Department of Cancer Research and Molecular Medicine, Faculty of Medicine, the FUGE Proteomics Node, Norwegian University of Science and Technology, N-7006 Trondheim, Norway.
DNA Repair (Amst). 2012 Jun 1;11(6):559-69. doi: 10.1016/j.dnarep.2012.03.006. Epub 2012 Apr 20.
In human cell nuclei, UNG2 is the major uracil-DNA glycosylase initiating DNA base excision repair of uracil. In activated B cells it has an additional role in facilitating mutagenic processing of AID-induced uracil at Ig loci and UNG-deficient patients develop hyper-IgM syndrome characterized by impaired class-switch recombination and disturbed somatic hypermutation. How UNG2 is recruited to either error-free or mutagenic uracil processing remains obscure, but likely involves regulated interactions with other proteins. The UNG2 N-terminal domain contains binding motifs for both proliferating cell nuclear antigen (PCNA) and replication protein A (RPA), but the relative contribution of these interactions to genomic uracil processing is not understood. Interestingly, a heterozygous germline single-nucleotide variant leading to Arg88Cys (R88C) substitution in the RPA-interaction motif of UNG2 has been observed in humans, but with unknown functional relevance. Here we demonstrate that UNG2-R88C protein is expressed from the variant allele in a lymphoblastoid cell line derived from a heterozygous germ line carrier. Enzyme activity as well as localization in replication foci of UNG2-R88C was similar to that of WT. However, binding to RPA was essentially abolished by the R88C substitution, whereas binding to PCNA was unaffected. Moreover, we show that disruption of the PCNA-binding motif impaired recruitment of UNG2 to S-phase replication foci, demonstrating that PCNA is a major factor for recruitment of UNG2 to unperturbed replication forks. Conversely, in cells treated with hydroxyurea, RPA mediated recruitment of UNG2 to stalled replication forks independently of functional PCNA binding. Modulation of PCNA- versus RPA-binding may thus constitute a functional switch for UNG2 in cells subsequent to genotoxic stress and potentially also during the processing of uracil at the immunoglobulin locus in antigen-stimulated B cells.
在人类细胞核中，UNG2 是启动尿嘧啶-DNA 糖基化酶修复尿嘧啶的主要尿嘧啶-DNA 糖苷酶。在活化的 B 细胞中，它在促进 AID 诱导的 Ig 基因座中尿嘧啶的诱变加工中具有额外的作用，UNG 缺陷患者会发展为高免疫球蛋白 M 综合征，其特征为类别转换重组受损和体细胞超突变紊乱。UNG2 如何被招募到无错误或诱变的尿嘧啶加工仍然不清楚，但可能涉及与其他蛋白质的调节相互作用。UNG2 N 端结构域包含与增殖细胞核抗原（PCNA）和复制蛋白 A（RPA）的结合基序，但这些相互作用对基因组尿嘧啶加工的相对贡献尚不清楚。有趣的是，在人类中观察到 UNG2 中 RPA 相互作用基序的 Arg88Cys（R88C）取代导致杂合生殖系单核苷酸变异，但功能相关性未知。在这里，我们证明 UNG2-R88C 蛋白从杂合生殖系载体衍生的淋巴母细胞系中的变体等位基因表达。UNG2-R88C 的酶活性以及在复制焦点中的定位与 WT 相似。然而，R88C 取代基本上消除了与 RPA 的结合，而对与 PCNA 的结合没有影响。此外，我们表明，破坏 PCNA 结合基序会损害 UNG2 向 S 期复制焦点的募集，这表明 PCNA 是将 UNG2 募集到未受干扰的复制叉的主要因素。相反，在用羟基脲处理的细胞中，RPA 介导 UNG2 向停滞的复制叉募集，而无需功能性 PCNA 结合。因此，PCNA 与 RPA 结合的调节可能构成细胞中 UNG2 在遗传毒性应激后的功能开关，并且在抗原刺激的 B 细胞中免疫球蛋白基因座中尿嘧啶处理时也可能如此。
