Protein Interactions in Antibody Diversity

在第一项研究中，DNA聚合酶i（Pol i）由于其低保真度而作为抗体基因中体细胞超突变的组分具有显著的兴趣。虽然在缺乏该酶的小鼠中对突变没有影响（菌株129，Poli 129/129），但我们假设表达但无催化活性的蛋白质可能揭示了Poli的作用。设计了在残基D126和E127处具有丙氨酸取代的突变体Pol i（Pol im），这消除了其在生化测定中的催化活性。构建敲入小鼠（Poli m/m），并检查生殖中心B细胞的突变。与Poli +/+小鼠相比，突变频率和谱没有差异。然而，因为我们之前已经证明Polzeta会产生串联突变，所以我们分析了它们的频率。令人惊讶的是，与Poli +/+和Poli 129/129小鼠相比，Poli m/m小鼠具有更少的连续突变，表明Pol im阻断Pol z接近错配末端。我们提出野生型Pol i与引物末端结合，需要被置换，以便Pol z可以合成。在第二项研究中，哺乳动物ATAD 5及其酵母同源物ELG 1负责从新合成的DNA中卸载PCNA。以前在HeLa和酵母细胞中的工作表明，ATAD 5蛋白水平的降低导致染色质结合的PCNA的积累，减缓细胞分裂和增加基因组不稳定性。在本研究中，使用来自杂合（Atad 5 +/m）小鼠的B细胞来检查细胞增殖降低对抗体多样性的影响。ATAD 5单倍不足并没有改变抗体基因体细胞高突变的频率或谱，表明DNA修复和易错DNA聚合酶eta的使用不受影响。然而，免疫的Atad 5 +/m小鼠血清IgG 1抗体降低，表明对类别转换重组的功能性影响。然后在离体刺激脾B细胞后检查这种改变的免疫应答的机制，其中Atad 5 +/m细胞在细胞周期的S期积累，并且与野生型细胞相比具有降低的增殖。这些单倍型不足的细胞经历了激活诱导的脱氨酶表达的显着下降，导致开关区DNA双链断裂和Igh基因座中的染色体间易位减少。类开关重组到几个同种型也减少了Atad 5 +/m细胞，虽然类型的末端连接途径没有受到影响。这些结果描述了DNA复制中的缺陷，其通过减少细胞分裂影响Igh重组。

In the first study, DNA polymerase iota (Pol i) has harbored significant interest as a component of somatic hypermutation in antibody genes because of its low fidelity. Although there was no effect on mutation in mice deficient for the enzyme (strain 129, Poli 129/129), we hypothesized that a protein that is expressed but catalytically inactive might unmask a role for Pol i. A mutant Pol i was designed with alanine substitutions at residues D126 and E127 (Pol im), which abolished its catalytic activity in biochemical assays. A knock-in mouse (Poli m/m) was constructed, and germinal center B cells were examined for mutations. Compared to Poli +/+ mice, the mutation frequency and spectra were not different. However, because we had previously shown that Pol zeta generates tandem mutations, we analyzed their frequency. Surprisingly, Poli m/m mice had fewer contiguous mutations compared to Poli +/+ and Poli 129/129 mice, suggesting that Pol im blocks access of Pol z to mismatched termini. We propose that wild type Pol i binds to a primer-terminus and needs to be displaced, so that Pol z can synthesize. In the second study, mammalian ATAD5 and its yeast homolog ELG1 are responsible for unloading PCNA from newly synthesized DNA. Prior work in HeLa and yeast cells showed that a decrease in ATAD5 protein levels resulted in accumulation of chromatin-bound PCNA, slowed cell division and increased genomic instability. In this study, B cells from heterozygous (Atad5+/m) mice were used to examine the effects of decreased cell proliferation on antibody diversity. ATAD5 haploinsufficiency did not change the frequency or spectrum of somatic hypermutation in antibody genes, indicating that DNA repair and error-prone DNA polymerase eta usage were unaffected. However, immunized Atad5+/m mice had decreased serum IgG1 antibodies, demonstrating a functional effect on class switch recombination. The mechanism of this altered immune response was then examined following ex vivo stimulation of splenic B cells, where Atad5+/m cells accumulated in S phase of the cell cycle and had reduced proliferation compared to wild type cells. These haploinsufficient cells underwent a significant decline in activation-induced deaminase expression, resulting in decreased switch region DNA double-strand breaks and inter-chromosomal translocations in the Igh locus. Class switch recombination to several isotypes was also reduced in Atad5+/m cells, although the types of end-joining pathways were not affected. These results describe a defect in DNA replication that affects Igh recombination via reduced cell division.