Bentley Johanne, Diggle Christine P, Harnden Patricia, Knowles Margaret A, Kiltie Anne E
Cancer Research UK Clinical Centre, St James's University Hospital, Leeds, LS9 7TF, UK.
Nucleic Acids Res. 2004 Oct 5;32(17):5249-59. doi: 10.1093/nar/gkh842. Print 2004.
In human cells DNA double strand breaks (DSBs) can be repaired by the non-homologous end-joining (NHEJ) pathway. In a background of NHEJ deficiency, DSBs with mismatched ends can be joined by an error-prone mechanism involving joining between regions of nucleotide microhomology. The majority of joins formed from a DSB with partially incompatible 3' overhangs by cell-free extracts from human glioblastoma (MO59K) and urothelial (NHU) cell lines were accurate and produced by the overlap/fill-in of mismatched termini by NHEJ. However, repair of DSBs by extracts using tissue from four high-grade bladder carcinomas resulted in no accurate join formation. Junctions were formed by the non-random deletion of terminal nucleotides and showed a preference for annealing at a microhomology of 8 nt buried within the DNA substrate; this process was not dependent on functional Ku70, DNA-PK or XRCC4. Junctions were repaired in the same manner in MO59K extracts in which accurate NHEJ was inactivated by inhibition of Ku70 or DNA-PK(cs). These data indicate that bladder tumour extracts are unable to perform accurate NHEJ such that error-prone joining predominates. Therefore, in high-grade tumours mismatched DSBs are repaired by a highly mutagenic, microhomology-mediated, alternative end-joining pathway, a process that may contribute to genomic instability observed in bladder cancer.
在人类细胞中，DNA双链断裂（DSB）可通过非同源末端连接（NHEJ）途径进行修复。在NHEJ缺陷的背景下，末端错配的DSB可通过一种易错机制连接，该机制涉及核苷酸微同源区域之间的连接。由人胶质母细胞瘤（MO59K）和尿路上皮（NHU）细胞系的无细胞提取物对具有部分不兼容3'突出端的DSB形成的大多数连接是准确的，并且是由NHEJ对错配末端进行重叠/填充产生的。然而，使用来自四种高级别膀胱癌组织的提取物对DSB进行修复，未形成准确的连接。连接是通过末端核苷酸的非随机缺失形成的，并且显示出倾向于在DNA底物内埋藏的8个核苷酸的微同源处退火；这个过程不依赖于功能性Ku70、DNA-PK或XRCC4。在通过抑制Ku70或DNA-PK（cs）使准确的NHEJ失活的MO59K提取物中，连接以相同的方式进行修复。这些数据表明，膀胱肿瘤提取物无法进行准确的NHEJ，从而易错连接占主导。因此，在高级别肿瘤中，错配的DSB通过高度诱变的、微同源介导的替代末端连接途径进行修复，这一过程可能导致在膀胱癌中观察到的基因组不稳定。
