Institute of Molecular Genetics IGM-CNR 'Luigi Luca Cavalli-Sforza', via Abbiategrasso 207, I-27100 Pavia, Italy.
Nucleic Acids Res. 2024 Oct 28;52(19):11641-11658. doi: 10.1093/nar/gkae731.
R-loops are three-stranded RNA-DNA hybrid structures that play important regulatory roles, but excessive or deregulated R-loops formation can trigger DNA damage and genome instability. Digestion of R-loops is mainly relying on the action of two specialized ribonucleases: RNaseH1 and RNaseH2. RNaseH2 is the main enzyme carrying out the removal of misincorporated rNMPs during DNA replication or repair, through the Ribonucleotide Excision Repair (RER) pathway. We have recently shown that the human RNA helicase DDX3X possessed RNaseH2-like activity, being able to substitute RNaseH2 in reconstituted RER reactions. Here, using synthetic R-loop mimicking substrates, we could show that human DDX3X alone was able to both displace and degrade the ssRNA strand hybridized to DNA. Moreover, DDX3X was found to physically interact with human RNaseH2. Such interaction suppressed the nuclease and helicase activities of DDX3X, but stimulated severalfold the catalytic activity of the trimeric RNaseH2, but not of RNaseH1. Finally, silencing of DDX3X in human cells caused accumulation of RNA-DNA hybrids and phosphorylated RPA foci. These results support a role of DDX3X as a scaffolding protein and auxiliary factor for RNaseH2 during R-loop degradation.
R 环是三链 RNA-DNA 杂交结构，在调节作用中具有重要意义，但过多或失调的 R 环形成会引发 DNA 损伤和基因组不稳定。R 环的消化主要依赖于两种专门的核糖核酸酶的作用：RNaseH1 和 RNaseH2。RNaseH2 是在 DNA 复制或修复过程中去除错误掺入的 rNMP 的主要酶，通过核苷酸切除修复 (RER) 途径。我们最近表明，人类 RNA 解旋酶 DDX3X 具有 RNaseH2 样活性，能够在重建的 RER 反应中替代 RNaseH2。在这里，使用合成的 R 环模拟底物，我们可以证明人类 DDX3X 本身能够置换和降解与 DNA 杂交的 ssRNA 链。此外，发现 DDX3X 与人类 RNaseH2 发生物理相互作用。这种相互作用抑制了 DDX3X 的核酸酶和解旋酶活性，但刺激了三聚体 RNaseH2 的催化活性几倍，但不刺激 RNaseH1 的催化活性。最后，在人类细胞中沉默 DDX3X 会导致 RNA-DNA 杂交体和磷酸化 RPA 焦点的积累。这些结果支持 DDX3X 作为 R 环降解过程中 RNaseH2 的支架蛋白和辅助因子的作用。
