Delagoutte E, Bertrand-Burggraf E, Lambert I B, Fuchs R P
U.P.R., CNRS, Strasborg/Illkirch-Graffenstaden, France.
J Mol Biol. 1996 Apr 19;257(5):970-6. doi: 10.1006/jmbi.1996.0216.
Previous in vivo studies involving sequence 5'-CCCG1G2G3-3' (SmaI site) have demonstrated that adducts of N-2-acetylaminofluorene (AAF) to any of the three guanine residues of the SmaI sequence induce, with different efficiencies, two classes of -1 frameshift events, namely -G and -C mutations, referred to as targeted and semitargeted mutations, respectively. It has been proposed that both events occur during replication as a consequence of slippage events involving slipped mutagenic intermediates (SMIs). In order to evaluate the potential role of the UvrABC excinuclease in frameshift mutagenesis, we have studied the interaction of this enzyme with DNA molecules mimicking SMIs in vitro. In all of our constructions, when present, the AAF adduct was located on the third guanine residue of the SmaI site (5'-CCCG1G2G3-3'). This strand was referred to as the top strand, the complementary strand being the bottom strand. Double-stranded heteroduplexes mimicking the targeted and semitargeted SMIs contained a deletion of a C and a G within the SmaI sequence in the bottom strand and were designated deltaC/3 and deltaG/3 when modified with the AAF on the third guanine residue in the top strand or deltaC/O and deltaG/O when unmodified. The modified homoduplex was designated SmaI/3. deltaC/O and deltaG/O were weakly recognized by UvrA2B, but not incised. All three AAF-modified substrates were recognized with similar efficiency and much more efficiently than unmodified heteroduplexes. With AAF-monomodified substrates, dissociation of UvrA2 from the UvrA2B-DNA complex occurred more readily in heteroduplexes than in the homoduplex. SmaI/3 and deltaC/3 were incised with equal efficiency, while deltaG/3 was less incised. The position of the AAF lesion dictated the position of the incised phosphodiester bonds, suggesting that the presence of a bulge can modulate the yield but not the incision pattern of AAF-modified substrates. The finding that UvrABC excinuclease acts on substrates that mimic SMIs suggests that the nucleotide excision repair pathway may help in fixing frameshift mutations before the following round of replication.
先前涉及序列5'-CCCG1G2G3-3'（SmaI位点）的体内研究表明，N-2-乙酰氨基芴（AAF）与SmaI序列的三个鸟嘌呤残基中任何一个形成的加合物会以不同效率诱导两类-1移码事件，即-G和-C突变，分别称为靶向突变和半靶向突变。有人提出这两种事件都发生在复制过程中，是涉及滑动诱变中间体（SMIs）的滑动事件的结果。为了评估UvrABC核酸外切酶在移码诱变中的潜在作用，我们在体外研究了该酶与模拟SMIs的DNA分子的相互作用。在我们所有的构建体中，如果存在AAF加合物，它位于SmaI位点（5'-CCCG1G2G3-3'）的第三个鸟嘌呤残基上。这条链称为顶链，互补链为底链。模拟靶向和半靶向SMIs的双链异源双链体在底链的SmaI序列内缺失一个C和一个G，当顶链的第三个鸟嘌呤残基用AAF修饰时称为deltaC/3和deltaG/3，未修饰时称为deltaC/O和deltaG/O。修饰的同源双链体称为SmaI/3。deltaC/O和deltaG/O被UvrA2B弱识别，但未被切割。所有三种AAF修饰的底物被识别的效率相似，且比未修饰的异源双链体更有效。对于AAF单修饰的底物，UvrA2从UvrA2B-DNA复合物中的解离在异源双链体中比在同源双链体中更容易发生。SmaI/3和deltaC/3被切割的效率相同，而deltaG/3被切割得较少。AAF损伤的位置决定了被切割的磷酸二酯键的位置，这表明凸起的存在可以调节AAF修饰底物的切割产量，但不能调节切割模式。UvrABC核酸外切酶作用于模拟SMIs的底物这一发现表明，核苷酸切除修复途径可能有助于在下一轮复制之前修复移码突变。
