Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church Street S.E. Minneapolis, Minneapolis, MN, 55455, USA.
College of Biological Engineering, Henan University of Technology, Zhengzhou, 450001, People's Republic of China.
Sci Rep. 2020 Jan 15;10(1):393. doi: 10.1038/s41598-019-56825-w.
Fowlpox virus resolvase (Fpr) is an endonuclease that cleaves a broad range of branched DNA structures, including the Holliday junction (HJ), with little sequence-specificity. To better understand the mechanisms underlying its relaxed substrate specificity, we determined the crystal structures of Fpr and that in a novel complex with HJ at 3.1-Å resolution. In the Fpr-HJ complex, two Fpr dimers use several distinct regions to interact with different DNA structural motifs, showing versatility in DNA-binding. Biochemical and solution NMR data support the existence of non-canonical modes of HJ interaction in solution. The binding of Fpr to various DNA motifs are mediated by its flat DNA-binding surface, which is centered on a short loop spanning K61 to I72 and flanked by longer α-helices at the outer edges, and basic side grooves near the dimer interface. Replacing the Fpr loop K61I72 with a longer loop from Thermus thermophilus RuvC (E71A87) endows Fpr with an enhanced selectivity toward HJ cleavage but with a target sequence preference distinct from that of RuvC, highlighting a unique role of this loop region in Fpr-HJ interaction. Our work helps explain the broad substrate selectivity of Fpr and suggests a possible mode of its association with poxvirus hairpin telomeres.
禽痘病毒核酸内切酶（Fpr）是一种内切核酸酶，能够切割广泛的分支 DNA 结构，包括 Holliday 连接点（HJ），但序列特异性低。为了更好地理解其松弛的底物特异性的机制，我们以 3.1-Å 的分辨率确定了 Fpr 及其与 HJ 的新型复合物的晶体结构。在 Fpr-HJ 复合物中，两个 Fpr 二聚体使用几个不同的区域与不同的 DNA 结构基序相互作用，显示出在 DNA 结合方面的多功能性。生化和溶液 NMR 数据支持在溶液中存在非经典的 HJ 相互作用模式。Fpr 与各种 DNA 基序的结合是由其平坦的 DNA 结合表面介导的，该表面集中在一个短环上，跨越 K61 到 I72，并由较长的α-螺旋在外边缘和二聚体界面附近的碱性侧槽环绕。用来自嗜热栖热菌 RuvC（E71A87）的更长环替换 Fpr 的环 K61I72，使 Fpr 对 HJ 切割具有增强的选择性，但与 RuvC 的靶序列偏好不同，突出了该环区域在 Fpr-HJ 相互作用中的独特作用。我们的工作有助于解释 Fpr 的广泛底物选择性，并提出了其与痘病毒发夹端粒结合的可能模式。
