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DNA molecules are polymers composed of basic repeating subunits of deoxyribonucleotides, which consist of the deoxyribose sugar, phosphate groups, and a nitrogenous base. They appear to fulfill all requirements necessary to maintain the genetic function of DNA. The five elements of nitrogen, phosphorus, carbon, hydrogen, and oxygen had been regarded as the canonical composition of DNA until the discovery of phosphorothioation, with a sixth element, sulfur, identified as an additional naturally occurring constituent on the DNA backbone, as a sequence-selective, stereospecific post-replicative modification governed by the dndgene cluster. Unlike any other DNA or RNA modification system, DNA phosphorothioation is the first-described physiological modification of the DNA sugar-phosphate backbone .
The physiological phosphorothioate modification is widespread in bacteria and occurs in diverse sequence contexts and frequencies in different bacterial genomes, implying a significant impact on bacteria . Recently, a counterpart phosphorothioate-dependent restriction system capable of protection against the invasion of unmodified foreign DNA was discovered to maintain the genetic stability of the phosphorothioate modified host . Another type IV endonuclease, ScoA3McrA, was found to be capable of specifically recognizing as well as cleaving phosphorothioate modified DNA . Interestingly, the gene sco4631, which code for ScoA3McrA, is unable to coexist with the dndgene cluster in the same host, causing immediate cell death . Here we summarize the discovery of this first reported physiological modification on the DNA backbone, and provide insights and perspectives into the biological functions of the phosphorothioate modification in prokaryotic physiology.
2. Discovery of phosphorothioation as an unusual post-replicative modification on the DNA backbone
The study of the physiological DNA phosphorothioation originated from an observation that an unusual DNA modification in Streptomyces lividansrenders DNA susceptible to in vitroTris-dependent double strand cleavage, resulting in a DNA degradation (Dnd) phenotype during conventional and pulsed-field gel electrophoresis . Zhou et al.demonstrated that such a Dnd phenotype was not due to nuclease contamination or improper in vitrogenetic manipulation, but instead, an unusual DNA modification . The modification sites are not randomly distributed in DNA. For instance, both plasmid pIJ101 and pIJ303 from Dnd+S. lividansunderwent site-specific cleavage during electrophoresis, giving particular fragment profiles [5, 6]. Ray et al.then verified that the Dnd phenotype depends on the cleavage activity of an oxidative Tris derivative generated in the electrophoretic buffer adjacent to the anode . In other words, the DNA isolated from S. lividansis intact, and the degradation only occurs during electrophoresis in the presence of oxidative Tris. Thiourea can react with the Tris derivative and thus inhibits the DNA scission. Alternatively, non-degradative electrophoresis of the DNA could also be achieved in a different buffer such as Hepes . Based on these observations, it was proposed that the DNA degradation was the consequence of a site-specific modification, which suffered cleavage by oxidative Tris resulting in degradation during electrophoresis [5, 7].
Dyson and Liang et al.later revealed that the modification required a conserved consensus sequence, as well as flanking sequences with potential for secondary structure(s) (section 3) [8, 9]. Meanwhile, no Tris-mediated scission was detected in single-stranded plasmid replication intermediates, supporting the post-replicative mechanism. The modifying reagents most probably acted post-replicatively on unmodified double-stranded DNA substrates .
The chemical nature of this unusual DNA modification is an intriguing question. Based on the information that two genes involved in this modification are related to sulfur transfer (section 2), Zhou et al.were prompted to conduct the 35S labeling experiment. Dnd+ strains of S. lividans, Streptomyces avermitilisNRRL8165, and Pseudomonas fluorescensPf0-1 were selected to propagate in media containing 35SO42-. Total genomic DNAs were prepared and analyzed on agarose gel followed by Southern blotting. 35S signals were detected in the DNA from three Dnd+ strains, but not in Dnd- mutant ZX1 or Streptomyces coelicolor. This feeding experiment set up a link between the unusual DNA modification and sulfur .
The chemical nature of this unusual DNA modification was eventually found to be a phosphorothioate modification of the DNA backbone by Wang et al. In this modification, the non-bridging oxygen of the backbone phosphate group is replaced by sulfur . Sequence specific phosphorothioate d(GPSA) and d(GPSG) were first detected in E. coliB7A and S. lividans, respectively. The discovery was based on the inability of nuclease P1 to cleave the phosphorothioate bond. Wang et al.fed Dnd+E. coliB7A with L-[35S]-cysteine to label the DNA . Enzymatic hydrolyzed and dephosphorylated nucleosides were resolved by liquid chromatography followed by scintillation counting to locate the 35S containing molecules. Mass spectrometric analysis of the 35S containing molecules revealed characteristic m/zof 597 accompanied by 446, 348, 152 and 136 fragments (Figure 1). 152 and 136 are characteristic m/zof guanine and adenine in positive mode, respectively. This suggests the presence of a G- and A-containing dinucleotide structure for the m/zof 597 molecular ion, with loss of guanine yielding the ion at m/zof 446. The 16-mass-unit increase over a canonical dG-dA dinucleotide (m/z581) is the exact mass difference between a sulfur and an oxygen atom. The putative dinucleotide species can survive the enzymatic digestion to single nucleosides, indicating nuclease resistance. These features suggested phosphorothioate-containing species shown in Figure 1. Enzymatic digestion with nuclease P1 followed by dephosphorylation with alkaline phosphatase yields phosphorothioate modified dinucleotides and canonical nucleosides. Wang et al.eventually corroborated the phosphorothioate structure in E. coliB7A as d(GPSA) in RP configuration by using synthetic d(GPSA) RP and d(GPSA) SP as references .
Remarkably, the phosphorothioate modification in S. lividansdisplayed different sequence selectivity as d(GPSG) RP. To date, a repertoire of phosphorothioate-containing sequences, including d(CPSC), d(GPST), d(APSC), and d(TPSC), have been discovered in diverse bacterial species . The substitution of sulfur creates a chiral center on the phosphate, resulting in two diastereoisomers, known as the RP and SP isomers. However, the physiological phosphorothioate modifications found in bacteria are all in the Rp configuration. DNA phosphorothioation thus represents a sequence-selective and stereo-specific physiological modification of the DNA backbone.
When phosphorothioate linked d(GPSA) RP from E. coliB7A was treated with activated Tris buffer in vitro, the cleavage of the phosphorothioate bond was detected with the observation of dG and dA, whereas regular d(GA) without phosphorothioate bond remained intact. Therefore, the phosphorothioate modification was verified as the molecular basis for the Dnd phenotype during electrophoresis .
3. The dndgene cluster is responsible for phosphorothioation
Evidence for a genetic link responsible for phosphorothioation came from the isolation of a mutant of S. lividans, ZX1, obtained by NTG ((N-methyl-N-nitro-N-nitrosoguanidine) mutagenesis . In comparison to the wild-type, ZX1 has a ca. 90 kb chromosomal deletion and loses the Dnd phenotype, suggesting that the endogenous genes related to the unusual DNA modification are located in this 90 kb fragment.
A set of 13 overlapping cosmids covering the 90 kb region deleted in ZX1 but present in the wild-type were constructed and aligned as shown in Figure 2. When transformed into ZX1, cosmid 16C3 (ZX1::16C3) could restore the Dnd phenotype of mutant ZX1, indicating that 16C3 harbored genes associated with the DNA modification. By subsequent sub-cloning and Dnd phenotypic tests, a 6,665 bp dndlocus containing five dndgenes was precisely localized on cosmid 16C3 .
The dndgene cluster in S. lividansconsists of five genes, dndABCDE. dndBCDEconstitute an operon, which is divergently transcribed from the dndAgene (Figure 2C) . The individual disruption of dndA, dndC, dndDor dndEabolishes phosphorothioation [10, 12]. dndAis predicted to encode a protein of 380 amino acids and homologous to cysteine desulfurase of IscS and NifS proteins in E. coli.Purified DndA protein is a pyridoxyl 5’-phosphate dependent homodimer and capable of catalyzing L-cysteine to produce elemental S and L-alanine. Cys327 in the C-terminal region of DndA is confirmed to be the active enzymatic center and surrounded by a consensus sequence of ATGSACTS . The mobilized elemental sulfur by DndA could subsequently involve the assembly of a [4Fe-4S] cluster in the DndC protein. DndC possesses ATP pyrophosphatase activity, catalyzing hydrolysis of ATP to AMP and pyrophosphate, and is predicted to have phosphoadenyl sulphate reductase activity . Meanwhile, DndC shares a unique adenylation specific P-loop motif of SGGKDS with SGGFDS in ThiI, an enzyme involved in the formation of 4-thiouridine in tRNAs.
DndD is homologous to the ATP-binding cassette (ABC) ATP-binding proteins and also shares extensive sequence similarity to the Structural Maintenance of Chromosomes (SMC) family of proteins associated with ATPase and DNA nicking activity. In addition, DndD possesses an ATP/GTP-binding Walker A motif (35-GLNGCGKT-42) and an ABC transporter family signature (556-LSAGERQLLAISLLW-570) . Yao et al.located an spfBCDEgene cluster in Dnd-phenotypic P. fluorescensPf0-1, which has an organization identical to that of dndBCDEin S. lividans1326. The spfBCDEcluster is essential for the Dnd phenotype in P. fluorescensPf0-1, and the putative SpfBCDE proteins exhibit 51%, 49%, 31% and 39% amino acid sequence homology to DndBCDE, respectively. SpfD, a DndD homolog, possesses an ATPase activity of 6.201 ± 0.695 units/mg and is proposed to provide the energy required in DNA phosphorothioation by hydrolyzing ATP .
DndE consists of merely 126 amino acids and shows 46% identity to a phosphoribosylaminoimidazole carboxylase (NCAIR synthetase) from Anabaena variabilisATCC 29413 . NCAIR synthetase is known to act at a condensing carboxylation step in purine biosynthesis .
Distinct from the others, the disruption of dndBdoes not abolish the Dnd phenotype, but instead it aggravates DNA degradation (Figure 2C). DndB shows 25% identity and 38% similarity to the ABC transporter ATPase from Sphingomonassp. SKA58, and 26% similarity to a DNA gyrase (GyrB) from Mycoplasmaputrefaciens. It also shows significant amino acid sequence homology to a group of putative transcriptional regulators. A run of 152 residues is 24% identical and 36% similar to the substrate-binding protein of an ABC transporter of Streptococcus pneumoniaeTIGE4. In addition, it is noticeable that the predicted DndB is likely to be a basic protein (pI: 8.79) under physiological conditions and would conceivably bind nucleic acids to mediate the modification frequency .
In the tRNA sulfur modification system, IscS converts L-cysteine to L-alanine and sulfane sulfur in the form of a cysteine persulfide in its active site. The generated sulfane sulfur is sequentially transferred to ThiI to continue catalyzing the biosynthesis of 4-thiouridine . Assembled by five Dnd proteins, the DNA phosphorothioation system appears to be more complicated than the tRNA sulfur modification system in accomplishing the sequence selective and stereo-specific sulfur substitution.
4. Widespread existence of phosphorothioation in bacteria
Homologous dndclusters are found in phylogenetically diverse bacterial species including Bacillus, Klebsiella, Enterobacter, Mycobacterium, Pseudomonas, Pseudoalteromonas, Roseobacter, Mesorhizobium, Serratia, Acinetobacter, Clostridium, as well as certain archaea, etc. . Moreover, dndgene homologues are also detected in oceanic metagenomes, including the Sargasso Sea, Roca Redonda, the gulf of Mexico, etc. . In some cases, dndAis not found adjacent to clustered dndBCDE. DndA is homologous to IscS, which usually has more than one copy in a genome. Therefore, an iscS homologue could be elsewhere in genomes and the cognate proteins may have served as functional homologues of DndA.
Apart from bacteria with a sequenced dndcluster, a large part of bacteria not previously known to possess dndclusters display the Dnd phenotype during electrophoresis. A survey on 74 actinomycetal strains from ecologically differentiated regions identified 5 Dnd+ strains . Genomic DNAs from 50% of the total of 69 tested Mycobacterium abscessusisolates degraded during pulse-field gel electrophoresis .
To investigate the phosphorothioate modification in diverse bacteria, Wang et al.developed a highly sensitive liquid chromatography-coupled electrospray ionization tandem quadrupole mass spectrometry technique (LC-MS/MS) that identifies phosphorothioate modifications at dinucleotide level . Due to the specific resistance of the phosphorothioate bond (RP) to nuclease P1, DNA harboring phosphorothioate sites generates nucleosides and phosphorothioate-linked dinucleotides upon digestion by nuclease P1 followed by dephosphorylation. As shown in Figure 3, quantification of phosphorothioate dinucleotides can be achieved using the non-physiological SP stereoisomer of d(GPSA) as an internal standard. This analytical approach makes it feasible to quantitatively screen all 16 phosphorothioate dinucleotides in DNA samples.
An extensive study of a collection of bacteria of variable origins and diverse habitats, including marine microbes Shewanella pealeanaATCC700345, Bermanella marisrubriRED65 and HahellachejuensisKCTC2396, anaerobic Geobacter uraniumreducensRf4, enterotoxigenic E. coliB7A and Salmonella entericaserovar Cerro 87, and one of the smallest known free-living bacteria CandidatusPelagibacter ubique strain HTCC1002, reveals the common possession of DNA phosphorothioate modifications in these taxonomically unrelated bacterial strains (Table 1). It is conceivable that the dnd-associated DNA phosphorothioation is ubiquitous in prokaryotes .
The study of representative strains from various habitats, environmental DNA samples and 63 Vibriostrains reveals that the phosphorothioate modification occurs in a characteristic manner. In S. enterica87, E. coliB7A and Vibrio1F267, the phosphorothioate modification occurs in d(GPST) and d(GPSA) at the ratio of 1:1. The marine bacteria B. marisrubriRED65 and H. chejuensisKCTC2396 possess d(GPSA) accompanied by barely detectable d(GPST). A pair of d(GPST) and d(GPSG) are simultaneously present in G. uraniumreducensRf4 and S. lividans1326, etc., but at levels that differed by two orders of magnitude. Three phosphorothioate contexts of d(CPSC), d(APSC) and d(TPSC) occur in Vibrio1C-10, ZF264, ZF29 and FF75, while the level of total phosphorothioation is almost 10 fold higher than that in other strains (Table 1) .
Wang et al. also analyzed the phosphorothioate modification in the environmental seawater from the Sargasso Sea and Oregon coast, leading to the discovery of phosphorothioate modifications of d(GPSA), d(GPST), d(GPSG) and d(CPSC) in these metagenomes . The Sargasso Sea is a low nutrient, low productivity, subtropical ocean gyre . The oceanic water DNA samples represent microbial communities, including uncultured microbes. Interestingly, phosphorothioates d(CPSC) and d(GPSA) were found throughout the water columns off the Oregon coast (5–40 m) and in the Sargasso Sea (0–200 m), while d(GPSG) was found only in deeper zones of the water column in both locations. More phosphorothioate sequence contexts and frequencies might be explored in the future.
5. Recognition sequences of the phosphorothioate modification
Authentic phosphorothioate modification in S. lividansrequires not only a conserved consensus sequence but also a considerable flanking sequence with the potential to form secondary structures . The investigations by two laboratories have demonstrated that phosphorothioate site selection requires recognition sequences. Dyson et al.and Liang et al.performed primer extension and cloning assays on the basis of Tris mediated DNA breakage, respectively, to localize the modification sites in S. lividans. At that time, the modification sites were proposed to be on closely opposed guanines on either strand of a stringently conserved 4 bp panlindromic core sequence of 5'-GGCC-3' in a region of 5'-c–cGGCCgccg-3' [8, 9]. It is clear now that the modification sites are actually phosphorothioation between two guanosines on both strands in S. lividans. Moreover, both groups confirmed that the phosphorothioate modification in S. lividansrequired a substantial portion of DNA sequences containing three 13 bp direct repeats. The central repeat contains the core sequence, while the left-hand and right-hand copies overlap two potential stem–loop structures (Figure 4). Deletion of either the left or right-hand repeat structures abolishes or alters modification within the core sequence .
Quantitative characterization of phosphorothioation in bacterial genomes provides an alternative way to predict the 4 bp core sequence for modification (Table 1). The predominant d(GPSG) in P. fluorescensPf0-1, G. uraniumreducensRf4, VibrioZS139 and Vibrio1F230 suggests a conserved palindromic 5'-GPSGCC-3' core sequence as it does in S. lividans. The 1:1 ratio of d(GPSA) and d(GPST) in E. coliB7A, S. enterica87 and Vibrio1F267 suggests the asymmetric complementary 5'-GPSAAC-3' and 5'-GPSTTC-3' core sequence. d(GPSA) is the major phosphorothioation in B. marisrubriRED65 and H. chejuensisKCTC2396, indicating the 5'-GPSATC-3' core sequence.
The quantification shows that the d(GPSG) modification of S. lividansoccurs at the frequency of 474 ± 39 every 106 nt, whereas there are 1.1 × 105 d(GG) available on the chromosome (the genomic sequence of S. lividansis not available and the statistical calculation of d(GG) is based on S. coelicolor) . Further statistical analysis revealed that even 4 nt 5'-GGCC-3' sequences still occur at too high frequency to serve as the consensus sequence. A 6 nt 5'-cGGCCg-3' with 2 bp extension, however, is more consistent with the phosphorothioate frequency on chromosomes. E. coliB7A, P. fluorescensPf0-1, VibrioZS139 and B. marisrubriRED65, etc., have phosphorothioate frequencies close to that of S. lividans, indicating the consensus sequence in these bacteria is longer than the proposed 4 bp [2, 8].
Another pattern of phosphorothioate modification is represented by d(CPSC) in Vibrio1C-10, ZF264, ZF29 and FF75. It leads to the proposal of 5'-CPSCGG-3' or 5'-GPSGCC-3' as core sequences. The frequency in genomic DNA, 1 site per 333-500 nt, agrees well with a 4 bp consensus sequence which has the theoretical frequency of once every 256 bp (44). Thus, the four Vibriostrains might have recognition mechanisms that are distinct from the former group [2, 8].
Apart from d(CPSC), phosphorothioate modified d(APSC) and d(TPSC) co-occur in the four Vibriostrains at low frequencies of 1-6 per 106 nt (Table 1). A similar situation holds for S. lividans, G. uraniumreducensRf4 and B. marisrubriRED65, etc., in which low levels of d(GPST) are detected. To explain the low phosphorothioate frequencies, the dndcluster from S. enterica87 was inserted to a low- and high-copy vector of pACYC184 and pBluescript SK+, respectively, generating pJTU1980 and pJTU1238. Both plasmids still confer host E. coliDH10B with d(GPST) and d(GPSA) modifications in a close 1:1 ratio. Moreover, the total phosphorothioate frequencies on chromosomes of DH10B(pJTU1980) and DH10B (pJTU1238) increased 1.5- and 2-fold in comparison to that of the original host S. enterica87. Remarkably, three more phosphorothioate modifications of d(CPSA), d(TPSA), and d(APSA) at low levels appeared due to the increased expression of the dndcluster. The low-frequency phosphorothioate modifications might result from relaxed DNA target recognition by Dnd proteins .
6. Phosphorothioation dependent restriction-modification system
After unveiling the chemical nature of the DNA phosphorothioate modification, an immediate question is what role this novel post-replicative DNA backbone modification plays. In bacteria, site-specific DNA modifications are often, but not always, associated with a sequence-specific endonuclease. The endonuclease is capable of making subtle distinctions between DNA molecules to prevent the invasion of foreign DNA from phage and plasmids that lack the specific DNA modification. For example, DNA methylation has been regarded as the classic restriction modification system. Because of the known resistance of phosphorothioate linkages to a variety of nuclease activities, as well as the post-replicative and site-specific nature of the modification, phosphorothioation of DNA could possibly function as a type of host defense mechanism, akin to restriction and modification systems .
Soon after the chemical nature of the Dnd modification was addressed, the dndcluster was found to constitute a host-specific phosphorothioation-restriction system along with an adjacent dptFGHcluster in S. enterica87 . A 15 kb DNA fragment from S. enterica87 conferred both host-specific phosphorothioation (dptBCDE) and restriction (dptFGH) in E. coli. The two clusters are divergently transcribed. In addition to four phosphorothioation-related genes, three genes are responsible for restriction activity in this DNA fragment, confirmed by gene deletion experiments. With at least 7 genes, phosphorothioation-restriction components seem to form a large complex. The dptFGHrestricts the invasion of non-phosphorothioate-modified pUC18 but not pUC18 with phosphorothioation. When transformed by pUC18 plasmid, S. enterica87 reproducibly yielded about 100 times fewer colonies with non-phosphorothioate pUC18 than with phosphorothioate pUC18. Plasmids from E. colithat had escaped restriction were no longer restricted in S. enterica87 . This observation is similar to the phenomenon leading to the discovery of restriction and modification systems in 1950s . Interestingly, once the modification cluster dptBCDEis disrupted, dptFGHloses the restriction function. The restriction genes dptFGHrequire the phosphorothioation genes dptBCDEto confer the restriction activity of S. enterica87 to ensure that the attack on invasive DNA occurs only when the host DNA is already protected by phosphorothioation .
On the basis of subunit composition, sequence recognition and cofactor requirement, the DNA phosphorothioate modification is close to Type I restriction modification systems but far more complicated. Homologous phosphorothioation-restriction genes were identified in 19 diverse bacteria strains (Figure 5), including phosphorothioate tested E. coliB7A, S. pealeanaATCC700345, B. marisrubriRED65, H. chejuensisKCTC2396, as well as E. cereusE33L, Vibrio choleraMZO-2, etc. E. coliB7A was confirmed to possess a similar phosphorothioation-restriction system by transformation experiments. Plasmids from dnd-XTG102 transformed E. coliB7A with 100-fold lower efficiency than phosphorothioate modified plasmid DNA from wild-type S. enterica87. However, which restriction genes are responsible for DNA cleavage site selection and DNA sequence specificity is not clear. Many bacteria possess only the homologous dndcluster without simultaneous dptFGHacross their genomes, suggesting that the phosphorothioate modification may act not only as a sort of protective system against infection by bacteriophages, but also as an epigenetic signal for new biological function(s) that need to be explored .
The quantification of phosphorothioation is also supportive for a restriction-modification system. Analysis of the quantitative data revealed that the levels of phosphorothioation were classified into three distinct levels: 2-3 per 103 nt, 3-8 per 104 nt, and 1-6 per 106 nt . Along with defined sequence contexts, the first two frequency ranges are consistent with a restriction-modification system with a 4-nt or 5-6 nt consensus sequence, respectively .
7. Cleavage of phosphorothioate DNA by type IV restriction endonuclease ScoMcrA
When the bi-functional plasmid pIJ699 was isolated from S. lividansand E. coli, only pIJ699 from S. lividansdegraded during electrophoresis, indicating that the Dnd phenotype selectively occurred in certain bacteria . This is consistent with the observation that dndis not present in S. coelicolorbut is in S. lividans, although chromosomes of the two strains share an almost identical DNA banding pattern upon enzymatic digestion [5, 6]. Most of the S. coelicolorand S. lividansDNA sequenced is similar or even identical. Interestingly, a type IV restriction endonuclease (ScoA3McrA) coded by gene sco4631in S. coelicolorcuts foreign DNA containing phosphorothioates. The search for a phosphorothioate-cutting enzyme in S. coelicolororiginated from the restriction to the dndgene cluster. Liu et al.tried to introduce the dndgene cluster from S. lividansinto its close relative S. coelicolor. However, they unexpectedly failed, while the same gene cluster with a single base insertion for a frame-shift mutation in dndEgene generated exoconjugants . This implied restriction towards phosphorothioate modification by S. coelicolor.
Comparison between the genome sequence of S. coelicolorand the dnd+ of S. avermitilis, revealed an endonuclease ScoA3McrA in S. coelicolorthat is absent in S. avermitilis. S. coelicolorlost its restriction to the dndgene cluster after disruption of ScoA3McrA. After integration of a vector containing ScoA3McrA into the genome, a dndmutant S. lividansHXY6 confers restriction toward the dndgene cluster. These knock-out and knock-in experiments confirmed the role of ScoA3McrA as the determinant of restriction of phosphorothioate in S. coelicolor.Moreover, in vitroin presence of Mn2+ and Co2+, the purified ScoA3McrA protein cleaved in vivophosphorothioated DNA as well as a synthesized 118 bp double strand DNA oligonucleotide bearing one phosphorothioate on each strand. ScoA3McrA specifically cleaves both the top and the bottom strand, and on both sides of the S-modification at multiple cleavage sites 16-28 nt away from the phosphorothioate sites. Liu et al.proposed that expression of the dndA-Egene cluster in S. coelicolorresulted in phosphorothioation of the host DNA. ScoA3McrA would then cleave the phosphorothioated host DNA near the modified sites and result in cell death as a cell suicide process. Purified ScoMcrA also cleaved Dcm-methylated DNA or Dcm-containing oligos 12-16 bp away from a C5mCWGG Dcm methylation site . ScoA3McrA thus builds an interesting link between phosphorothioation and methylation.
8. Phylogenetic relationship and evolutionary path of dndgenes
The phylogeny of Dnd from 12 bacteria shows strong correlation between phosphorothioate modifications and four Dnd proteins (Figure 6). With the exception of CandidatusPelagibacter ubique, the other 11 strains are well classified based on DNA phosphorothioate sequence contexts and frequencies. Results suggest the diversification of DNA phosphorothioate modifications depends on Dnd protein sequence but not on the phylogenetic descent of the bacteria strains. Furthermore, using phylogenetic analysis based on Dnd proteins and 16S rRNA, Wang et al.found the Dnd phylogenies do not follow their corresponding species tree. This is clearly seen in three Vibrioisolates (ZS139, 1F230, and 1F267) which are phylogenetically incoherent in all four DndBCDE proteins. The phylogenetic differentiation of the Vibrioisolates suggests horizontal gene transfer of dndclusters facilitated by genomic islands in evolution [2, 17].
Sequence analysis reveals that the ca. 90 kb fragment containing the dndcluster in S. lividansis indeed a genomic island with precise length of 92,770 bp . The G+C content of the genomic island is 67.8%, lower than the average for S. coelicolorof 72.1%, indicating the dndsystem may have originated from elsewhere. Genomic islands are discrete DNA segments, which differ among closely related strains. It explains why the dndcluster occurs in S. lividansand S. avermitilis, but not in S. coelicolor, a close relative of S. lividanseven at genomic sequence level. Genomic islands play a role in the evolution, diversification and adaption of microbes as they are involved in the dissemination of variable genes, including antibiotic resistance and virulence genes, as well as catabolic genes .
Active genomic island transfer has been reported in some cases. For instance, the PAPI-1 pathogenicity island in P. aeruginosawas shown to transfer from a donor strain into P. aeruginosastrains . ICEHin1056, an integrative and conjugative element from Haemophilus influenzae, proceeds conjugative transfer between two H. influenzaestrains. Moreover, ICEclcof Pseudomonassp. strain B13 can self-transfer to P. putida, Cupriviadus necatoror P. aeruginosaat similar frequencies . He et al.demonstrated that the 93 kb genomic island in S. lividanswas capable of spontaneous excision from the chromosome at a level of 0.016%-0.027%. However, exposure to MNNG (N-methyL-N'-nitro-N-nitrosoguanidine) can increase the excision frequency by at least five fold. The excised island loses its capabilities of inter and even intra-species transmission between Streptomycesstrains. This genomic island may have lost genes required for its transfer during evolution in order to maintain a relatively stable inheritance with the host .
Besides S. lividans, 11 additional dnd+ bacteria were analyzed by He et al.Remarkably, all dndclusters lie on mobile genetic elements based on the characteristic features of G+C content, dinucleotide bias, direct repeats, and possession of intergrase and/or transposase (Figure 7). Ten of them lie within chromosomal genomic islands and one on a large plasmid. This indicates the dissemination of dndgenes in evolution and explains the ubiquitous occurrence of dndclusters in taxonomically unrelated bacteria. It is still unclear how the dndclusters evolved and disseminated across different bacterial species. He et al.suggested that the dndcluster might be organized into a functional locus on a conjugative plasmid or other mobile element in very ancient times followed by extensive dissemination and diversification over the eons .
Ou et al.organized available data from experimental and bioinformatics analysis of the DNA phosphorothioation to assemble a dndDBdatabase . It contains detailed phosphorothioation-related information including the Dnd phenotype, dndgene clusters, genomic islands harboring dndgenes, and Dnd proteins and conserved domains. The dndDBdatabase provides a useful tool to effectively combine and interlink the genetics, biochemistry and functional aspects of dndsystems and related genomic islands.
Chemically synthesized phosphorothioate internucleotide bonds had been in use for decades prior to the discovery of the physiological phosphorothioate modification in bacteria. Enzymes like snake venom phosphodiesterase, nuclease S1 and nuclease P1 recognize RP and SP phosphorothioate isomers differently, hydrolyzing one isomer more efficiently than the other. Therefore, phosphorothioate isomers have been utilized widely to elucidate the stereochemical action of different enzymes . Other nucleases such as DNase I, DNase II, staphylococcal nuclease and spleen phosphodiesterase are unable to hydrolyse the internucleotidic linkage of either phosphorothioate diastereomer . The significantly increased resistance of phosphorothioate linkage to nuclease hydrolysis inspired the extensive application of phosphorothioate oligonucleotide analogues in antisense therapy to treat a broad range of diseases, including viral infections, cancer and inflammatory diseases. It has been more than a decade since the approval of the first antisense drug Vitravene in 1998 by the FDA. The synthetic 21-mer oligonucleotide with phosphorothioate linkage is used in the treatment of cytomegalovirus retinitis (CMV) in immunocompromised patients, including those with AIDS .
Phosphorothioates can be introduced into oligonucleotides and DNA by both chemical synthesis and enzymatic polymerization. Currently, phosphorothioate-modified oligonucleotides are available via the oxathiaphospholane method, in which nucleoside 3′-O-(2-thio-1,3,2-oxathiaphospholane) derivatives are used as monomers. This method generates a racemic mixture of RP and SP stereoisomers at a close 1:1 ratio. The access to stereospecific phosphorothioate bearing oligonucleotides is still severely limited despite the considerable efforts that have been made . Interestingly, the SP diastereomer of dNTPαS can be accepted as a substrate by E. coliDNA polymerase I, and may be employed in polymerization reactions to produce phosphorothioate linkages of the RP configuration . This is consistent with the physiological configuration of phosphorothioation. However, the phosphorothioate modification modified by the dndgenes is post-replicative, requiring conserved core sequences and flanking sequences.
The desulfurization of phosphorothioate to a phosphate bond is an easy process. However, the reverse phosphorothioation is thought to be energetically uphill. It agrees well with the observed role of DndD which acts as an ATPase. Friz Eckstein proposed that the phosphorothioate modification might first require the activation of target phosphodiester bonds by alkylation, acylation, adenylation or phosphorylation followed by the successive substitution by a nucleophilic sulfur . It is still unclear how the five Dnd proteins cooperate together to use L-cysteine and SO42- as sulfur sources and transfer the sulfur to the DNA backbone sequence selectively and Rp specifically. Although the biochemical activity of several Dnd proteins has been assayed, additional insights are still needed to elucidate the role of each Dnd protein in the DNA phosphorothioation pathway and the interaction between Dnd proteins and target DNA regions. The dndgene cluster is widespread in diverse and distantly related bacteria, however, a complete set of dndhomologs has not yet been found in eukaryotes.
Most of the commonly found structural changes in DNA are due to methylation of particular bases. In some viral DNAs, certain bases may be hydroxymethylated or glucosylated [33-35]. DNA phosphorothioation apparently is an unprecedented physiological modification, which renders DNA susceptible to Tris derivative leading to the characteristic Dnd phenotype.
It is such a surprise to find out that nature can synthesize a phosphorothioate-containing DNA backbone using the dndABCDE genes. Particularly, it is interesting that the modification occurs in a sequence-selective and stereo-specific manner. The discovery of physiological DNA phosphorothioation has revolutionized our view of the composition and structure of DNA, opening a new window that will stimulate research into novel aspects of DNA.
Lianrong Wang, Shi Chen and Zixin Deng (August 1st 2011). Phosphorothioation: An Unusual Post-Replicative Modification on the DNA Backbone, DNA Replication - Current Advances, Herve Seligmann, IntechOpen, DOI: 10.5772/22509. Available from:
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