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A single active site in the mariner transposase cleaves DNA strands of opposite polarity.

ABSTRACT: The RNase H structural fold defines a large family of nucleic acid metabolizing enzymes that catalyze phosphoryl transfer reactions using two divalent metal ions in the active site. Almost all of these reactions involve only one strand of the nucleic acid substrates. In contrast, cut-and-paste transposases cleave two DNA strands of opposite polarity, which is usually achieved via an elegant hairpin mechanism. In the mariner transposons, the hairpin intermediate is absent and key aspects of the mechanism by which the transposon ends are cleaved remained unknown. Here, we characterize complexes involved prior to catalysis, which define an asymmetric pathway for transpososome assembly. Using mixtures of wild-type and catalytically inactive transposases, we show that all the catalytic steps of transposition occur within the context of a dimeric transpososome. Crucially, we find that each active site of a transposase dimer is responsible for two hydrolysis and one transesterification reaction at the same transposon end. These results provide the first strong evidence that a DDE/D active site can hydrolyze DNA strands of opposite polarity, a mechanism that has rarely been observed with any type of nuclease.

SUBMITTER: Claeys Bouuaert C

PROVIDER: S-EPMC5714172 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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A single active site in the mariner transposase cleaves DNA strands of opposite polarity.

Claeys Bouuaert Corentin C Chalmers Ronald R

Nucleic acids research 20171101 20

The RNase H structural fold defines a large family of nucleic acid metabolizing enzymes that catalyze phosphoryl transfer reactions using two divalent metal ions in the active site. Almost all of these reactions involve only one strand of the nucleic acid substrates. In contrast, cut-and-paste transposases cleave two DNA strands of opposite polarity, which is usually achieved via an elegant hairpin mechanism. In the mariner transposons, the hairpin intermediate is absent and key aspects of the m ...[more]

PMID: 29036477

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Project description:Chlamydiales species are obligate intracellular bacteria lacking a classical peptidoglycan sacculus but relying on peptidoglycan synthesis for cytokinesis. While septal peptidoglycan biosynthesis seems to be regulated by MreB actin and its membrane anchor RodZ rather than FtsZ tubulin in Chlamydiales, the mechanism of peptidoglycan remodeling is poorly understood. An amidase conserved in Chlamydiales is able to cleave peptide stems in peptidoglycan, but it is not clear how peptidoglycan glycan strands are cleaved since no classical lytic transglycosylase is encoded in chlamydial genomes. However, a protein containing a SpoIID domain, known to possess transglycosylase activity in Bacillus subtilis, is conserved in Chlamydiales We show here that the SpoIID homologue of the Chlamydia-related pathogen Waddlia chondrophila is a septal peptidoglycan-binding protein. Moreover, we demonstrate that SpoIID acts as a lytic transglycosylase on peptidoglycan and as a muramidase on denuded glycan strands in vitro As SpoIID-like proteins are widespread in nonsporulating bacteria, SpoIID might commonly be a septal peptidoglycan remodeling protein in bacteria, including obligate intracellular pathogens, and thus might represent a promising drug target.IMPORTANCEChlamydiales species are obligate intracellular bacteria and important human pathogens that have a minimal division machinery lacking the proteins that are essential for bacterial division in other species, such as FtsZ. Chlamydial division requires synthesis of peptidoglycan, which forms a ring at the division septum and is rapidly turned over. However, little is known of peptidoglycan degradation, because many peptidoglycan-degrading enzymes are not encoded by chlamydial genomes. Here we show that an homologue of SpoIID, a peptidoglycan-degrading enzyme involved in sporulation of bacteria such as Bacillus subtilis, is expressed in Chlamydiales, localizes at the division septum, and degrades peptidoglycan in vitro, indicating that SpoIID is not only involved in sporulation but also likely implicated in division of some bacteria.

Project description:BackgroundFormamidopyrimidine-DNA glycosylase (Fpg) removes abundant pre-mutagenic 8-oxoguanine (oxoG) bases from DNA through nucleophilic attack of its N-terminal proline at C1' of the damaged nucleotide. Since oxoG efficiently pairs with both C and A, Fpg must excise oxoG from pairs with C but not with A, otherwise a mutation occurs. The crystal structures of several Fpg-DNA complexes have been solved, yet no structure with A opposite the lesion is available.ResultsHere we use molecular dynamic simulation to model interactions in the pre-catalytic complex of Lactococcus lactis Fpg with DNA containing oxoG opposite C or A, the latter in either syn or anti conformation. The catalytic dyad, Pro1-Glu2, was modeled in all four possible protonation states. Only one transition was observed in the experimental reaction rate pH dependence plots, and Glu2 kept the same set of interactions regardless of its protonation state, suggesting that it does not limit the reaction rate. The adenine base opposite oxoG was highly distorting for the adjacent nucleotides: in the more stable syn models it formed non-canonical bonds with out-of-register nucleotides in both the damaged and the complementary strand, whereas in the anti models the adenine either formed non-canonical bonds or was expelled into the major groove. The side chains of Arg109 and Phe111 that Fpg inserts into DNA to maintain its kinked conformation tended to withdraw from their positions if A was opposite to the lesion. The region showing the largest differences in the dynamics between oxoG:C and oxoG:A substrates was unexpectedly remote from the active site, located near the linker joining the two domains of Fpg. This region was also highly conserved among 124 analyzed Fpg sequences. Three sites trapping water molecules through multiple bonds were identified on the protein-DNA interface, apparently helping to maintain enzyme-induced DNA distortion and participating in oxoG recognition.ConclusionOverall, the discrimination against A opposite to the lesion seems to be due to incorrect DNA distortion around the lesion-containing base pair and, possibly, to gross movement of protein domains connected by the linker.

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A single active site in the mariner transposase cleaves DNA strands of opposite polarity.

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A single active site in the mariner transposase cleaves DNA strands of opposite polarity.

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