Tebbs Robert S, Hinz John M, Yamada N Alice, Wilson James B, Salazar Edmund P, Thomas Cynthia B, Jones Irene M, Jones Nigel J, Thompson Larry H
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, P.O. Box 808, Livermore, CA 94551-0808, USA.
DNA Repair (Amst). 2005 Jan 2;4(1):11-22. doi: 10.1016/j.dnarep.2004.06.013.
The Fanconi anemia (FA) proteins overlap with those of homologous recombination through FANCD1/BRCA2, but the biochemical functions of other FA proteins are largely unknown. By constructing and characterizing a null fancg mutant (KO40) of hamster CHO cells, we show that FancG protects cells against a broad spectrum of genotoxic agents. KO40 is consistently hypersensitive to both alkylating agents that produce monoadducts and those that produce interstrand crosslinks. KO40 cells were no more sensitive to mitomycin C (3x) and diepoxybutane (2x) than to 6-thioguanine (5x), ethylnitrosourea (3x), or methyl methanesulfonate (MMS) (3x). These results contrast with the pattern of selective sensitivity to DNA crosslinking agents seen historically with cell lines from FA patients. The hypersensitivity of KO40 to MMS was not associated with a higher level of initial DNA single-strand breaks; nor was there a defect in removing MNU-induced methyl groups from DNA. Both control and MMS-treated synchronized G1-phase KO40 cells progressed through S phase at a normal rate but showed a lengthening of G2 phase compared with wild type. MMS-treated and untreated early S-phase KO40 cells had increased levels of Rad51 foci compared with wild type. Asynchronous KO40 treated with ionizing radiation (IR) exhibited a normal Rad51 focus response, consistent with KO40 having only slight sensitivity to killing by IR. The plating efficiency and doubling time of KO40 cells were nearly normal, and they showed no increase in spontaneous chromosomal aberrations or sister chromatid exchanges. Collectively, our results do not support a role for FancG during DNA replication that deals specifically with processing DNA crosslinks. Nor do they suggest that the main function of the FA protein "pathway" is to promote efficient homologous recombination. We propose that the primary function of FA proteins is to maintain chromosomal continuity by stabilizing replication forks that encounter nicks, gaps, or replication-blocking lesions.
范可尼贫血（FA）蛋白通过FANCD1/BRCA2与同源重组蛋白重叠，但其他FA蛋白的生化功能大多未知。通过构建和鉴定仓鼠CHO细胞的fancg基因敲除突变体（KO40），我们发现FancG可保护细胞免受多种基因毒性剂的损伤。KO40对产生单加合物的烷化剂和产生链间交联的烷化剂均持续高度敏感。KO40细胞对丝裂霉素C（3倍）和二环氧丁烷（2倍）的敏感性并不高于对6-硫鸟嘌呤（5倍）、乙基亚硝基脲（3倍）或甲基磺酸甲酯（MMS）（3倍）的敏感性。这些结果与FA患者细胞系历史上对DNA交联剂的选择性敏感模式形成对比。KO40对MMS的高度敏感性与初始DNA单链断裂水平较高无关；从DNA中去除MNU诱导的甲基也没有缺陷。对照和经MMS处理的同步化G1期KO40细胞均以正常速率进入S期，但与野生型相比，G2期延长。与野生型相比，经MMS处理和未处理的早期S期KO40细胞中Rad51灶的水平增加。用电离辐射（IR）处理的异步KO40表现出正常的Rad51灶反应，这与KO40对IR杀伤仅具有轻微敏感性一致。KO40细胞的接种效率和倍增时间几乎正常，且自发染色体畸变或姐妹染色单体交换没有增加。总体而言，我们的结果不支持FancG在专门处理DNA交联的DNA复制过程中发挥作用。它们也没有表明FA蛋白“途径”的主要功能是促进有效的同源重组。我们提出，FA蛋白的主要功能是通过稳定遇到切口、间隙或复制阻断损伤的复制叉来维持染色体的连续性。
