Bennett R A
Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Mol Cell Biol. 1999 Mar;19(3):1800-9. doi: 10.1128/MCB.19.3.1800.
The recently sequenced Saccharomyces cerevisiae genome was searched for a gene with homology to the gene encoding the major human AP endonuclease, a component of the highly conserved DNA base excision repair pathway. An open reading frame was found to encode a putative protein (34% identical to the Schizosaccharomyces pombe eth1(+) [open reading frame SPBC3D6.10] gene product) with a 347-residue segment homologous to the exonuclease III family of AP endonucleases. Synthesis of mRNA from ETH1 in wild-type cells was induced sixfold relative to that in untreated cells after exposure to the alkylating agent methyl methanesulfonate (MMS). To investigate the function of ETH1, deletions of the open reading frame were made in a wild-type strain and a strain deficient in the known yeast AP endonuclease encoded by APN1. eth1 strains were not more sensitive to killing by MMS, hydrogen peroxide, or phleomycin D1, whereas apn1 strains were approximately 3-fold more sensitive to MMS and approximately 10-fold more sensitive to hydrogen peroxide than was the wild type. Double-mutant strains (apn1 eth1) were approximately 15-fold more sensitive to MMS and approximately 2- to 3-fold more sensitive to hydrogen peroxide and phleomycin D1 than were apn1 strains. Elimination of ETH1 in apn1 strains also increased spontaneous mutation rates 9- or 31-fold compared to the wild type as determined by reversion to adenine or lysine prototrophy, respectively. Transformation of apn1 eth1 cells with an expression vector containing ETH1 reversed the hypersensitivity to MMS and limited the rate of spontaneous mutagenesis. Expression of ETH1 in a dut-1 xthA3 Escherichia coli strain demonstrated that the gene product functionally complements the missing AP endonuclease activity. Thus, in apn1 cells where the major AP endonuclease activity is missing, ETH1 offers an alternate capacity for repair of spontaneous or induced damage to DNA that is normally repaired by Apn1 protein.
在最近测序的酿酒酵母基因组中搜索与编码主要人类脱嘌呤嘧啶内切酶的基因具有同源性的基因，该酶是高度保守的DNA碱基切除修复途径的一个组成部分。发现一个开放阅读框编码一种推定蛋白（与粟酒裂殖酵母eth1(+) [开放阅读框SPBC3D6.10]基因产物有34%的同一性），其347个残基的片段与脱嘌呤嘧啶内切酶的核酸外切酶III家族同源。在野生型细胞中，暴露于烷基化剂甲磺酸甲酯（MMS）后，ETH1的mRNA合成相对于未处理细胞诱导增加了6倍。为了研究ETH1的功能，在野生型菌株和由APN1编码的已知酵母脱嘌呤嘧啶内切酶缺陷的菌株中对开放阅读框进行了缺失。eth1菌株对MMS、过氧化氢或博来霉素D1杀伤的敏感性并不更高，而apn1菌株对MMS的敏感性比野生型高约3倍，对过氧化氢的敏感性比野生型高约10倍。双突变菌株（apn1 eth1）对MMS的敏感性比apn1菌株高约15倍，对过氧化氢和博来霉素D1的敏感性比apn1菌株高约2至3倍。与野生型相比，在apn1菌株中消除ETH1也分别使回复到腺嘌呤或赖氨酸原养型所确定的自发突变率增加了9倍或31倍。用含有ETH1的表达载体转化apn1 eth1细胞可逆转对MMS的超敏感性并限制自发诱变率。ETH1在dut-1 xthA3大肠杆菌菌株中的表达表明该基因产物在功能上补充了缺失的脱嘌呤嘧啶内切酶活性。因此，在缺少主要脱嘌呤嘧啶内切酶活性的apn1细胞中，ETH1提供了一种替代能力，用于修复通常由Apn1蛋白修复的DNA自发或诱导损伤。
