Moore Anthony F T, Berhie Yepeth, Weislow Isaac S, Koculi Eda
Department of Chemistry, University of Central Florida, 4111 Libra Drive, Physical Sciences, Orlando, FL 32816-2366.
Department of Chemistry and Biochemistry, The University of Texas at El Paso, 500 W University Ave, Chemistry and Computer Science, El Paso, TX, 79902-5802.
bioRxiv. 2024 Jan 10:2024.01.09.573566. doi: 10.1101/2024.01.09.573566.
DDX1 is a human protein which belongs to the DEAD-box protein family of enzymes and is involved in various stages of RNA metabolism from transcription to decay. Many members of the DEAD-box family of enzymes use the energy of ATP binding and hydrolysis to perform their cellular functions. On the other hand, a few members of the DEAD-box family of enzymes bind and/or hydrolyze other nucleotides in addition to ATP. Furthermore, the ATPase activity of DEAD-box family members is stimulated differently by nucleic acids of various structures. The identity of the nucleotides that the DDX1 hydrolyzes and the structure of the nucleic acids upon which it acts in the cell remain largely unknown. Identifying the DDX1 protein's substrates is important for deciphering the molecular roles of DDX1 in cells. Here we identify the nucleic acid sequences and structures supporting the nucleotide hydrolysis activity of DDX1 and its nucleotide specificity. Our data demonstrate that the DDX1 protein hydrolyzes only ATP and deoxy-ATP in the presence of RNA. The ATP hydrolysis activity of DDX1 is stimulated by multiple molecules: single-stranded RNA molecules as short as ten nucleotides, a blunt-ended double-stranded RNA molecule, a hybrid of a double-stranded DNA-RNA molecule, and a single-stranded DNA molecule. Under our experimental conditions, the single-stranded DNA molecule stimulates the ATPase activity of DDX1 at a significantly reduced extent when compared to the other investigated RNA constructs or the hybrid double-stranded DNA/RNA molecule.
DDX1是一种人类蛋白质，属于DEAD-box蛋白家族的酶类，参与从转录到衰变的RNA代谢的各个阶段。DEAD-box酶家族的许多成员利用ATP结合和水解的能量来执行其细胞功能。另一方面，DEAD-box酶家族的一些成员除了ATP之外，还能结合和/或水解其他核苷酸。此外，不同结构的核酸对DEAD-box家族成员的ATP酶活性的刺激方式不同。DDX1水解的核苷酸的身份以及它在细胞中作用的核酸结构在很大程度上仍然未知。确定DDX1蛋白的底物对于解读DDX1在细胞中的分子作用很重要。在这里，我们确定了支持DDX1核苷酸水解活性及其核苷酸特异性的核酸序列和结构。我们的数据表明，DDX1蛋白在RNA存在的情况下仅水解ATP和脱氧ATP。DDX1的ATP水解活性受到多种分子的刺激：短至十个核苷酸的单链RNA分子、平端双链RNA分子、双链DNA-RNA分子杂交体和单链DNA分子。在我们的实验条件下，与其他研究的RNA构建体或双链DNA/RNA杂交分子相比，单链DNA分子对DDX1的ATP酶活性的刺激程度显著降低。
