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  • The unique ability of R PabI to specifically excise adenine

    2022-05-13

    The unique ability of R.PabI to specifically excise MMP-2 Inhibitor II from a palindromic sequence is based on a novel structural architecture that is distinct not only from other DNA glycosylases but also from other proteins [88]. In the absence of DNA, R.PabI forms a dimer with a central, highly twisted β-sheet, composed of six β-strands from each subunit. This structural element is the defining feature of the HALFPIPE superfamily and plays a crucial role in substrate recognition, providing much of the positively charged DNA-binding interface 88., 90.. In the presence of nonspecific DNA, R.PabI dimers associate to form a homotetramer that fully encircles the DNA duplex (Figure 6B) [94]. The interface between dimers is small, consisting only of four salt bridges between Arg70 and Asp71. Nonetheless, tetramerization appears to facilitate the search for GTAC sequences because mutation of Arg70 decreased the relative rate of adenine excision in the presence of increasing amounts of nonspecific DNA, and also reduced the proportion of higher oligomeric species bound to DNA lacking the recognition sequence [94]. As in other complexes between DNA glycosylases and DNA [9], nearly all DNA interactions in the tetrameric search complex are nonspecific and are mediated by hydrogen bonds between R.PabI and phosphoryl groups in the DNA backbone [94]. Perturbation of the duplex in this complex is modest, consisting of a 20° bend along the helical axis and a slight widening of the minor groove. However, upon locating the recognition sequence, R.PabI transitions to a far more disruptive binding mode in which one of the two dimers is displaced as the duplex is bent by nearly 90° and the minor groove is stretched until the GTAC base pairs are pulled apart (Figure 6C) [90]. This highly distorted DNA conformation is stabilized by insertion of Gln155 and Arg156 into the melted region of the duplex (Figure 6C). The unpaired nucleotides themselves form a large number of contacts with the remaining R.PabI dimer, including 13 sequence-specific hydrogen bonds with each strand in the palindromic sequence (Figure 6D). Such a large number of contacts is possible because both the 2′-deoxyguanosine nucleotide and the excised adenine nucleobase in the product complex are pulled into adjacent but separate nucleobase binding pockets (Figure 6C,E). Only the adenine binding pocket possesses the catalytic residues necessary for base excision. Asp214, which forms a hydrogen bond MMP-2 Inhibitor II with the AP site in the product complex (Figure 6E), is positioned to stabilize the sugar as positive charge develops during cleavage of the glycosidic bond, while also preorganizing the water nucleophile. The manner in which the nucleobase is activated for excision is less apparent (Box 1). No obvious general acid is present in the active site. Nevertheless, N7 of adenine appears to form a hydrogen bond with the backbone carbonyl of Ile66, which would require N7 to be protonated (Figure 6E). Similarly to the plant toxin ricin, which excises adenine from 28S rRNA 95., 96., R.PabI may select for substrates that are protonated at N7 before entering the nucleobase binding pocket. However, given the high acidity of N7-protonated adenosine (pKa = 2.2) [97], the fraction of adenine likely to be activated for excision at neutral pH is exceedingly small. Conceivably, R.PabI – as well as ricin – could facilitate protonation before or during flipping of the substrate into the active site. R.PabI and its homologs are the only DNA glycosylases known to function in a restriction-modification system, a role that requires sequence-specific excision of unmodified nucleobases [90]. The specificity that R.PabI exhibits for GTAC sequences comes from a large number of sequence-specific contacts present in a homodimeric structure, which is ideally suited for recognition of palindromic sequences. The residues involved in these contacts are highly conserved among homologs, suggesting that all enzymes in the HALFPIPE superfamily recognize GTAC sequences. This is in stark contrast to other DNA glycosylases, which display little to no sequence selectivity and only form sequence-specific contacts with the lesion and, in some instances, the nucleotide complementary to the lesion [9]. As with restriction endonucleases, the sequence-specific activity of R.PabI has potential biotechnology applications. These applications could be expanded by modulating the specificity of the enzyme through mutation of residues involved in DNA sequence recognition. However, it is currently unclear what features of the recognition sequence induce the tetrameric search complex to further remodel the DNA substrate and transition to the dimeric excision complex.