Somatic hypermutation is initiated by activation-induced cytidine deaminase (AID), and occurs in several kilobases of DNA around rearranged immunoglobulin variable (V) genes and switch (S) sites before constant genes. AID deaminates cytosine to uracil, which can produce mutations of C:G nucleotide pairs, and the mismatch repair protein Msh2 participates in generating substitutions of downstream A:T pairs. Msh2 is always found as a heterodimer with either Msh3 or Msh6, so it is important to know which one is involved. Therefore, we sequenced V and S regions from Msh3- and Msh6-deficient mice and compared mutations to those from wild-type mice. Msh6-deficient mice had fewer substitutions of A and T bases in both regions and reduced heavy chain class switching, whereas Msh3-deficient mice had normal antibody responses. This establishes a role for the Msh2-Msh6 heterodimer in hypermutation and switch recombination. When the positions of mutation were mapped, several focused peaks were found in Msh6 −/− clones, whereas mutations were dispersed in Msh3 −/− and wild-type clones. The peaks occurred at either G or C in WGCW motifs (W = A or T), indicating that C was mutated on both DNA strands. This suggests that AID has limited entry points into V and S regions in vivo, and subsequent mutation requires Msh2-Msh6 and DNA polymerase.

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.

Hypermutation in immunoglobulin genes produces a high frequency of substitutions of all four bases, which are likely generated by low-fidelity DNA polymerases. Indeed, humans deficient for DNA polymerase (pol) have decreased substitutions of A⅐T base pairs in variable and switch regions. To study the role of pol in a genetically tractable system, we created mice lacking pol . B cells from Polh ؊/؊ mice produced normal amounts of IgG, indicating that pol does not affect class switch recombination. Similar to their human counterparts, variable and switch regions from Polh ؊/؊ mice had fewer substitutions of A⅐T base pairs and correspondingly more mutations of C⅐G base pairs, which firmly establishes a central role for pol in hypermutation. Notably, the location and types of substitutions differ markedly from those in Msh6 ؊/؊ clones, which also have fewer A⅐T mutations. The data suggest that pol preferentially synthesizes a repair patch on the nontranscribed strand, whereas MSH6 functions to generate the patch.class switch recombination ͉ somatic hypermutation ͉ low-fidelity DNA polymerase ͉ mismatch repair I mmunoglobulin (Ig) genes undergo a high frequency of somatic hypermutation during B cell activation. The mutations, which are predominantly nucleotide substitutions, are introduced into Ϸ2-kb regions of DNA containing variable (V) region genes and switch (S) regions that precede each constant region gene. Mutations in V gene exons can generate high-affinity antibodies that bind to antigen, whereas mutations in S region introns are linked to class switch recombination that produces different isotypes (reviewed in refs. 1-3). Hypermutation and switching are both initiated by the activation-induced cytidine deaminase protein (4, 5), which deaminates dC to dU in DNA (6-8). The dU lesion likely generates mutation in two phases, as originally proposed by Neuberger and colleagues (6). In phase 1, the dU lesion could generate mutations of C bases, or of G bases if C is deaminated on the complementary strand. For example, dU could remain in the DNA and be copied by a high-fidelity DNA polymerase (pol) to produce transitions of C⅐G. Alternatively, dU could be removed by uracil DNA glycosylase, and the abasic site could be copied or repaired by a low-fidelity pol to produce transitions and transversions of C⅐G.In phase 2, the dU lesion could initiate a repair patch and generate mutations of neighboring bases, including A and T. In this pathway, the MSH2-MSH6 heterodimer (9-11) could bind to the U⅐G mispair (12, 13), an endonuclease could nick the DNA, and exonuclease 1 (14) would create a gap near the original dU lesion. The gap would then be filled in by a low-fidelity pol, and the types of substitutions would reflect the specificity of the pol. A plethora of pols have been studied for their role in hypermutation in animals and cells:

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Activation-induced cytosine deaminase preferentially deaminates C in DNA on the nontranscribed strand in vitro, which theoretically should produce a large increase in mutations of C during hypermutation of immunoglobulin genes. However, a bias for C mutations has not been observed among the mutations in variable genes. Therefore, we examined mutations in the μ and γ switch regions, which can form stable secondary structures, to look for C mutations. To further simplify the pattern, mutations were studied in the absence of DNA polymerase (pol) η, which may produce substitutions of nucleotides downstream of C. DNA from lymphocytes of patients with xeroderma pigmentosum variant (XP-V) disease, whose polymerase η is defective, had the same frequency of switching to all four γ isotypes and hypermutation in μ-γ switch sites (0.5% mutations per basepair) as control subjects. There were fewer mutations of A and T bases in the XP-V clones, similar to variable gene mutations from these patients, which confirms that polymerase η produces substitutions opposite A and T. Most importantly, the absence of polymerase η revealed an increase in C mutations on the nontranscribed strand. This data shows for the first time that C is preferentially mutated in vivo and pol η generates hypermutation in the μ and γ switch regions.

Almost 150 years after the first autologous blood transfusion was reported, intraoperative blood salvage has become an important method of blood conservation. The primary goal of autologous transfusion is to reduce or avoid allogeneic red blood cell transfusion and the associated risks and costs. Autologous salvaged blood does not result in immunological challenge and its consequences, provides a higher quality red blood cell that has not been subjected to the adverse effects of blood storage, and can be more cost-effective than allogeneic blood when used for carefully selected surgical patients. Cardiac, orthopaedic and vascular surgery procedures with large anticipated blood loss can clearly benefit from the use of cell salvage. There are safety concerns in cases with gross bacterial contamination. There are theoretical safety concerns in obstetrical and cancer surgery; however, careful cell washing as well as leucoreduction filters makes for a safer autologous transfusion in these circumstances. Further studies are needed to determine whether oncologic outcomes are impacted by transfusing salvaged blood during cancer surgery. In this new era of patient blood management, where multimodal methods of reducing dependence on allogeneic blood are becoming commonplace, autologous blood salvage remains a valuable tool for perioperative blood conservation. Future studies will be needed to best determine how and when cell salvage should be utilized along with newer blood conservation measures.

The endocellular microbe Wolbachia pipientis infects a wide variety of invertebrate species, in which its presence is closely linked to a form of reproductive failure termed cytoplasmic incompatibility (CI). CI renders infected males unable to father offspring when mated to uninfected females. Because CI can dramatically affect fitness in natural populations, mechanisms that abate CI can have equally large impacts on fitness. We have discovered that repeated copulation by Wolbachia-infected male Drosophila simulans significantly diminishes CI. Repeated copulation does not prevent Wolbachia from populating developing spermatids, but may reduce the time during spermatogenesis when Wolbachia can express CI. This restoration of fertility in premated infected males could have important implications for Wolbachia transmission and persistence in nature and for its exploitation as an agent of biological pest control.

Genetic inactivation of the genes encoding several low-fidelity DNA polymerases indicates that DNA polymerase ζ inserts tandem double-base substitutions in the immunoglobulin variable region in mouse B cells.