Bridges B A, Woodgate R, Ruiz-Rubio M, Sharif F, Sedgwick S G, Hübscher U
MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton, Great Britain.
Mutat Res. 1987 Dec;181(2):219-26. doi: 10.1016/0027-5107(87)90099-6.
UV mutagenesis in E. coli is believed to occur in two discrete steps. The second step involves continued DNA synthesis beyond a blocking lesion in the template strand. This bypass step requires induced levels of umuD and umuC gene products and activated recA protein. DNA polymerase III may be involved since a dnaE mutator strain (believed to have defective base selection) is associated with enhanced UV mutagenesis in conjunction with a genetic background permitting the bypass step. In non-UV-mutable umu and lexA strains, UV mutagenesis can be demonstrated if delayed photoreversal is given. This is interpreted as indicating that an earlier misincorporation step can occur in such strains but the resulting mutations do not survive because the bypass step is blocked. The misincorporation step does not require any induced SOS gene products and can occur either at the replication fork or during repair replication following excision of a DNA lesion. Neither a dnaE mutator gene (leading to a defective alpha subunit of DNA polymerase III holoenzyme) nor a mutD5 mutator gene (leading to a defective epsilon proofreading subunit) had any effect on the misincorporation step. Although this is consistent with DNA polymerase III holoenzyme not being involved in the misincorporation step, other interpretations involving the inhibition of epsilon proofreading activity by recA protein are possible. In vitro studies are reported in which sites of termination of synthesis by DNA polymerase III holoenzyme on UV-irradiated M13 mp8 DNA were examined in the presence of inhibitors of the 3'-5' proofreading exonuclease (including recA protein). No evidence was found for incorporation of bases opposite photoproducts suggesting that either inhibition is more complete in the cell and/or that other factors are involved in the misincorporation step.
大肠杆菌中的紫外线诱变被认为分两个不同步骤发生。第二步涉及在模板链上的阻断损伤处继续进行DNA合成。这个绕过步骤需要诱导水平的umuD和umuC基因产物以及激活的recA蛋白。DNA聚合酶III可能参与其中，因为一个dnaE突变菌株（被认为碱基选择有缺陷）在允许绕过步骤的遗传背景下与增强的紫外线诱变相关。在非紫外线可诱变的umu和lexA菌株中，如果给予延迟光逆转，就可以证明紫外线诱变。这被解释为表明在这些菌株中可能发生更早的错配步骤，但由于绕过步骤被阻断，产生的突变无法存活。错配步骤不需要任何诱导的SOS基因产物，可以在复制叉处或DNA损伤切除后的修复复制过程中发生。dnaE突变基因（导致DNA聚合酶III全酶的α亚基有缺陷）和mutD5突变基因（导致ε校对亚基有缺陷）对错配步骤都没有任何影响。虽然这与DNA聚合酶III全酶不参与错配步骤一致，但涉及recA蛋白抑制ε校对活性的其他解释也是可能的。本文报道了体外研究，其中在3'-5'校对核酸外切酶抑制剂（包括recA蛋白）存在的情况下，检测了DNA聚合酶III全酶在紫外线照射的M13 mp8 DNA上的合成终止位点。没有发现碱基掺入光产物对面的证据，这表明要么在细胞中的抑制更完全，要么在错配步骤中涉及其他因素。
