Karanja Kenneth K, Lee Eu Han, Hendrickson Eric A, Campbell Judith L
Braun Laboratories; California Institute of Technology; Pasadena, CA USA.
Department of Biochemistry, Molecular Biology, and Biophysics; University of Minnesota; Minneapolis, MN USA.
Cell Cycle. 2014;13(10):1540-50. doi: 10.4161/cc.28476. Epub 2014 Mar 12.
FANCD2 is required for the repair of DNA damage by the FA (Fanconi anemia) pathway, and, consequently, FANCD2-deficient cells are sensitive to compounds such as cisplatin and formaldehyde that induce DNA:DNA and DNA:protein crosslinks, respectively. The DNA2 helicase/nuclease is required for RNA/DNA flap removal from Okazaki fragments during DNA replication and for the resection of DSBs (double-strand breaks) during HDR (homology-directed repair) of replication stress-induced damage. A knockdown of DNA2 renders normal cells as sensitive to cisplatin (in the absence of EXO1) and to formaldehyde (even in the presence of EXO1) as FANCD2(-/-) cells. Surprisingly, however, the depletion of DNA2 in FANCD2-deficient cells rescues the sensitivity of FANCD2(-/-) cells to cisplatin and formaldehyde. We previously showed that the resection activity of DNA2 acts downstream of FANCD2 to insure HDR of the DSBs arising when replication forks encounter ICL (interstrand crosslink) damage. The suppression of FANCD2(-/-) by DNA2 knockdowns suggests that DNA2 and FANCD2 also have antagonistic roles: in the absence of FANCD2, DNA2 somehow corrupts repair. To demonstrate that DNA2 is deleterious to crosslink repair, we used psoralen-induced ICL damage to trigger the repair of a site-specific crosslink in a GFP reporter and observed that "over-resection" can account for reduced repair. Our work demonstrates that excessive resection can lead to genome instability and shows that strict regulatory processes have evolved to inhibit resection nucleases. The suppression of FANCD2(-/-) phenotypes by DNA2 depletion may have implications for FA therapies and for the use of ICL-inducing agents in chemotherapy.
范可尼贫血（FA）途径修复DNA损伤需要FANCD2，因此，FANCD2缺陷型细胞对顺铂和甲醛等化合物敏感，顺铂和甲醛分别诱导DNA:DNA和DNA:蛋白质交联。DNA2解旋酶/核酸酶在DNA复制过程中从冈崎片段去除RNA/DNA瓣以及在复制应激诱导损伤的同源定向修复（HDR）过程中切除双链断裂（DSB）时是必需的。敲低DNA2会使正常细胞对顺铂（在缺乏EXO1的情况下）和甲醛（即使在存在EXO1的情况下）的敏感性与FANCD2（-/-）细胞相同。然而，令人惊讶的是，在FANCD2缺陷型细胞中耗尽DNA2可挽救FANCD2（-/-）细胞对顺铂和甲醛的敏感性。我们之前表明，DNA2的切除活性在FANCD2下游起作用，以确保复制叉遇到链间交联（ICL）损伤时产生的DSB的HDR。DNA2敲低对FANCD2（-/-）的抑制表明DNA2和FANCD2也具有拮抗作用：在没有FANCD2的情况下，DNA2以某种方式破坏修复。为了证明DNA2对交联修复有害，我们使用补骨脂素诱导的ICL损伤来触发GFP报告基因中位点特异性交联的修复，并观察到“过度切除”可解释修复减少的原因。我们的工作表明，过度切除会导致基因组不稳定，并表明已经进化出严格的调节过程来抑制切除核酸酶。DNA2耗尽对FANCD2（-/-）表型的抑制可能对FA疗法以及化疗中使用ICL诱导剂有影响。
