Bj Rås Karine Ø, Sousa Mirta M L, Sharma Animesh, Fonseca Davi M, S Gaard Caroline K, Bj Rås Magnar, Otterlei Marit
Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), N-7491 Trondheim, Norway.
The Central Norway Regional Health Authority, N-7501 Stj⊘rdal, Norway.
Nucleic Acids Res. 2017 Aug 21;45(14):8291-8301. doi: 10.1093/nar/gkx476.
Base lesions in DNA can stall the replication machinery or induce mutations if bypassed. Consequently, lesions must be repaired before replication or in a post-replicative process to maintain genomic stability. Base excision repair (BER) is the main pathway for repair of base lesions and is known to be associated with DNA replication, but how BER is organized during replication is unclear. Here we coupled the iPOND (isolation of proteins on nascent DNA) technique with targeted mass-spectrometry analysis, which enabled us to detect all proteins required for BER on nascent DNA and to monitor their spatiotemporal orchestration at replication forks. We demonstrate that XRCC1 and other BER/single-strand break repair (SSBR) proteins are enriched in replisomes in unstressed cells, supporting a cellular capacity of post-replicative BER/SSBR. Importantly, we identify for the first time the DNA glycosylases MYH, UNG2, MPG, NTH1, NEIL1, 2 and 3 on nascent DNA. Our findings suggest that a broad spectrum of DNA base lesions are recognized and repaired by BER in a post-replicative process.
DNA中的碱基损伤会使复制机制停滞，若绕过这些损伤则会诱发突变。因此，损伤必须在复制前或复制后过程中得到修复，以维持基因组稳定性。碱基切除修复（BER）是修复碱基损伤的主要途径，已知其与DNA复制相关，但尚不清楚BER在复制过程中是如何组织的。在这里，我们将iPOND（新生DNA上蛋白质的分离）技术与靶向质谱分析相结合，这使我们能够检测新生DNA上BER所需的所有蛋白质，并监测它们在复制叉处的时空协调。我们证明，在未受应激的细胞中，XRCC1和其他BER/单链断裂修复（SSBR）蛋白在复制体中富集，这支持了复制后BER/SSBR的细胞能力。重要的是，我们首次在新生DNA上鉴定出DNA糖基化酶MYH、UNG2、MPG、NTH1、NEIL1、2和3。我们的研究结果表明，在复制后过程中，BER可识别并修复广泛的DNA碱基损伤。
