Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
Biochem Biophys Res Commun. 2010 Feb 5;392(2):140-4. doi: 10.1016/j.bbrc.2009.12.167. Epub 2010 Jan 6.
REV1 is a member of the Y-family DNA polymerases, but is atypical in utilizing only dCTP with a preference for guanine (G) as the template. Crystallography of the REV1-DNA-dCTP ternary complex has revealed a unique mechanism by which template G is evicted from the DNA helix and incoming dCTP is recognized by an arginine residue in an alpha-loop, termed the N-digit. To better understand functions of its individual amino acid residues, we made a series of mutant human REV1 proteins. We found that R357 and L358 play vital roles in template binding. Furthermore, extensive mutation analysis revealed a novel function of R357 for substrate discrimination, in addition to previously proposed specific interaction with incoming dCTP. We found that the binding pocket for dCTP of REV1 has also significant but latent affinity for dGTP. The results suggest that the positive charge on R357 could prevent interaction with dGTP. We propose that both direct and indirect mechanisms mediated by R357 ensure specificity for dCTP.
REV1 是 Y 家族 DNA 聚合酶的成员，但它是一种非典型的酶，只利用 dCTP，并且偏爱以鸟嘌呤 (G) 为模板。REV1-DNA-dCTP 三元复合物的晶体学揭示了一种独特的机制，通过该机制，模板 G 从 DNA 螺旋中排出，而进入的 dCTP 被称为 N 位的α环中的精氨酸残基识别。为了更好地理解其单个氨基酸残基的功能，我们构建了一系列突变的人类 REV1 蛋白。我们发现 R357 和 L358 在模板结合中起着至关重要的作用。此外，广泛的突变分析揭示了 R357 对于底物识别的新功能，除了先前提出的与进入的 dCTP 的特异性相互作用。我们发现 REV1 的 dCTP 结合口袋对 dGTP 也有显著但潜在的亲和力。结果表明，R357 上的正电荷可以防止与 dGTP 的相互作用。我们提出，R357 介导的直接和间接机制都确保了对 dCTP 的特异性。
