Miller Adam S, Daley James M, Pham Nhung Tuyet, Niu Hengyao, Xue Xiaoyu, Ira Grzegorz, Sung Patrick
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.
Genes Dev. 2017 Mar 1;31(5):503-510. doi: 10.1101/gad.295659.116. Epub 2017 Mar 23.
DNA double-strand break repair by homologous recombination entails nucleolytic resection of the 5' strand at break ends. Dna2, a flap endonuclease with 5'-3' helicase activity, is involved in the resection process. The Dna2 helicase activity has been implicated in Okazaki fragment processing during DNA replication but is thought to be dispensable for DNA end resection. Unexpectedly, we found a requirement for the helicase function of Dna2 in end resection in budding yeast cells lacking exonuclease 1. Biochemical analysis reveals that ATP hydrolysis-fueled translocation of Dna2 on ssDNA facilitates 5' flap cleavage near a single-strand-double strand junction while attenuating 3' flap incision. Accordingly, the ATP hydrolysis-defective dna2-K1080E mutant is less able to generate long products in a reconstituted resection system. Our study thus reveals a previously unrecognized role of the Dna2 translocase activity in DNA break end resection and in the imposition of the 5' strand specificity of end resection.
通过同源重组进行的DNA双链断裂修复需要在断裂末端对5'链进行核酸酶切除。Dna2是一种具有5'-3'解旋酶活性的瓣状内切核酸酶，参与切除过程。Dna2解旋酶活性与DNA复制过程中的冈崎片段加工有关，但被认为对DNA末端切除是可有可无的。出乎意料的是，我们发现在缺乏核酸外切酶1的芽殖酵母细胞中，末端切除需要Dna2的解旋酶功能。生化分析表明，由ATP水解驱动的Dna2在单链DNA上的易位促进了单链-双链交界处附近的5'瓣切割，同时减弱了3'瓣切割。因此，ATP水解缺陷型dna2-K1080E突变体在重组切除系统中产生长产物的能力较弱。我们的研究因此揭示了Dna2转位酶活性在DNA断裂末端切除以及在赋予末端切除的5'链特异性方面以前未被认识的作用。
