Smith C A, Wang M, Jiang N, Che L, Zhao X, Taylor J S
Department of Chemistry, Washington University, St. Louis, Missouri 6330, USA.
Biochemistry. 1996 Apr 2;35(13):4146-54. doi: 10.1021/bi951975c.
The mutations spectra of cis-syn, trans-syn-I, (6-4), and Dewar pyrimidone photoproducts of the TT site of AATTAA and TATTAT in the (-) strand of a heteroduplex M13 vector were obtained in an excision and photoreversal repair deficient Escherichia coli host under SOS conditions. Oligonucleotides containing site-specific photoproducts were annealed to a complementary uracil-containing (+) strand that contained one or more unique pairs of nucleotide mismatches and used to prime (-) strand synthesis with a DNA polymerase and dNTPs. Following DNA synthesis, the reaction mixtures were incubated with T4 DNA ligase and ATP and then used to transfect SOS-induced competent CSRO6F' cells (uvrA6 and phr-1). The transfectants were plated, gridded, and probed by oligonucleotides specific for progeny of the (-) and (+) strands. Individual progeny of the photoproduct-containing (-) strands were plaque purified and sequenced by the dideoxy method. The cis-syn and trans-syn-I dimers were found not to be very mutagenic (<9%), the Dewar product more so (<33%), and the (6-4) product the most mutagenic (<73%). The mutation spectra were similar to those previously reported for the same photoproducts of the TT site of AGTTGG in the (+) strand of an M13 vector [Lawrence, C. W., et al. (1990) Mol. Gen Genet. 222, 166-168; LeClerc, J. E., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9685-9689] except that -1 deletion mutations were not observed for the trans-syn-I photoproducts, and a lower frequency of 3'-T-->C mutations was observed for the (6-4) photoproduct. Evidence that a small percentage of (+) strand repair of a double mismatch to the 3'-side of the photoproduct. Evidence that a small percentage of (+) strand repair of a double mismatch to the 3'-side was obtained from transfection experiments in which a second double mismatch was introduced opposite or flanking the photoproduct. Analysis of the minor tandem mutations induced by the (6-4) and Dewar products suggests that the SOS polymerase complex is able to elongate what amounts to double mismatches opposite these photoproducts and is consistent with the action of a highly processive polymerase that lacks proofreading ability.
在SOS条件下，于切除修复和光逆转修复缺陷的大肠杆菌宿主中，获得了异源双链M13载体（-）链上AATTAA和TATTAT的TT位点的顺式-顺式、反式-顺式-I、（6-4）和杜瓦嘧啶酮光产物的突变谱。将含有位点特异性光产物的寡核苷酸与含有一个或多个独特核苷酸错配的互补含尿嘧啶（+）链退火，并用DNA聚合酶和dNTPs引发（-）链合成。DNA合成后，将反应混合物与T4 DNA连接酶和ATP一起孵育，然后用于转染SOS诱导的感受态CSRO6F'细胞（uvrA6和phr-1）。将转染子铺板、网格化，并用针对（-）链和（+）链后代的寡核苷酸进行探测。对含光产物的（-）链的单个后代进行噬菌斑纯化，并通过双脱氧法进行测序。发现顺式-顺式和反式-顺式-I二聚体的诱变率不高（<9%），杜瓦产物的诱变率较高（<33%），而（6-4）产物的诱变率最高（<73%）。该突变谱与先前报道的M13载体（+）链上AGTTGG的TT位点相同光产物的突变谱相似[劳伦斯，C.W.等人（1990年）《分子遗传学与基因组学》222卷，第166 - 168页；勒克莱尔，J.E.等人（1991年）《美国国家科学院院刊》88卷，第9685 - 9689页]，只是反式-顺式-I光产物未观察到-1缺失突变，且（6-4）光产物的3'-T→C突变频率较低。有证据表明，双错配向光产物3'-侧的（+）链修复占比小。通过在光产物相对或侧翼引入第二个双错配的转染实验，获得了双错配向3'-侧的（+）链修复占比小的证据。对（6-4）和杜瓦产物诱导的微小串联突变的分析表明，SOS聚合酶复合物能够延伸相当于这些光产物对面的双错配，这与缺乏校对能力的高度持续合成的聚合酶的作用一致。
