Jia Xiaomeng, Inman James T, Singh Anupam, Patel Smita S, Wang Michelle D
Howard Hughes Medical Institute, Cornell University, Ithaca, NY 14853, USA.
Department of Physics & LASSP, Cornell University, Ithaca, NY 14853, USA.
bioRxiv. 2024 Oct 10:2024.10.09.617451. doi: 10.1101/2024.10.09.617451.
Replication of DNA requires the parental DNA to be unwound to allow the genetic information to be faithfully duplicated by the replisome. While this function is usually shared by a host of proteins in the replisome, notably DNA polymerase (DNAP) and helicase, the consequence of DNAP synthesizing DNA while decoupled from helicase remains not well understood. The unwinding of downstream DNA poses significant stress to DNAP, and the interaction between DNAP and the replication fork may affect replication restart. In this work, we examined the consequences of DNAP working against the stress of the DNA replication fork. We found that prolonged exposure of DNAP to the stress of the replication fork inactivates replication. Surprisingly, replication inactivation was often accompanied by a strong DNAP interaction with the leading and lagging strands at the fork, locking the fork in place. We demonstrated that fork locking is a consequence of DNAP forward translocation, and the exonuclease activity of DNAP, which allows DNAP to move in reverse, is essential in protecting the fork from inactivation. Furthermore, we found the locking configuration is not reversible by the subsequent addition of helicase. Collectively, this study provides a deeper understanding of the DNAP-fork interaction and mechanism in keeping the replication fork active during replication stress.
DNA复制需要解开亲代DNA，以便复制体忠实地复制遗传信息。虽然复制体中的许多蛋白质通常共同承担这一功能，特别是DNA聚合酶（DNAP）和解旋酶，但DNAP在与解旋酶分离的情况下合成DNA的后果仍未得到充分理解。下游DNA的解旋给DNAP带来了巨大压力，DNAP与复制叉之间的相互作用可能会影响复制重新启动。在这项工作中，我们研究了DNAP抵抗DNA复制叉压力的后果。我们发现，DNAP长时间暴露于复制叉的压力会使复制失活。令人惊讶的是，复制失活常常伴随着DNAP与复制叉处的前导链和后随链强烈相互作用，从而将复制叉锁定在原位。我们证明，复制叉锁定是DNAP向前易位的结果，而DNAP的核酸外切酶活性允许DNAP反向移动，这对于保护复制叉不被失活至关重要。此外，我们发现后续添加解旋酶并不能使锁定构型逆转。总的来说，这项研究为DNAP与复制叉的相互作用以及在复制压力期间保持复制叉活跃的机制提供了更深入的理解。
