In molecular evolution analyses, genomic DNA sequence information is usually represented in the form of 4 bases (ATGC). However, research since the turn of the century has revealed the importance of epigenetic genome modifications, such as DNA base methylation, which can now be decoded using advanced sequence technologies. Here we provide an integrated framework for analyzing molecular evolution of nucleotide substitution, methylation, and demethylation using an expanded nucleotide code that incorporates different types of methylated bases. As a first attempt, we analysed substitution rates between bases, both unmethylated and methylated ones. As the model methylomes, we chose those of Helicobacter pylori, a unicellular bacterium with the largest known repertoire of sequence-specific DNA methyltransferases. We found that the demethylation rates are remarkably high while the methylation rates are comparable with the substitution rates between unmethylated bases. We found that the ribosomal proteins known for sequence conservation showed high methylation and demethylation frequencies, whereas the genes for DNA methyltransferases themselves showed low methylation and demethylation frequencies compared to base substitution. This study represents the first step toward molecular evolutionary epigenomics, which, we expect, would contribute to understanding epigenome evolution.

Many prokaryotes, including members of the Neisseria species, lack MutH and cannot employ methyl-directed DNA mismatch repair (MMR). The nick on the daughter strand is created by the endonuclease activity present in the C-terminal domain (CTD) of the MutL homodimer. MutL-CTD is known to interact with the processivity-clamp. The crystal structure of the homodimeric MutL-CTD from Neisseria (NgoL-CTD) in complex with homodimeric processivity-clamp (Nβ-Clamp) shows that each NgoL-CTD monomer binds to a Nβ-Clamp monomer through the conserved motif III (517QHLLIP522). The structure and allied biochemical studies plus in vivo growth assays conducted with wild-type (wt) plus mutant proteins shows that the endonuclease dimer sits transversely across the C-terminal face of the Nβ-Clamp ring. The comparison of the structure with that of the partial prokaryotic replisome suggests that the relative orientation of DNA, Nβ-Clamp, and NgoL-CTD may direct the daughter strand towards one of the active sites in endonuclease homodimer. Nicking assays conducted with wt and mutant NgoL-CTD in the presence and absence of Nβ-Clamp support this inference. Overall, our studies posit that strand discrimination in non-methyl-directed MMR is achieved through a structural strategy involving the β-Clamp which is distinct from the chemical strategy employed in prokaryotes like Escherichia coli.

UvrD helicase and RecJ exonuclease play a critical role in DNA repair and recombination process thereby involved in the maintenance of the genomic integrity. In addition to DNA repair pathways, UvrD helicase plays an important role in phase variation and maintenance of virulence in pathogenic bacteria while RecJ is a single-stranded DNA -specific 5'-3' exonuclease activity responsible for generating a long 3'ssDNA gap for DNA resynthesis by DNA ploymerases in mismatch repair (MMR). In spite of being vital for performing these functions, there have been few reports on the mismatch repair pathway in pathogenic bacteria and particularly the interplay of mismatch repair proteins in methylation independent mismatch repair. Purified UvrD helicase from Neisseria gonorrhoeae (FA1090) (NgoUvrD) exhibits 3'-5' polarity on ssDNA and unwinds blunt end duplex DNA as well as different DNA substrates with overhangs. While NgoUvrD binds to Ni2+, Mg2+. Mn2+, Zn2+ and Ca2+, only Mg2+ and Mn2+ support the helicase activity as well as ATPase activity. Interestingly, Zn2+ inhibits both the helicase as well as ATPase activity. ssDNA binding to NgoUvrD abrogates the inhibition by Zn2+. This study, for the first time reveals a unique role of zinc in regulating UvrD helicase activity in N. gonorrhoeae. RecJ exonuclease from Neisseria gonorrhoeae is a 566 amino acid protein that contains the characteristic motifs conserved among all RecJ homologs. Site-directed mutagenesis in the conserved DHH motif abrogated enzymatic activity in D160A and H161A mutants. Interestingly, substitution of histidine 161 with alanine or serine residues enhanced RecJ exonuclease activity while the corresponding mutation in other bacterial RecJs abrogated the activity. NgoRecJ degrades double-stranded DNA with 2, 4, 6 and 8 nucleotide 5' overhang substrates unlike E. coli RecJ which degrades ssDNA with 6-nts overhang. In the present investigation we have studied the interaction between UvrD helicase and RecJ proteins participating in methylation-independent MMR pathway. Our studies highlight novel properties of NgoUvrD and NgoRecJ proteins and specific interaction between these proteins which could play in genome maintenance, pathogenesis and virulence of Neisseria gonorrhoeae.

Helicobacter pylori (H. pylori), a gastric pathogen with high genetic variability and a unique niche, causes peptic ulcers and gastric cancer. Natural transformation contributes to the genetic variability of H. pylori. To date, protein acetylation and the associated acetyltransferase(s) have not been reported in this bacterium. Here, we report protein acetylation in H. pylori and identify a putative protein acetyltransferase, HP0935, capable of acetylating amino acids and proteins, including DNA processing protein A (DprA), which is involved in natural transformation. HP0935 acetylates residue K133 in DprA, which is important for DNA binding, thus is likely to regulate natural transformation. We determined the crystal structures of HP0935 in its apo form and in complex with acetyl-coenzyme A (ACO) to 2.00 Å and 2.40 Å resolution, respectively. Structural analysis revealed a conformational change in the substrate-binding loops, α1-α2 and β6-β7, upon ACO binding. The structural comparison showed that HP0935 differs from other protein acetyltransferases in the length and orientation of these loops. Molecular dynamics simulation data suggest that these loops are highly dynamic, and ACO binding could affect their dynamics. Given that several proteins may undergo acetylation in H. pylori and the fact that HP0935 is the only known protein acetyltransferase, the loop dynamics are likely to facilitate the acceptance of multiple substrates by HP0935. Structure-based mutational analysis showed that no general base is required for the enzymatic activity. However, a conserved catalytic water molecule at the active site is likely to serve the purpose. Furthermore, the general acid Y127 is essential for enzymatic activity.

Helicobacter pylori (H. pylori) seems to play causative roles in gastric cancers. H. pylori has also been detected in established gastric cancers. How the presence of H. pylori modulates immune response to the cancer is unclear. The cytotoxicity of natural killer (NK) cells, toward infected or malignant cells, is controlled by the repertoire of activating and inhibitory receptors expressed on their surface. Here, we studied H. pylori-induced changes in the expression of ligands, of activating and inhibitory receptors of NK cells, in the gastric adenocarcinoma AGS cells, and their impacts on NK cell responses. AGS cells lacked or had low surface expression of the class I major histocompatibility complex (MHC-I) molecules HLA-E and HLA-C-ligands of the major NK cell inhibitory receptors NKG2A and killer-cell Ig-like receptor (KIR), respectively. However, AGS cells had high surface expression of ligands of activating receptors DNAM-1 and CD2, and of the adhesion molecules LFA-1. Consistently, AGS cells were sensitive to killing by NK cells despite the expression of inhibitory KIR on NK cells. Furthermore, H. pylori enhanced HLA-C surface expression on AGS cells. H. pylori infection enhanced HLA-C protein synthesis, which could explain H. pylori-induced HLA-C surface expression. H. pylori infection enhanced HLA-C surface expression also in the hepatoma Huh7 and HepG2 cells. Furthermore, H. pylori-induced HLA-C surface expression on AGS cells promoted inhibition of NK cells by KIR, and thereby protected AGS cells from NK cell cytotoxicity. These results suggest that H. pylori enhances HLA-C expression in host cells and protects them from the cytotoxic attack of NK cells expressing HLA-C-specific inhibitory receptors.

S-adenosylmethionine (SAM) is a ubiquitous co-factor that serves as a donor for methylation reactions and additionally serves as a donor of other functional groups such as amino and ribosyl moieties in a variety of other biochemical reactions. Such versatility in function is enabled by the ability of SAM to be recognized by a wide variety of protein molecules that vary in their sequences and structural folds. To understand what gives rise to specific SAM binding in diverse proteins, we set out to study if there are any structural patterns at their binding sites. A comprehensive analysis of structures of the binding sites of SAM by all-pair comparison and clustering, indicated the presence of 4 different site-types, only one among them being well studied. For each site-type we decipher the common minimum principle involved in SAM recognition by diverse proteins and derive structural motifs that are characteristic of SAM binding. The presence of the structural motifs with precise three-dimensional arrangement of amino acids in SAM sites that appear to have evolved independently, indicates that these are winning arrangements of residues to bring about SAM recognition. Further, we find high similarity between one of the SAM site types and a well known ATP binding site type. We demonstrate using in vitro experiments that a known SAM binding protein, HpyAII.M1, a type 2 methyltransferase can bind and hydrolyse ATP. We find common structural motifs that explain this, further supported through site-directed mutagenesis. Observation of similar motifs for binding two of the most ubiquitous ligands in multiple protein families with diverse sequences and structural folds presents compelling evidence at the molecular level in favour of convergent evolution.

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the β-glucocerebrosidase (GCase) GBA gene, which result in macrophage dysfunction. CRISPR (clustered regularly interspaced short palindromic repeats) editing of the homozygous L444P (1448T→C) GBA mutation in type 2 GD (GBA-/-) human-induced pluripotent stem cells (hiPSCs) yielded both heterozygous (GBA+/-) and homozygous (GBA+/+) isogenic lines. Macrophages derived from GBA-/-, GBA+/- and GBA+/+ hiPSCs showed that GBA mutation correction restores normal macrophage functions: GCase activity, motility, and phagocytosis. Furthermore, infection of GBA-/-, GBA+/- and GBA+/+ macrophages with the Mycobacterium tuberculosis H37Rv strain showed that impaired mobility and phagocytic activity were correlated with reduced levels of bacterial engulfment and replication suggesting that GD may be protective against tuberculosis.

Helicobacter pyloris (H.pylori) a Gram-negative bacterium whose infection of the human stomach triggers a chronic gastritis that can evolve into a series of severe pathologies such as gastroduodenal ulcers and cancer. H.pylori infects more than 50% of world population. Its amazing diversity and variability are major contributors to this success by allowing the emegence of new alleles. The bacterium lacks SOS response, DNA mismatch repair system, has a huge repertoire of restriction-modification systems and is highly competent. These characteristics compelled us to investigate its all-possible regulatory system (s). Acetylation-a post-translational modification has been proven to be a major regulatory machinery in prokaryotic physiology Acetylome analysis of different strains of H. pylori showed the presence of prominent and strain specific protein acetylation. Mass-spectrometry based analysis showed that acetylated proteins participate in diverse biological processes such as metabolic pathways, transcription, translation, cell signalling and motility. In addition, acetylome analysis showed that two Cag pathogenicity island proteins. Cag 7, Cag 14, UvrD helicase involved in nucleotide excision repair, DNA methyltransferase- HPyAVIA, and DprA- involved in Natural Transformation process were acetylated. HP0935, a putative N-acetyltransferase belonging to the GNAT superfamily was identified as possible protein acetyltransferase. It was observed that HP0935 acetylates L-amino acids such as arginine and methionine at Nα amino group and lysine at both Nα and Nε amino group. Mutational and pH analysis of HP0935 suggest that a possible catalytic mechanism involves deprotonation of amino group of substrates by conserved water molecule followed by nucleophilic attack of deprotonated amino group on Ac-CoA. Furthermore, Tyr127 act as a general acid that protonates the leaving thiolate anion of Coenzyme A. It was observed that ATPase and helicase activity of HPUvrD was regulated by both enzymatic (HP0935) and non-enzymatic (Ac-CoA) acetylation while the methyltransferase activity of M. HPyAVIA was enhanced only by enzymatic (HP0935) acetylation. These observations suggest acetylation of several key proteins plays a role in pathogenesis and in Natural transformation of H. pylori.

AbstractGaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the β-glucocerebrosidase (GCase)GBAgene, which result in macrophage dysfunction. To investigate whether correction ofGBAmutations restores normal function to Gaucher macrophages, we performed CRISPR editing of homozygous L444P (1448T→C)GBAmutation in Type 2 GD (GBA-/-) hiPSCs, which yielded both heterozygous (GBA+/-) and homozygous (GBA+/+) isogenic lines. Macrophages derived fromGBA-/-,GBA+/- andGBA+/+ hiPSCs, were compared for GCase enzymatic activity, motility, and phagocytosis, all of which showed thatGBAmutation correction restores normal macrophage functions. Furthermore, we investigated whether lysosomal disorders drive susceptibility toMycobacterium tuberculosis, by infectingGBA-/-,GBA+/- andGBA+/+ macrophages with the virulent H37Rv lab strain. The results showed that impaired mobility and phagocytic activity of Gaucher macrophages, correlated with reduced levels of TB engulfment and TB multiplication, supporting the hypothesis that GD may be protective against tuberculosis.

Programmable nucleases-ZFNs, TALENs and CRISPR-Cas9-have equipped scientists with an unprecedented ability to modify cells and organisms almost at will, with great implications across life sciences: biology, agriculture, ecology and medicine. Nucleases-based genome editing (aka gene editing) depends on cellular responses to a targeted double-strand break (DSB). The first truly targetable reagents were zinc finger nucleases (ZFNs) showing that arbitrary DNA sequences within a mammalian genome, could be addressed by protein engineering, ushering in the era of genome editing. ZFNs that are fusions of zinc finger proteins (ZFPs) and FokI cleavage domain, resulted from the basic research on Type IIs FokI restriction enzyme, which showed a bipartite structure with a separable DNA-binding domain and a non-specific cleavage domain. Studies on 3-finger ZFNs established that the preferred substrates were paired binding sites, which doubled the size of the target recognition sequence from 9 to 18 bp that is large enough to specify a unique genomic locus in plant and mammalian cells, including human cells. Subsequently, a ZFN-induced DSB was shown to stimulate homologous recombination in frog eggs. Transcription activator-like effector nucleases (TALENs) that are based on bacterial TALEs fused to FokI cleavage domain expanded the capability. ZFNs and TALENs have been successfully used to modify a multitude of recalcitrant organisms and cell types that were unapproachable previously attesting to the success of protein engineering, long before the arrival of CRISPR. The recent technique to deliver a targeted DSB to cellular genomes are RNA-guided nucleases as exemplified by the Type II prokaryotic CRISPR-Cas9 system. Unlike ZFNs and TALENs that use protein motifs for DNA sequence recognition, CRISPR-Cas9 depends on RNA-DNA recognition. The advantages of the CRISPR-Cas9 system, which include ease of RNA design for new targets and dependence on a single constant Cas9 protein, have led to its wide adoption by research labs around the world. The 2020 Nobel Prize for Chemistry was awarded to Jennifer Doudna and Emmanualle Charpentier for harnessing CRISPR-Cas9 system to provide a simplified technique for genome editing. The programmable nucleases have also been shown to cut at off-target sites with mutagenic consequences, which is a serious concern for human therapeutic applications. Therefore, applications of genome editing technologies to human therapeutics will ultimately depend on risk versus benefit analysis and informed consent

The mismatch repair system (MMR) ensures the stability of genetic information during DNA replication in almost all organisms. Mismatch repair is initiated after recognition of a non-canonical nucleotide pair by the MutS protein and the formation of a complex between MutS and MutL. Eukaryotic and most bacterial MutL homologs function as endonucleases that introduce a single-strand break in the daughter strand of the DNA, thus activating the repair process. However, many aspects of the functioning of this protein remain unknown. We studied the ATPase and DNA binding functions of the MutL protein from the pathogenic bacterium Neisseria gonorrhoeae (NgoMutL), which exhibits endonuclease activity. For the first time, the kinetic parameters of ATP hydrolysis by the full-length NgoMutL protein were determined. Its interactions with single- and double-stranded DNA fragments of various lengths were studied. NgoMutL was shown to be able to efficiently form complexes with DNA fragments that are longer than 40 nucleotides. Using modified DNA duplexes harboring a 2-pyridyldisulfide group on linkers of various lengths, we obtained NgoMutL conjugates with DNA for the first time. According to these results, the Cys residues of the wild-type protein are located at a distance of approximately 18–50 Å from the duplex. The efficiency of the affinity modification of Cys residues in NgoMutL with reactive DNAs was shown to decrease in the presence of ATP or its non-hydrolyzable analog, as well as ZnCl2, in the reaction mixture. We hypothesize that the conserved Cys residues of the C-terminal domain of NgoMutL, which are responsible for the coordination of metal ions in the active center of the protein, are involved in its interaction with DNA. This information may be useful in reconstruction of the main stages of MMR in prokaryotes that are different from γ-proteobacteria, as well as in the search for new targets for drugs against N. gonorrhoeae.

Helicobacter pylori is a carcinogenic bacterium that is responsible for 5.5% of all human gastric cancers. H. pylori codes for an unusually large number of restriction-modification (R-M) systems and several of them are strain-specific and phase-variable. HpyAII is a novel Type IIs phase-variable restriction endonuclease present in 26695 strain of H. pylori. We show that HpyAII prefers two-site substrates over one-site substrates for maximal cleavage activity. HpyAII is less stringent in metal ion requirement and shows higher cleavage activity with Ni2+ over Mg2+. Mutational analysis of the putative residues of the HNH motif of HpyAII confirms that the protein has an active HNH site for the cleavage of DNA. However, mutation of the first Histidine residue of the HNH motif to Alanine does not abolish the enzymatic activity, but instead causes loss of fidelity compared with wildtype HpyAII. Previous studies have shown that mutation of the first Histidine residue of the HNH motif of all other known HNH motif motif-containing enzymes completely abolishes enzymatic activity. We found, in the case of HpyAII, mutation of an active site residue leads to the loss of endonuclease fidelity. The present study provides further insights into the evolution of restriction enzymes.

DNA topoisomerases play a crucial role in maintaining DNA superhelicity, thereby regulating various cellular processes. Unlike most other species, the human pathogen Helicobacter pylori has only two topoisomerases, Topoisomerase I and DNA gyrase, the physiological roles of which remain to be explored. Interestingly, there is enormous variability among the C-terminal domains (CTDs) of Topoisomerase I across bacteria. H. pylori Topoisomerase I (HpTopoI) CTD harbors four zinc finger motifs (ZFs). We show here that sequential deletion of the third and/or fourth ZFs had only a marginal effect on the HpTopoI activity, while deletion of the second, third and fourth ZFs severely reduced DNA relaxation activity. Deletion of all ZFs drastically hampered DNA binding and thus abolished DNA relaxation. Surprisingly, mutagenesis of the annotated active site tyrosine residue (Y297 F) did not abrogate the enzyme activity and HpTopoI CTD alone (spanning the four ZFs) showed DNA relaxation activity. Additionally, a covalent linkage between the DNA and HpTopoI CTD was identified. The capacity of HpTopoI CTD to complement Escherichia coli topA mutant strains further supported the in vitro observations. Collectively these results imply that not all ZFs are dispensable for HpTopoI activity and unveil the presence of additional non-canonical catalytic site(s) within the enzyme.

As no crystal structure of full-size MutL bound to DNA has been obtained up to date, in the present work we used crosslinking and Förster resonance energy transfer (FRET) assays for probing the putative DNA-binding center of MutL from Escherichia coli. Several single-cysteine MutL variants (scMutL) were used for site-specific crosslinking or fluorophore modification. The crosslinking efficiency between scMutL proteins and mismatched DNA modified with thiol-reactive probes correlated with the distances from the Cys residues to the DNA calculated from a model of MutS–MutL–DNA complex. FRET-based investigation of DNA binding with different scMutL variants clearly showed that the highest signals were detected for the variants MutL(T218C) and MutL(A251C) indicating closeness of the positions 218 and 251 to DNA in the MutL–DNA complex. Indeed, the Cys218 and Cys251 of scMutL were crosslinked to the reactive DNA with the highest yield demonstrating their proximity to DNA in the MutL–DNA complex. The presence of MutS increased the yield of conjugate formation between the MutL variants and the modified DNA due to tighter MutL–DNA interactions caused by MutS binding to MutL.

An international consortium of scientists has embarked on the total design and synthesis of all the 16 yeast chromosomes of the laboratory organism, Saccharomyces cerevisiae . Once constructed, the 16 synthetic chromosomes will be consolidated into a single yeast strain along with a new 17th yeast chromosome called the “neochromosome” which contains all the tRNA genes, to generate a designer eukaryotic genome, Sc2.0. The key criterion for the stream-lined yeast (Sc2.0) is that it should retain the same cell fitness and phenotype of the wild-type (Sc1.0), but show increased genetic stability and flexibility to enable future studies. All the 16 synthetic yeast chromosomes have been designed using BioStudio , an open-source framework that was developed specifically to design and construct chromosome-size fragments in silico. The completely redesigned Sc2.0 genome is a highly modified version of the S. cerevisiae genome, with a reduction in the size of ∼1.1 million base pairs, which is about 8% of the native genome. In 2017, the Sc2.0 consortium reported the complete synthesis and assembly of 6.5 individual yeast chromosomes in discrete strains and showed consolidation of 2.5 synthetic chromosomes (synIII/synVI/synIXR) into a single yeast strain that bodes well for the successful completion of the Sc2.0 genome.

Diketone DNA derivatives have been proposed to modify the guanidine group of Arg in proteins. The β-diketo group at the C2' atom of the sugar phosphate moiety has been introduced in DNA by acylation of oligonucleotide precursors, i.e., DNA fragments containing 2'-amino-2'-deoxyuridine, which have been synthesized by the chemical automatic synthesis. Water-soluble N-[3-(dimethylamino)propyl]-N′-ethylcarbodiimide (EDC) and 4,6-dioxoheptanoic acid have been used in the reaction. The ability of oligodeoxyribonucleotides containing the 2'-β-diketo group to react with guanidine, Nα-Boc-L-arginine, and Nα-Dns-L-arginine has been demonstrated. The introduction of this modification into one of the strands of the 15-base pair DNA duplex has been shown to lead to its destabilization. The conjugate formation of MutS and MutL proteins from the E. coli mismatch repair system with 17-base pair DNA duplexes containing the 2'-deoxy-2'-(4,6-dioxoheptylamido)uridine residue has been detected for the first time. To increase the selectivity of the DNA ligands containing the β-diketo group in the reaction with the Arg residues of proteins, we have proposed to treat the reaction mixture with hydroxylamine. This treatment leads to the cleavage of Schiff bases, which are formed with the involvement of lysine residues.