Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.
Proc Natl Acad Sci U S A. 2013 Aug 13;110(33):E3090-9. doi: 10.1073/pnas.1304231110. Epub 2013 Jul 29.
Base oxidation by endogenous and environmentally induced reactive oxygen species preferentially occurs in replicating single-stranded templates in mammalian genomes, warranting prereplicative repair of the mutagenic base lesions. It is not clear how such lesions (which, unlike bulky adducts, do not block replication) are recognized for repair. Furthermore, strand breaks caused by base excision from ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would generate double-strand breaks during replication, which are not experimentally observed. NEIL1, whose deficiency causes a mutator phenotype and is activated during the S phase, is present in the DNA replication complex isolated from human cells, with enhanced association with DNA in S-phase cells and colocalization with replication foci containing DNA replication proteins. Furthermore, NEIL1 binds to 5-hydroxyuracil, the oxidative deamination product of C, in replication protein A-coated ssDNA template and inhibits DNA synthesis by DNA polymerase δ. We postulate that, upon encountering an oxidized base during replication, NEIL1 initiates prereplicative repair by acting as a "cowcatcher" and preventing nascent chain growth. Regression of the stalled replication fork, possibly mediated by annealing helicases, then allows lesion repair in the reannealed duplex. This model is supported by our observations that NEIL1, whose deficiency slows nascent chain growth in oxidatively stressed cells, is stimulated by replication proteins in vitro. Furthermore, deficiency of the closely related NEIL2 alone does not affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of NEIL1-mediated prereplicative repair of oxidized bases in the replicating strand, with NEIL2 providing a backup function.
内源性和环境诱导的活性氧优先在哺乳动物基因组中复制的单链模板上进行基底氧化，这就需要在复制前修复这些诱变碱基损伤。目前尚不清楚如何识别这些损伤（与大体积加合物不同，这些损伤不会阻止复制）进行修复。此外，DNA 糖苷酶（包括 Nei 样（NEIL）1）从 ssDNA 中碱基切除会在复制过程中产生双链断裂，但这种断裂在实验中并未观察到。NEIL1 的缺乏会导致突变体表型，并且在 S 期被激活，它存在于从人细胞中分离出的 DNA 复制复合物中，在 S 期细胞中与 DNA 的结合增强，并与含有 DNA 复制蛋白的复制焦点共定位。此外，NEIL1 结合到复制蛋白 A 覆盖的 ssDNA 模板中 C 的氧化脱氨产物 5-羟尿嘧啶，并抑制 DNA 聚合酶 δ的 DNA 合成。我们假设，在复制过程中遇到氧化碱基时，NEIL1 通过充当“挡车器”来启动复制前修复，防止新生链的生长。停滞的复制叉的倒退，可能由退火解旋酶介导，然后允许在重新退火的双链体中修复损伤。我们的观察结果支持了这一模型，即在氧化应激细胞中，NEIL1 的缺乏会减缓新生链的生长，但其在体外受到复制蛋白的刺激。此外，单独缺乏密切相关的 NEIL2 不会影响链延伸，但 NEIL1/2 联合缺乏会进一步抑制 DNA 复制。这些结果支持了 NEIL1 介导的复制链中氧化碱基的复制前修复机制，NEIL2 提供了备份功能。
