The Drosophila melanogaster PIF1 Helicase Promotes Survival During Replication Stress and Processive DNA Synthesis During Double-Strand Gap Repair
The Drosophila melanogaster PIF1 Helicase Promotes Survival During Replication Stress and Processive DNA Synthesis During Double-Strand Gap Repair
复制标题
DOI：
10.1534/genetics.119.302665
复制
发表时间：
2019-11-01
期刊：
影响因子：
3.3
通讯作者：
McVey, Mitch
中科院分区：
文献类型：
--
作者：
Kocak, Ece;Dykstra, Sarah;McVey, Mitch
PIF1是一种5 ‘到3 ’ DNA解旋酶，可以解开双链DNA并破坏核酸-蛋白质复合物。在酿酒酵母中，Pif1在断裂诱导复制过程中对线粒体和核基因组的维持、端粒长度调节、g -四重体结构的解绕以及DNA合成等方面发挥着重要作用。这些功能中的一些（但不是全部）与其他真核生物共享。为了深入了解PIF1的进化保守功能，我们在黑腹果蝇中创建了PIF1无效突变体，并评估了它们在整个发育过程中的表型。我们发现pif1突变体幼虫暴露于高浓度的羟基脲中，而不是其他DNA损伤剂，其成虫存活率降低。缺乏PIF1的胚胎在早期核分裂期间不能有效地分离染色体，这与DNA复制缺陷一致。此外，在后生动物中，BRCA2蛋白是稳定停滞的复制叉所必需的，BRCA2蛋白的缺失会导致缺乏PIF1或聚合酶δ亚基POL32的三龄幼虫的合成致死。有趣的是，pif1突变体在双链间隙修复过程中合成DNA的能力降低，但仅在缺乏POL32的情况下。总之，这些结果支持了一个模型，即果蝇PIF1在复制胁迫期间与POL32一起起作用，但在双链间隙修复期间独立于POL32促进合成。
PIF1 is a 5 ' to 3 ' DNA helicase that can unwind double-stranded DNA and disrupt nucleic acid-protein complexes. In Saccharomyces cerevisiae, Pif1 plays important roles in mitochondrial and nuclear genome maintenance, telomere length regulation, unwinding of G-quadruplex structures, and DNA synthesis during break-induced replication. Some, but not all, of these functions are shared with other eukaryotes. To gain insight into the evolutionarily conserved functions of PIF1, we created pif1 null mutants in Drosophila melanogaster and assessed their phenotypes throughout development. We found that pif1 mutant larvae exposed to high concentrations of hydroxyurea, but not other DNA damaging agents, experience reduced survival to adulthood. Embryos lacking PIF1 fail to segregate their chromosomes efficiently during early nuclear divisions, consistent with a defect in DNA replication. Furthermore, loss of the BRCA2 protein, which is required for stabilization of stalled replication forks in metazoans, causes synthetic lethality in third instar larvae lacking either PIF1 or the polymerase delta subunit POL32. Interestingly, pif1 mutants have a reduced ability to synthesize DNA during repair of a double-stranded gap, but only in the absence of POL32. Together, these results support a model in which Drosophila PIF1 functions with POL32 during times of replication stress but acts independently of POL32 to promote synthesis during double-strand gap repair.