Ferrari M E, Fang J, Lohman T M
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Biophys Chem. 1997 Feb 28;64(1-3):235-51. doi: 10.1016/s0301-4622(96)02223-5.
E. coli SSB tetramer binds with high affinity and cooperatively to single-stranded (ss) DNA and functions in replication, recombination and repair. Curth et al. (Biochemistry, 32 (1993) 2585-2591) have shown that a mutant SSB protein, in which Trp-54 has been replaced by Ser (W54S) in each subunit, binds preferentially to ss-polynucleotides in the (SSB)35 mode in which only 35 nucleotides are occluded per tetramer under conditions in which wild-type (wt) SSB binds in its (SSB)65 mode. The W54S mutant also displays increased UV sensitivity and slow growth phenotypes, suggesting defects in vivo in both repair and replication (Carlini et al. (Molecular Microbiology, 10 (1993) 1067)). We have characterized the energetics of SSBW54S binding to poly(dT) as well as short oligodeoxyribonucleotides (dA(pA)69, dT(pT)34, dC(pC)34) to determine the basis for this dramatic change in binding mode preference. We find that the W54S mutant remains a stable tetramer; however, its affinity for ss-DNA as well as both the intra-tetramer negative cooperativity and its inter-tetramer positive cooperativity in the (SSB)35 mode (omega 35) are altered significantly compared to wtSSB. The increased intra-tetramer negative cooperativity makes it more difficult for ss-DNA to bind the third and fourth subunits of the W54S tetramer, explaining the increased stability of the (SSB)35 mode in complexes with poly(dT). When bound to dA(pA)69 in the (SSB)35 mode, W54S tetramer also displays a dramatically lower inter-tetramer positive cooperativity (omega 35 = 77(+/-20)) than wtSSB (omega 35 > or = 10(5)) as well as a significantly lower affinity for ss-DNA. These results indicate that a single amino acid change can dramatically influence the ability of SSB tetramers to bind in the different SSB binding modes. The altered ss-DNA properties of the W54S SSB mutant are probably responsible for the observed defects in replication and repair and support the proposal that the different SSB binding modes may function selectively in replication, recombination and/or repair.
大肠杆菌单链结合蛋白（SSB）四聚体以高亲和力协同结合单链（ss）DNA，并在复制、重组和修复过程中发挥作用。柯思等人（《生物化学》，32卷（1993年），2585 - 2591页）表明，一种突变型SSB蛋白，其每个亚基中的色氨酸 - 54（Trp - 54）已被丝氨酸取代（W54S），在野生型（wt）SSB以其（SSB）65模式结合的条件下，优先以（SSB）35模式结合ss - 多核苷酸，在该模式下每个四聚体仅封闭35个核苷酸。W54S突变体还表现出对紫外线敏感性增加和生长缓慢的表型，表明在体内修复和复制方面存在缺陷（卡里尼等人（《分子微生物学》，10卷（1993年），1067页））。我们已经对SSBW54S与聚（dT）以及短寡脱氧核糖核苷酸（dA（pA）69、dT（pT）34、dC（pC）34）结合的能量学进行了表征，以确定这种结合模式偏好发生巨大变化的基础。我们发现W54S突变体仍然是一个稳定的四聚体；然而，与wtSSB相比，其对ss - DNA的亲和力以及在（SSB）35模式下的四聚体内负协同性和四聚体间正协同性都发生了显著改变。四聚体内增加的负协同性使得ss - DNA更难结合W54S四聚体的第三个和第四个亚基，这解释了在与聚（dT）形成的复合物中（SSB）35模式稳定性的增加。当以（SSB）35模式与dA（pA）69结合时，W54S四聚体还表现出比wtSSB（ω35≥10^5）显著更低的四聚体间正协同性（ω35 = 77（±20））以及对ss - DNA的亲和力显著降低。这些结果表明，单个氨基酸的变化可以显著影响SSB四聚体以不同SSB结合模式结合的能力。W54S SSB突变体改变的ss - DNA特性可能是观察到的复制和修复缺陷的原因，并支持了不同SSB结合模式可能在复制、重组和/或修复中选择性发挥作用的提议。
