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abstracts
Restriction-modification systems consist of genes that encode a restriction enzyme and a cognate modification methyltransferase. It was believed that restriction enzymes are sequence-specific endonucleases that introduce double-strand breaks at specific sites by catalyzing the cleavages of phosphodiester bonds. R.PabI is a type II restriction enzyme from a hyperthermophilic archaea Pyrococcus abyssi that recognizes 5'-GTAC-3' sequence and cleaves DNA duplexes without the addition of a divalent cation. The structural and mutational analyses of R.PabI in our previous work showed that R.PabI forms a homodimer and has a novel DNA-binding fold called a "half-pipe," which consists of a highly curved anti-parallel β-sheet. Because the structure of R.PabI shares no structural similarity to any other protein, the structural basis of the sequence-recognition and DNA-cleavage mechanisms remained unclear. In this study, we report the crystal structure of R.PabI in complex with a DNA duplex containing the R.PabI recognition sequence. The structure of the R.PabI-DNA complex shows that R.PabI unwinds a DNA duplex at a 5'-GTAC-3' site and flips the guanine and adenine bases out of the DNA helix to recognize the sequence. The electron-density map of the R.PabI-DNA complex shows that R.PabI releases adenine bases from the R.PabI recognition sequence. Biochemical assays using HPLC and MALDI-TOF MS spectrometry also support the observation that R.PabI releases adenine bases by hydrolysis. These results show that R.PabI is not an endonuclease but a sequence-specific adenine DNA glycosylase. R.PabI is the first example of a restriction enzyme that shows DNA glycosylase activity. Mutational analysis reveals the active site of the adenine DNA glycosylase activity of R.PabI. The two opposing apurinic/apyrimidinic (AP) sites generated by R.PabI are cleaved by heat promoted β elimination and/or by endogenous AP endonucleases of host cells to introduce a double-strand break.
abstracts
The terpenoid, small-compound strigolactones (SLs) are plant hormones that regulate plant shoot branching, which is an important agronomic trait that determines crop yields. An α/β-hydrolase protein, DWARF14 (D14), has been recognized to be an essential component of plant SL signaling. Recently, it has been demonstrated that D14 interacts with a gibberellin (GA)-signaling repressor SLR1 in an SL-dependent manner [1], which suggests that SLR1 mediates crosstalk between the SL and GA signalings in the regulation of plant shoot branching. Although D14 functions in SL perception to promote the interaction with SLR1, its molecular mechanism remains unclear. Here, we report the crystal structure of D14 in the complex with 5-hydroxy-3-methylbutenolide (D-OH), which is a reaction product of SLs. The structure was solved at a 2.10-Å resolution when an SL synthetic analogue, (–)-ent-2'-epi-GR7, was soaked into D14 crystals [1]. In the complex structure, D-OH was located at a site far from the catalytic residues including H297 and appeared to function as a plug for the catalytic cavity to induce an overall hydrophobic surface with a hydrophilic patch between the two α-helices in the cap structure of D14. In the binding site, the indole amine of Trp205 formed a hydrogen bond with the oxygen atom of the C2' hydroxy group, which arose from the catalytic reaction of D14, instead of a water molecule in the structure of apo D14. In addition, the side chain of Phe245 moved 1.3 Å toward D-OH. Mutational analyses of D14 showed that the interaction between D14 and SLR1 required an enzymatic activity of D14 and the residues Trp205 and Phe245 were essential for the SL-dependent SLR1-binding of D14. These results suggest that the D14–D-OH complex mediates the interaction with SLR1 in which the D-OH-induced surface and/or structural change is crucial.
