Abstract
The ribonuclease (RNase) H family of enzymes catalyze the specific cleavage of RNA strands of RNA/DNA hybrid duplexes and play an important role in DNA replication and repair. Since the first report of the crystal structure of RNase HI, its catalytic mechanisms, which require metal ions, have been discussed based on numerous structural and functional analyses, including X-ray crystallography. In contrast, the function of the conserved histidine residue (His124 in Escherichia coli) in the flexible loop around the active site remains poorly understood, although an important role was suggested by NMR analyses. Here, novel high-resolution X-ray crystal structures of E. coli RNase HI are described, with a particular focus on the interactions of divalent cations with His124 oriented towards the active site. The enzyme-Mg2+ complex contains two metal ions in the active site, one of which has previously been observed. The second ion lies alongside the first and binds to His124 in an octahedral coordination scheme. In the enzyme-Zn2+ complex a single metal ionwas found to bind to the active site, showing a tetrahedral coordination geometry with the surrounding atoms, including His124. These results provide structural evidence that His124 plays a crucial role in the catalytic activity of RNase HI by interacting weakly and transiently with metal ions in the catalytic center.
Highlights
The ribonuclease H (RNase H) family of non-sequencespecific endonucleases hydrolyze the RNA strand of RNA/ DNA hybrids (Majorek et al, 2014; Hyjek et al, 2019)
E. coli RNase HI was expressed in the rnhA-deficient strain MIC3001 [FÀ, supE44, supF58, lacY1 or6, trpR55, galK2, galT22, metB1, hsdR14, rnhA339::cat, recB270] with a heat-inducible expression system based on pR and pL promoters (Itaya & Crouch, 1991; Kanaya et al, 1993)
The column was washed with 20 mM Tris–HCl pH 7.0, 100 mM NaCl, 1 mM EDTA, Zengwei Liao et al Conserved histidine in ribonuclease HI 391
Summary
The ribonuclease H (RNase H) family of non-sequencespecific endonucleases hydrolyze the RNA strand of RNA/ DNA hybrids (Majorek et al, 2014; Hyjek et al, 2019). Members of the RNase H family can be found in almost all organisms. They are mainly divided into two groups: RNase HI and RNase HII (or type 1 and type 2 in eukaryotes). Their overall amino-acid sequences and tertiary structures are dissimilar, RNase HI and RNase HII share conserved active-center residues. RNase HI was concluded to have essential functions in nucleic acid metabolism, including removal of the RNA primers during replication (Holmes et al, 2015; Al-Behadili et al, 2018) and the regulation of R-loop homeostasis (Broccoli et al, 2004; Cheng et al, 2021). In the development of anti-HIV drugs in particular, remarkable efforts have been made to investigate the polymerase domain of HIV-1 reverse transcriptase (RT), despite the persisting
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