In eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA. The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, 115 amino acids in Drosophila melanogaster (Dm). Molecular modeling predicted this extended linker to fold into a structure similar to the conserved HBD. Based on a deletion series, both the catalytic domain and the conserved HBD were required for high-affinity binding to heteroduplex substrates, whilst loss of the novel HBD led to a ∼90% drop in K[cat] with a decreased K[M], and a large increase in the stability of the RNA/DNA hybrid-enzyme complex, supporting a bipartite binding model in which the second HBD facilitates processivity. Shotgun proteomics following in vivo crosslinking identified single-stranded DNA-binding proteins from both nuclear and mitochondrial compartments, respectively RpA-70 and mtSSB, as prominent interaction partners of Dm RNase H1. However, we were not able to document direct and stable interactions with mtSSB when the proteins were co-overexpressed in S2 cells, and functional interactions between them in vitro were minor.

中文翻译：

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第二杂合结合结构域调节果蝇核糖核酸酶H1的活性。

在真核生物中，核糖核酸酶H1（RNase H1）参与核和线粒体DNA中RNA / DNA杂合体的加工和去除。该酶在果蝇（Dmophila melanogaster，Dm）中包含一个C端催化结构域和一个N端杂化结合结构域（HBD），由可变长度的115个氨基酸的接头隔开）。分子模型预测该延伸的接头折叠成类似于保守的HBD的结构。基于缺失序列，高亲和力结合异源双链体底物需要催化结构域和保守的HBD，而新型HBD的丢失导致K [cat]下降约90％，而K [M]降低，并大大提高了RNA / DNA杂化酶复合物的稳定性，从而支持了第二HBD促进合成能力的二分结合模型。以下鸟枪蛋白质组学体内交联识别单链DNA结合来自两个核和线粒体区室，分别RPA-70和mtSSB蛋白质，作为突出相互作用配偶DMRNase H1。但是，我们不能够记录直接和稳定的互动与mtSSB当蛋白质共过表达的S2细胞，以及它们之间功能的相互作用在体外是次要的。