Correction to: Mutations in the nucleotide binding and hydrolysis domains of helicobacter pylori MutS2 lead to altered biochemical activities and inactivation of its in vivo function.

Following publication of the original article [1], the authors notified us of an error in the presentation of Fig. 6G.

Kinetic and catalytic properties of M.HpyAXVII, a phase-variable DNA methyltransferase from Helicobacter pylori.

The bacterium Helicobacter pylori is one of the most common infectious agents found in the human stomach. H. pylori has an unusually large number of DNA methyltransferases (MTases), prompting speculation that they may be involved in the cancerization of epithelial cells. The mod-4a/4b locus, consisting of the hp1369 and hp1370 ORFs, encodes for a truncated and inactive MTase in H. pylori strain 26695. However, slipped-strand synthesis within the phase-variable polyguanine tract in hp1369 results in expression of an active HP1369-1370 fusion N6-adenine methyltransferase, designated M.HpyAXVII. Sequence analysis of the mod-4a/4b locus across 74 H. pylori strain genomes has provided insights into the regulation of M.HpyAXVII expression. To better understand the role of M.HpyAXVII in the H. pylori biology, here we cloned and overexpressed the hp1369-70 fusion construct in Escherichia coli BL21(DE3) cells. Results from size-exclusion chromatography and multi-angle light scattering (MALS) analyses suggested that M.HpyAXVII exists as a dimer in solution. Kinetic studies, including product and substrate inhibition analyses, initial velocity dependence between substrates, and isotope partitioning, suggested that M.HpyAXVII catalyzes DNA methylation in an ordered Bi Bi mechanism in which the AdoMet binding precedes DNA binding and AdoMet's methyl group is then transferred to an adenine within the DNA recognition sequence. Altering the highly conserved catalytic motif (DPP(Y/F)) as well as the AdoMet-binding motif (FXGXG) by site-directed mutagenesis abolished the catalytic activity of M.HpyAXVII. These results provide insights into the enzyme kinetic mechanism of M.HpyAXVII. We propose that AdoMet binding conformationally "primes" the enzyme for DNA binding.

Asymmetric DNA methylation by dimeric EcoP15I DNA methyltransferase.

EcoP15I DNA methyltransferase (M.EcoP15I) recognizes short asymmetric sequence, 5'-CAGCAG-3', and methylates the second adenine only on one strand of the double-stranded DNA (dsDNA). In vivo, this methylation is sufficient to protect the host DNA from cleavage by the cognate restriction endonuclease, R.EcoP15I, because of the stringent cleavage specificity requirements. Biochemical and structural characterization support the notion that purified M.EcoP15I exists and functions as dimer. However, the exact role of dimerization in M.EcoP15I reaction mechanism remains elusive. Here we engineered M.EcoP15I to a stable monomeric form and studied the role of dimerization in enzyme catalyzed methylation reaction. While the monomeric form binds single-stranded DNA (ssDNA) containing the recognition sequence it is unable to methylate it. Further we show that, while the monomeric form has AdoMet binding and Mg(2+) binding motifs intact, optimal dsDNA binding required for methylation is dependent on dimerization. Together, our biochemical data supports a unique subunit organization for M.EcoP15I to catalyze the methylation reaction.

Mutations in the nucleotide binding and hydrolysis domains of Helicobacter pylori MutS2 lead to altered biochemical activities and inactivation of its in vivo function.

BACKGROUND: Helicobacter pylori MutS2 (HpMutS2), an inhibitor of recombination during transformation is a non-specific nuclease with two catalytic sites, both of which are essential for its anti-recombinase activity. Although HpMutS2 belongs to a highly conserved family of ABC transporter ATPases, the role of its ATP binding and hydrolysis activities remains elusive. RESULTS: To explore the putative role of ATP binding and hydrolysis activities of HpMutS2 we specifically generated point mutations in the nucleotide-binding Walker-A (HpMutS2-G338R) and hydrolysis Walker-B (HpMutS2-E413A) domains of the protein. Compared to wild-type protein, HpMutS2-G338R exhibited ~2.5-fold lower affinity for both ATP and ADP while ATP hydrolysis was reduced by ~3-fold. Nucleotide binding efficiencies of HpMutS2-E413A were not significantly altered; however the ATP hydrolysis was reduced by ~10-fold. Although mutations in the Walker-A and Walker-B motifs of HpMutS2 only partially reduced its ability to bind and hydrolyze ATP, we demonstrate that these mutants not only exhibited alterations in the conformation, DNA binding and nuclease activities of the protein but failed to complement the hyper-recombinant phenotype displayed by mutS2-disrupted strain of H. pylori. In addition, we show that the nucleotide cofactor modulates the conformation, DNA binding and nuclease activities of HpMutS2. CONCLUSIONS: These data describe a strong crosstalk between the ATPase, DNA binding, and nuclease activities of HpMutS2. Furthermore these data show that both, ATP binding and hydrolysis activities of HpMutS2 are essential for the in vivo anti-recombinase function of the protein.

The nuclease activities of both the Smr domain and an additional LDLK motif are required for an efficient anti-recombination function of Helicobacter pylori†MutS2.

Helicobacter pylori, a human pathogen, is a naturally and constitutively competent bacteria, displaying a high rate of intergenomic recombination. While recombination events are essential for evolution and adaptation of H. pylori to dynamic gastric niches and new hosts, such events should be regulated tightly to maintain genomic integrity. Here, we analyze the role of the nuclease activity of MutS2, a protein that limits recombination during transformation in H. pylori. In previously studied MutS2 proteins, the C-terminal Smr domain was mapped as the region responsible for its nuclease activity. We report here that deletion of Smr domain does not completely abolish the nuclease activity of HpMutS2. Using bioinformatics analysis and mutagenesis, we identified an additional and novel nuclease motif (LDLK) at the N-terminus of HpMutS2 unique to Helicobacter and related ε-proteobacterial species. A single point mutation (D30A) in the LDLK motif and the deletion of Smr domain resulted in ∼ 5-10-fold loss of DNA cleavage ability of HpMutS2. Interestingly, the mutant forms of HpMutS2 wherein the LDLK motif was mutated or the Smr domain was deleted were unable to complement the hyper-recombination phenotype of a mutS2(-) strain, suggesting that both nuclease sites are indispensable for an efficient anti-recombinase activity of HpMutS2.