Guillet Marie, Van Der Kemp Patricia Auffret, Boiteux Serge
CEA, DSV Département de Radiobiologie et Radiopathologie, UMR 217 CNRS Radiobiologie Moléculaire et Cellulaire, BP 6, 92265 Fontenay aux Roses, France.
Nucleic Acids Res. 2006 Apr 14;34(7):2056-66. doi: 10.1093/nar/gkl139. Print 2006.
We identified a viable allele (dut1-1) of the DUT1 gene that encodes the dUTPase activity in Saccharomyces cerevisiae. The Dut1-1 protein possesses a single amino acid substitution (Gly82Ser) in a conserved motif nearby the active site and exhibits a greatly reduced dUTPase activity. The dut1-1 single mutant exhibits growth delay and cell cycle abnormalities and shows a strong spontaneous mutator phenotype. All phenotypes of the dut1-1 mutant are suppressed by the simultaneous inactivation of the uracil DNA N-glycosylase, Ung1. However, the ung1 dut1-1 double mutant accumulates uracil in its genomic DNA. The viability of the dut1-1 mutant is greatly impaired by the simultaneous inactivation of AP endonucleases. These data strongly suggest that the phenotypes of the dut1-1 mutant result from the incorporation of dUMPs into DNA subsequently converted into AP sites. The analysis of the dut1-1 strain mutation spectrum showed that cytosines are preferentially incorporated in front of AP sites in a Rev3-dependent manner during translesion synthesis. These results point to a critical role of the Dut1 protein in the maintenance of the genetic stability. Therefore, the normal cellular metabolism, and not only its byproducts, is an important source of endogenous DNA damage and genetic instability in eukaryotic cells.
我们在酿酒酵母中鉴定出了DUT1基因的一个有功能的等位基因（dut1-1），该基因编码dUTPase活性。Dut1-1蛋白在活性位点附近的一个保守基序中有一个单氨基酸替换（Gly82Ser），并且其dUTPase活性大幅降低。dut1-1单突变体表现出生长延迟和细胞周期异常，并呈现出强烈的自发突变体表型。dut1-1突变体的所有表型都被尿嘧啶DNA N-糖基化酶Ung1的同时失活所抑制。然而，ung1 dut1-1双突变体在其基因组DNA中积累了尿嘧啶。AP核酸内切酶的同时失活极大地损害了dut1-1突变体的生存能力。这些数据有力地表明，dut1-1突变体的表型是由于dUMP掺入DNA随后转化为AP位点所致。对dut1-1菌株突变谱的分析表明，在跨损伤合成过程中，胞嘧啶以Rev3依赖的方式优先掺入到AP位点之前。这些结果表明Dut1蛋白在维持遗传稳定性中起关键作用。因此，正常的细胞代谢，而不仅仅是其副产物，是真核细胞内源性DNA损伤和遗传不稳定的一个重要来源。
