Production of UV-induced frameshift mutations in vitro by DNA polymerases deficient in 3'-->5' exonuclease activity.
Production of UV-induced frameshift mutations in vitro by DNA polymerases deficient in 3'-->5' exonuclease activity.
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缺乏 3-->5 核酸外切酶活性的 DNA 聚合酶在体外产生紫外线诱导的移码突变。
DOI：
10.1006/jmbi.1994.1437
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发表时间：
1994
影响因子：
5.6
通讯作者：
Strauss,B
中科院分区：
文献类型：
--
作者：
Sagher,D;Turkington,E;Acharya,S;Strauss,B
为了研究UV损伤转化为移码突变和碱基置换突变，通过添加额外的嘧啶或通过在β-半乳糖苷酶互补区域内构建无义密码子并在其之前运行嘧啶来改变M13 mp2噬菌体DNA。将正常序列5′ GTC GTT TTA CAA 3′改为GTC GTT T TTA CAA（MIDT）或GTC GTT C TTA CAA（MIDC）以研究移码，并将其改为GTC GTT CTT TAA（OCHRE）以研究赭石密码子（TAA）的回复突变。去除环丁烷二聚体与光裂合酶beforein vitrosynthesis没有很大影响突变频率，虽然治疗导致显着增加的DNA合成的野生型T7 DNA聚合酶的UV照射基板。Δ28 T7 DNA聚合酶产生的框内赭石序列GTT CTT TAA的逆转主要是通过TAA密码子中的碱基替换。大约一半的E。coliKfexo-enzyme ochre回复突变体具有TTA缺失。五个突变T7 DNA聚合酶与不同的核酸外切酶活性的回复突变频率，相关性更好地与发表的值的酶速度比与核酸外切酶活性或测量旁路合成。我们的数据表明，校对活动的损失增加的频率UV诱导的移码，但缺乏这样的活动是不够的，他们的生产。我们认为，当病变对面的核苷酸添加缓慢时，移码发生得更频繁。相同的病变可以产生不同的突变谱，这取决于聚合酶。
In order to study the conversion of UV lesions into frameshift and base substitution mutations, M13mp2 phage DNA was altered by the addition of extra pyrimidines, or by construction of a nonsense codon preceded by a run of pyrimidines within the β-galactosidase complementing region. The normal sequence 5′ GTC GTT TTA CAA 3′ was changed to GTC GTT T TTA CAA (MIDT) of GTC GTT C TTA CAA (MIDC) to study frameshifts an to GTC GTT CTT TAA (OCHRE) to study reversion of the ochre (TAA) codon.Escherichia colipol I Kf and T7 DNA polymerase mutant enzymes devoid of 3′ → 5′ exonuclease activity produce UV-induced revertants at higher frequency than did their exonuclease proficient counterparts. Removal of cyclobutane dimers with photolyase beforein vitrosynthesis did not greatly affect mutant frequency although which treatment led to significantly increased DNA synthesis by the wild-Type T7 DNA polymerase on UV-irradiated substrate. Reversions of the in frame ochre sequence GTT CTT TAA produced by the Δ28 T7 DNA polymerase were mainly by base substitution in the TAA codon. About half of theE. coliKfexo-enzyme ochre revertants had a TTA deletion. Five mutant T7 DNA polymerases with varying exonuclease activity gave revertant frequencies that correlated better with published values of enzyme velocity than with exonuclease activity or with measured bypass synthesis. Our data indicate that loss of proofreading activity increase the frequency of UV-induced frameshifts, but lack of such activity is not sufficient for their production. We suggest that frameshifts occur more frequently when nucleotide addition opposite the lesion is slow. The same lesion can give rise to a different spectrum of mutations depending on the polymerase.