McEachern M J, Filutowicz M, Helinski D R
Proc Natl Acad Sci U S A. 1985 Mar;82(5):1480-4. doi: 10.1073/pnas.82.5.1480.
Plasmid pMM3 is a pBR322 derivative carrying the gamma origin of replication of the naturally occurring plasmid R6K. We have produced a gamma-origin mutant bank of this plasmid using the single-strand-specific mutagen sodium bisulfite. Members of this bank contain single or multiple mutations in the seven direct repeats and the flanking sequences in the gamma origin. Three mutants with defective gamma origins have been isolated from this mutant bank. Two of these direct repeat mutants, gamma 117 and gamma 120, are unable to replicate and also have lost the ability to bind the R6K initiation protein pi in vitro at one of the seven 22-base-pair direct repeats within their respective origins. Precise deletion of the damaged repeat of either of these mutants restores origin function, suggesting that the primary defect of these mutants involves a disruption of the normal spacing of pi binding and flanking sequences within the gamma origin. The third mutant, gamma 111, binds pi normally but replicates at a greatly reduced copy number due to a mutation near the seventh repeat. This mutation falls within a short sequence that appears to be conserved among a number of other plasmids that contain direct repeats within their origins of replication.
质粒pMM3是一种pBR322衍生物，携带天然存在的质粒R6K的γ复制起点。我们使用单链特异性诱变剂亚硫酸氢钠构建了该质粒的γ起点突变文库。该文库的成员在γ起点的七个直接重复序列及其侧翼序列中含有单个或多个突变。已从该突变文库中分离出三个γ起点有缺陷的突变体。其中两个直接重复突变体γ117和γ120无法复制，并且在体外也失去了在其各自起点内七个22碱基对直接重复序列之一处结合R6K起始蛋白π的能力。精确缺失这两个突变体中任何一个受损的重复序列均可恢复起点功能，这表明这些突变体的主要缺陷涉及γ起点内π结合和侧翼序列正常间距的破坏。第三个突变体γ111正常结合π，但由于第七个重复序列附近的突变，其复制拷贝数大大降低。该突变位于一个短序列内，该短序列在许多其他在复制起点内含有直接重复序列的质粒中似乎是保守的。
