density in DNA flanking both sides of a 1-kb repetitive sequence that forms the core of the switch region. The pileup of polymerases was similar in unstimulated and stimulated cells from Ung−/− and Aid−/−Ung−/− mice but was absent in cells from mice with a deletion of the switch region. For mutations, DNA was sequenced from Ung−/− B cells stimulated in vivo. Surprisingly, mutations of A nucleotides, which are incorporated by DNA polymerase η, decreased 10-fold before the repetitive sequence, suggesting that the polymerase was less active in this region. We propose that altered DNA structure in the switch region pauses RNA polymerase II and limits access of DNA polymerase η during hypermutation.

Cockayne syndrome is a neurodegenerative accelerated aging disorder caused by mutations in the CSA or CSB genes. Although the pathogenesis of Cockayne syndrome has remained elusive, recent work implicates mitochondrial dysfunction in the disease progression. Here, we present evidence that loss of CSA or CSB in a neuroblastoma cell line converges on mitochondrial dysfunction caused by defects in ribosomal DNA transcription and activation of the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1). Indeed, inhibition of ribosomal DNA transcription leads to mitochondrial dysfunction in a number of cell lines. Furthermore, machine-learning algorithms predict that diseases with defects in ribosomal DNA (rDNA) transcription have mitochondrial dysfunction, and, accordingly, this is found when factors involved in rDNA transcription are knocked down. Mechanistically, loss of CSA or CSB leads to polymerase stalling at non-B DNA in a neuroblastoma cell line, in particular at G-quadruplex structures, and recombinant CSB can melt G-quadruplex structures. Indeed, stabilization of G-quadruplex structures activates PARP1 and leads to accelerated aging in Caenorhabditis elegans. In conclusion, this work supports a role for impaired ribosomal DNA transcription in Cockayne syndrome and suggests that transcription-coupled resolution of secondary structures may be a mechanism to repress spurious activation of a DNA damage response.Cockayne syndrome | aging | polymerase I transcription | nucleolus | CSA | CSB

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