Singer B, Chavez F, Goodman M F, Essigmann J M, Dosanjh M K
Lawrence Berkeley Laboratory, University of California, Berkeley 94720.
Proc Natl Acad Sci U S A. 1989 Nov;86(21):8271-4. doi: 10.1073/pnas.86.21.8271.
O6-Methylguanine (m6G) was incorporated site-specifically into two 25-base oligonucleotides differing only in the nucleotide on the 3' side of the modified base. Templates were primed with oligonucleotides terminating one or two bases prior to the site at which incorporation kinetics were to be investigated. Escherichia coli DNA polymerase I (Klenow fragment) was used to determine the apparent Km and relative Vmax of incorporation of either dCTP or dTTP opposite m6G or G. These data were used to calculate the relative frequency of incorporation opposite the m6G or the unmodified G. When the sequence was 3'-Cm6G-5', there was a 6- to 7-fold preference for formation of a m6G.T pair compared with m6G.C. The m6G.T frequency, based on Vmax/Km, was at least 50-fold greater than that of a G.T pair at the same site. Changing the sequence to 3'-Tm6G-5' had a marked effect on both Km and Vmax of pairs containing m6G and on the incorporation frequency of T opposite m6G, which was then only slightly favored over m6G.C. When replication was started directly opposite m6G, the kinetics appeared unaffected. These data indicate that the frequency of incorporation of C or T opposite m6G in a DNA template is dependent on the flanking neighbors and that a change of even a single base at the 3' position can have a major effect on mutagenic efficiency. Replication using Drosophila Pol alpha gave the same values for relative frequencies. Pairing of either C or T with m6G on the primer terminus did not significantly inhibit extension of the next normal base pair, in contrast to terminal mismatches of unmodified bases. It is concluded that, in the absence of repair, m6G can exhibit widely differing mutation frequencies which, in these experiments, can be as high as 85% of the replicated base. This variation in frequency of changed pairing could contribute to the occurrence of mutational "'hot spots" after replication of damaged DNA.
O6-甲基鸟嘌呤（m6G）被位点特异性地掺入到两条25个碱基的寡核苷酸中，这两条寡核苷酸仅在修饰碱基3'侧的核苷酸上有所不同。模板用在要研究掺入动力学的位点之前终止一个或两个碱基的寡核苷酸进行引发。使用大肠杆菌DNA聚合酶I（Klenow片段）来确定与m6G或G相对应的dCTP或dTTP掺入的表观Km和相对Vmax。这些数据用于计算与m6G或未修饰的G相对应的掺入相对频率。当序列为3'-Cm6G-5'时，与m6G.C相比，形成m6G.T对的偏好性高6至7倍。基于Vmax/Km的m6G.T频率比同一位点的G.T对至少高50倍。将序列改变为3'-Tm6G-5'对含有m6G的碱基对的Km和Vmax以及与m6G相对应的T的掺入频率都有显著影响，此时T相对于m6G.C仅略有偏好。当复制直接从m6G相对的位置开始时，动力学似乎未受影响。这些数据表明，DNA模板中与m6G相对应的C或T的掺入频率取决于侧翼相邻碱基，并且即使在3'位置改变单个碱基也会对诱变效率产生重大影响。使用果蝇Polα进行复制得到的相对频率值相同。与未修饰碱基的末端错配相反，引物末端的C或T与m6G配对不会显著抑制下一个正常碱基对的延伸。可以得出结论，在没有修复的情况下，m6G可以表现出广泛不同的突变频率，在这些实验中，该频率可高达复制碱基的85%。配对改变频率的这种变化可能导致受损DNA复制后突变“热点”的出现。
