Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA, 91010-3000, USA.
J. Craig Venter Institute, San Diego, CA, 92037, USA.
Sci Rep. 2020 Oct 12;10(1):17033. doi: 10.1038/s41598-020-74095-9.
The synthesis of mitochondrial DNA (mtDNA) is a complex process that involves the formation and resolution of unusual nucleic acid structures, such as RNA:DNA hybrids. However, little is known about the enzymes that regulate these processes. RECQ4 is a DNA replication factor important for mtDNA maintenance, and here, we unveil a role of human RECQ4 in regulating the formation and resolution of mitochondrial RNA:DNA hybrids. Mitochondrial membrane protein p32 can block mtDNA synthesis by restricting RECQ4 mitochondrial localization via protein-protein interaction. We found that the interaction with p32 was disrupted not only by the previously reported cancer-associated RECQ4 mutation, del(A420-A463), but also by a clinical mutation of the adjacent residue, P466L. Surprisingly, although P466L mutant was present in the mitochondria at greater levels, unlike del(A420-A463) mutant, it failed to enhance mtDNA synthesis due to the accumulation of RNA:DNA hybrids throughout the mtDNA. Biochemical analysis revealed that P466L mutation enhanced RECQ4 annealing activity to generate RNA:DNA hybrids at the same time reduced its unwinding activity to resolve this structure. Hence, P466L mutation led to a reduced efficiency in completing mtDNA synthesis due to unresolved RNA:DNA hybrids across mtDNA.
线粒体 DNA（mtDNA）的合成是一个复杂的过程，涉及到形成和解决不寻常的核酸结构，如 RNA:DNA 杂交。然而，对于调节这些过程的酶知之甚少。RECQ4 是一种对 mtDNA 维持很重要的 DNA 复制因子，在这里，我们揭示了人类 RECQ4 在调节线粒体 RNA:DNA 杂交的形成和解决中的作用。线粒体膜蛋白 p32 可以通过蛋白-蛋白相互作用限制 RECQ4 的线粒体定位来阻止 mtDNA 的合成。我们发现，与 p32 的相互作用不仅被先前报道的与癌症相关的 RECQ4 突变 del(A420-A463)破坏，而且被相邻残基 P466L 的临床突变破坏。令人惊讶的是，尽管 P466L 突变体在线粒体中存在的水平更高，但与 del(A420-A463)突变体不同，由于整个 mtDNA 中 RNA:DNA 杂交的积累，它未能增强 mtDNA 的合成。生化分析表明，P466L 突变增强了 RECQ4 的退火活性，同时降低了其解链活性以解决这种结构。因此，由于 mtDNA 上未解决的 RNA:DNA 杂交，P466L 突变导致 mtDNA 合成的效率降低。
