Cisneros-Aguirre Metztli, Lopezcolorado Felicia Wednesday, Ping Xiaoli, Chen Ruby, Stark Jeremy M
Department of Cancer Genetics and Epigenetics, Beckman Research Institute of the City of Hope, 1500 E Duarte Road, Duarte, CA 91010, USA.
Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, 1500 E Duarte Road, Duarte, CA 91010, USA.
iScience. 2025 May 22;28(6):112722. doi: 10.1016/j.isci.2025.112722. eCollection 2025 Jun 20.
A key step of canonical-nonhomologous end joining is synapsis of DNA double-strand break (DSB) ends, which appears to be mediated by both the DNA-PKcs dimer and XLF homodimer. We have examined this process by monitoring end joining (EJ) of blunt Cas9 DSBs, focusing on the Ku-binding factors PAXX and MRI. We found that PAXX and/or MRI are dispensable for such EJ. However, PAXX becomes important for blunt DSB EJ with disruption of either DNA-PKcs or XLF, whereas MRI becomes important only with XLF disruption. In contrast, while DNA-PKcs also suppresses short deletion mutations with microhomology, this effect is not magnified with PAXX loss. Finally, XLF loss causes an increase in larger deletions compared to DNA-PKcs inhibition, which is magnified with loss of MRI. We suggest that PAXX promotes DSB end synapsis in a manner that is partially redundant with DNA-PKcs and XLF, whereas MRI is a backup factor for XLF-mediated EJ.
经典非同源末端连接的一个关键步骤是DNA双链断裂（DSB）末端的联会，这似乎由DNA-PKcs二聚体和XLF同二聚体共同介导。我们通过监测平端Cas9 DSB的末端连接（EJ）来研究这一过程，重点关注Ku结合因子PAXX和MRI。我们发现，PAXX和/或MRI对于此类EJ是可有可无的。然而，当DNA-PKcs或XLF受到破坏时，PAXX对于平端DSB的EJ变得重要，而MRI仅在XLF受到破坏时才变得重要。相比之下，虽然DNA-PKcs也能抑制具有微同源性的短缺失突变，但这种效应不会因PAXX缺失而增强。最后，与DNA-PKcs抑制相比，XLF缺失会导致更大缺失的增加，而MRI缺失会使这种增加更加明显。我们认为，PAXX以一种与DNA-PKcs和XLF部分冗余的方式促进DSB末端联会，而MRI是XLF介导的EJ的备用因子。
