Levy M Z, Allsopp R C, Futcher A B, Greider C W, Harley C B
Department of Biochemistry, McMaster University Hamilton, Ontario, Canada.
J Mol Biol. 1992 Jun 20;225(4):951-60. doi: 10.1016/0022-2836(92)90096-3.
Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this "end-replication" problem. Immortal eukaryotic cells, including transformed human cells, apparently use telomerase, an enzyme that elongates telomeres, to overcome incomplete end-replication. However, telomerase has not been detected in normal somatic cells, and these cells lose telomeres with age. Therefore, to better understand the consequences of incomplete replication, we modeled this process for a population of dividing cells. The analysis suggests four things. First, if single-stranded overhangs generated by incomplete replication are not degraded, then mean telomere length decreases by 0.25 of a deletion event per generation. If overhangs are degraded, the rate doubles. Data showing a decrease of about 50 base-pairs per generation in fibroblasts suggest that a full deletion event is 100 to 200 base-pairs. Second, if cells senesce after 80 doublings in vitro, mean telomere length decreases about 4000 base-pairs, but one or more telomeres in each cell will lose significantly more telomeric DNA. A checkpoint for regulation of cell growth may be signalled at that point. Third, variation in telomere length predicted by the model is consistent with the abrupt decline in dividing cells at senescence. Finally, variation in length of terminal restriction fragments is not fully explained by incomplete replication, suggesting significant interchromosomal variation in the length of telomeric or subtelomeric repeats. This analysis, together with assumptions allowing dominance of telomerase inactivation, suggests that telomere loss could explain cell cycle exit in human fibroblasts.
由于DNA聚合酶需要一个不稳定的引物来启动单向5'-3'合成，除非有特殊机制绕过这个“末端复制”问题，否则每个模板链3'端的一些碱基不会被复制。包括转化的人类细胞在内的永生真核细胞显然利用端粒酶（一种延长端粒的酶）来克服不完全末端复制。然而，在正常体细胞中未检测到端粒酶，并且这些细胞会随着年龄增长而丢失端粒。因此，为了更好地理解不完全复制的后果，我们对一群分裂细胞的这个过程进行了建模。分析表明了四点。第一，如果不完全复制产生的单链悬端不被降解，那么平均端粒长度每代减少0.25个缺失事件。如果悬端被降解，速率会加倍。显示成纤维细胞每代减少约50个碱基对的数据表明一个完全缺失事件为100到200个碱基对。第二，如果细胞在体外加倍80次后衰老，平均端粒长度减少约4000个碱基对，但每个细胞中的一个或多个端粒将丢失显著更多的端粒DNA。此时可能会发出细胞生长调控的检查点信号。第三，模型预测的端粒长度变化与衰老时分裂细胞的突然减少一致。最后，末端限制片段长度的变化不能完全由不完全复制来解释，这表明端粒或亚端粒重复序列长度存在显著的染色体间变化。该分析以及允许端粒酶失活占主导地位的假设表明，端粒丢失可以解释人类成纤维细胞的细胞周期退出。
