DNA metabarcoding reveals ecological patterns and driving mechanisms of archaeal, bacterial, and eukaryotic communities in sediments of the Sansha Yongle Blue Hole.

The Sansha Yongle Blue Hole (SYBH) is the world's deepest marine blue hole with unique physicochemical characteristics. However, our knowledge of the biodiversity and community structure in SYBH sediments remains limited, as past studies have mostly focused on microbial communities in the water column. Here, we collected sediment samples from the aerobic zone (3.1 to 38.6 m) and the deep anaerobic zone (150 m, 300 m) of the SYBH and extracted DNA to characterize the archaeal, bacterial, and eukaryotic communities inhabiting these sediments. Our results showed that the archaeal and bacterial communities were dominated by Thaumarchaeota and Proteobacteria, respectively. The dominant taxa of eukaryotes in different sites varied greatly, mainly including Phaeophyceae, Annelida, Diatomea and Arthropoda. All three examined domains showed clear vertical distributions and significant differences in community composition between the aerobic and anaerobic zones. Sulfide played a prominent role in structuring the three domains, followed by salinity, nitrous oxide, pH, temperature and dissolved oxygen, all of which were positively correlated with the turnover component, the main contributor to beta diversity. Neutral community model revealed that stochastic processes contributed to more than half of the community variations across the three domains. Co-occurrence network showed an equal number of positive and negative interactions in the archaeal network, while positive interactions accounted for ~ 80% in the bacterial and eukaryotic networks. Our findings reveal the ecological features of prokaryotes and eukaryotes in SYBH sediments and shed new light on community dynamics and survival strategies in the special environment of marine blue holes.

The Hypersaline Archaeal Histones HpyA and HstA Are DNA Binding Proteins That Defy Categorization According to Commonly Used Functional Criteria.

Histone proteins are found across diverse lineages of Archaea, many of which package DNA and form chromatin. However, previous research has led to the hypothesis that the histone-like proteins of high-salt-adapted archaea, or halophiles, function differently. The sole histone protein encoded by the model halophilic species Halobacterium salinarum, HpyA, is nonessential and expressed at levels too low to enable genome-wide DNA packaging. Instead, HpyA mediates the transcriptional response to salt stress. Here we compare the features of genome-wide binding of HpyA to those of HstA, the sole histone of another model halophile, Haloferax volcanii. hstA, like hpyA, is a nonessential gene. To better understand HpyA and HstA functions, protein-DNA binding data (chromatin immunoprecipitation sequencing [ChIP-seq]) of these halophilic histones are compared to publicly available ChIP-seq data from DNA binding proteins across all domains of life, including transcription factors (TFs), nucleoid-associated proteins (NAPs), and histones. These analyses demonstrate that HpyA and HstA bind the genome infrequently in discrete regions, which is similar to TFs but unlike NAPs, which bind a much larger genomic fraction. However, unlike TFs that typically bind in intergenic regions, HpyA and HstA binding sites are located in both coding and intergenic regions. The genome-wide dinucleotide periodicity known to facilitate histone binding was undetectable in the genomes of both species. Instead, TF-like and histone-like binding sequence preferences were detected for HstA and HpyA, respectively. Taken together, these data suggest that halophilic archaeal histones are unlikely to facilitate genome-wide chromatin formation and that their function defies categorization as a TF, NAP, or histone. IMPORTANCE Most cells in eukaryotic species-from yeast to humans-possess histone proteins that pack and unpack DNA in response to environmental cues. These essential proteins regulate genes necessary for important cellular processes, including development and stress protection. Although the histone fold domain originated in the domain of life Archaea, the function of archaeal histone-like proteins is not well understood relative to those of eukaryotes. We recently discovered that, unlike histones of eukaryotes, histones in hypersaline-adapted archaeal species do not package DNA and can act as transcription factors (TFs) to regulate stress response gene expression. However, the function of histones across species of hypersaline-adapted archaea still remains unclear. Here, we compare hypersaline histone function to a variety of DNA binding proteins across the tree of life, revealing histone-like behavior in some respects and specific transcriptional regulatory function in others.

Chromosome segregation in Archaea: SegA- and SegB-DNA complex structures provide insights into segrosome assembly.

Genome segregation is a vital process in all organisms. Chromosome partitioning remains obscure in Archaea, the third domain of life. Here, we investigated the SegAB system from Sulfolobus solfataricus. SegA is a ParA Walker-type ATPase and SegB is a site-specific DNA-binding protein. We determined the structures of both proteins and those of SegA-DNA and SegB-DNA complexes. The SegA structure revealed an atypical, novel non-sandwich dimer that binds DNA either in the presence or in the absence of ATP. The SegB structure disclosed a ribbon-helix-helix motif through which the protein binds DNA site specifically. The association of multiple interacting SegB dimers with the DNA results in a higher order chromatin-like structure. The unstructured SegB N-terminus plays an essential catalytic role in stimulating SegA ATPase activity and an architectural regulatory role in segrosome (SegA-SegB-DNA) formation. Electron microscopy results also provide a compact ring-like segrosome structure related to chromosome organization. These findings contribute a novel mechanistic perspective on archaeal chromosome segregation.

Hydrogel composite of lanthanum and Halorubrum ejinoor sp. cell lysate as an adsorbing material.

OBJECTIVES: Although halophilic archaea are rich in natural environments, their biotechnological applications are not as prevalent as those of other extremophiles, such as thermophiles and alkaliphiles. This study presents an simple method to prepare a hydrogel composite using crude cell lysate of a halophilic archaea, Halorubrum ejinoor sp. (H.e.) which was isolated from a saline lake in Inner Mongolia, China. Furthermore, formation mechanism and potential applications of the hydrogel as an adsorbing material are discussed. RESULTS: Halorubrum ejinoor sp. (H.e.) cell lysate was firstly prepared by adding pure water onto the H.e. cell pellet, followed by a short incubation at 60 °C. The cell lysate was injected into different metal ion (or H+) solutions to obtain the hydrogel composite. It was observed that H+, Fe3+, La3+, Cu2+, and Ca2+ induced gelation of the cell lysate, while Fe2+, Co2+, Ni2+, Mg2+, Na+, and K+ did not. DNA and extracellular polysaccharides (EPS) in the H.e. cell lysate were found to be responsible for the gelation reaction. These results suggest that DNA and EPS should be crosslinked by metal ions (or H+) and form a networked structure in which the metal ion (or H+) serves as an anchor point. Potential application of the hydrogel as an adsorbing material was explored using La3+-induced H.e. hydrogel composite. The hydrogel composite can adsorb the fluoride, phosphate and DNA-binding carcinogenic agents, such as acridine orange. CONCLUSIONS: The simplicity and cost effectiveness of the preparation method might make H.e. hydrogel a promising adsorbing material. This work is expected to expand the technical applications of haloarchaea.

Structural basis for recognition of distinct deaminated DNA lesions by endonuclease Q.

Spontaneous deamination of DNA cytosine and adenine into uracil and hypoxanthine, respectively, causes C to T and A to G transition mutations if left unrepaired. Endonuclease Q (EndoQ) initiates the repair of these premutagenic DNA lesions in prokaryotes by cleaving the phosphodiester backbone 5' of either uracil or hypoxanthine bases or an apurinic/apyrimidinic (AP) lesion generated by the excision of these damaged bases. To understand how EndoQ achieves selectivity toward these structurally diverse substrates without cleaving undamaged DNA, we determined the crystal structures of Pyrococcus furiosus EndoQ bound to DNA substrates containing uracil, hypoxanthine, or an AP lesion. The structures show that substrate engagement by EndoQ depends both on a highly distorted conformation of the DNA backbone, in which the target nucleotide is extruded out of the helix, and direct hydrogen bonds with the deaminated bases. A concerted swing motion of the zinc-binding and C-terminal helical domains of EndoQ toward its catalytic domain allows the enzyme to clamp down on a sharply bent DNA substrate, shaping a deep active-site pocket that accommodates the extruded deaminated base. Within this pocket, uracil and hypoxanthine bases interact with distinct sets of amino acid residues, with positioning mediated by an essential magnesium ion. The EndoQ-DNA complex structures reveal a unique mode of damaged DNA recognition and provide mechanistic insights into the initial step of DNA damage repair by the alternative excision repair pathway. Furthermore, we demonstrate that the unique activity of EndoQ is useful for studying DNA deamination and repair in mammalian systems.

VOLN27B: A New Head-Tailed Halovirus Isolated from an Underground Salt Crystal and Infecting Halorubrum.

A novel halovirus, VOLN27B, was isolated from a drill core sample taken at a depth of approximately 430 m, from a layer formed during the Cretaceous period (Anhui, China). VOLN27B infects the halophilic archaeon Halorubrum sp. LN27 and has a head-tailed morphotype with a contractile tail, typical of myoviruses. The average head diameter is 64 ± 2.0 nm, and uncontracted tails are 15 ± 1.0 × 65 ± 2.0 nm. The latent period is about 10 h. The maturing time of VOLN27B in cells of Halorubrum sp. LN27 was nearly 8 h. The adsorption time of VOLN27B on cells of Halorubrum sp. LN27 was less than 1 min. Virus particles are unstable at pH values less than 5 or when the NaCl concentration is below 12% (w/v). VOLN27B and Halorubrum sp. LN27 were recovered from the same hypersaline environment and provide a new virus-host system in haloarchaea.

A rod conformation of the Pyrococcus furiosus Rad50 coiled coil.

The Rad50-Mre11 nuclease complex plays a vital role in DNA repair in all domains of life. It recognizes and processes DNA double-strand breaks. Rad50 proteins fold into an extended structure with a 20 to 60 nm long coiled coil connecting a globular ABC ATPase domain with a zinc hook dimerization domain. A published structure of an archaeal Rad50 zinc hook shows coiled coils pointing away from each other. Here we present the crystal structure of an alternate conformation displaying co-aligned coiled coils. Archaeal Rad50 may thus switch between rod-shaped and ring-like conformations as recently proposed for a bacterial homolog.

Mechanical and structural properties of archaeal hypernucleosomes.

Many archaea express histones, which organize the genome and play a key role in gene regulation. The structure and function of archaeal histone-DNA complexes remain however largely unclear. Recent studies show formation of hypernucleosomes consisting of DNA wrapped around an 'endless' histone-protein core. However, if and how such a hypernucleosome structure assembles on a long DNA substrate and which interactions provide for its stability, remains unclear. Here, we describe micromanipulation studies of complexes of the histones HMfA and HMfB with DNA. Our experiments show hypernucleosome assembly which results from cooperative binding of histones to DNA, facilitated by weak stacking interactions between neighboring histone dimers. Furthermore, rotational force spectroscopy demonstrates that the HMfB-DNA complex has a left-handed chirality, but that torque can drive it in a right-handed conformation. The structure of the hypernucleosome thus depends on stacking interactions, torque, and force. In vivo, such modulation of the archaeal hypernucleosome structure may play an important role in transcription regulation in response to environmental changes.

Archaea: The Final Frontier of Chromatin.

The three domains of life employ various strategies to organize their genomes. Archaea utilize features similar to those found in both eukaryotic and bacterial chromatin to organize their DNA. In this review, we discuss the current state of research regarding the structure-function relationships of several archaeal chromatin proteins (histones, Alba, Cren7, and Sul7d). We address individual structures as well as inferred models for higher-order chromatin formation. Each protein introduces a unique phenotype to chromatin organization, and these structures are put into the context of in vivo and in vitro data. We close by discussing the present gaps in knowledge that are preventing further studies of the organization of archaeal chromatin, on both the organismal and domain level.

Structural Studies of HNA Substrate Specificity in Mutants of an Archaeal DNA Polymerase Obtained by Directed Evolution.

Archaeal DNA polymerases from the B-family (polB) have found essential applications in biotechnology. In addition, some of their variants can accept a wide range of modified nucleotides or xenobiotic nucleotides, such as 1,5-anhydrohexitol nucleic acid (HNA), which has the unique ability to selectively cross-pair with DNA and RNA. This capacity is essential to allow the transmission of information between different chemistries of nucleic acid molecules. Variants of the archaeal polymerase from Thermococcus gorgonarius, TgoT, that can either generate HNA from DNA (TgoT_6G12) or DNA from HNA (TgoT_RT521) have been previously identified. To understand how DNA and HNA are recognized and selected by these two laboratory-evolved polymerases, we report six X-ray structures of these variants, as well as an in silico model of a ternary complex with HNA. Structural comparisons of the apo form of TgoT_6G12 together with its binary and ternary complexes with a DNA duplex highlight an ensemble of interactions and conformational changes required to promote DNA or HNA synthesis. MD simulations of the ternary complex suggest that the HNA-DNA hybrid duplex remains stable in the A-DNA helical form and help explain the presence of mutations in regions that would normally not be in contact with the DNA if it were not in the A-helical form. One complex with two incorporated HNA nucleotides is surprisingly found in a one nucleotide-backtracked form, which is new for a DNA polymerase. This information can be used for engineering a new generation of more efficient HNA polymerase variants.

Two conformations of DNA polymerase D-PCNA-DNA, an archaeal replisome complex, revealed by cryo-electron microscopy.

BACKGROUND: DNA polymerase D (PolD) is the representative member of the D family of DNA polymerases. It is an archaea-specific DNA polymerase required for replication and unrelated to other known DNA polymerases. PolD consists of a heterodimer of two subunits, DP1 and DP2, which contain catalytic sites for 3'-5' editing exonuclease and DNA polymerase activities, respectively, with both proteins being mutually required for the full activities of each enzyme. However, the processivity of the replicase holoenzyme has additionally been shown to be enhanced by the clamp molecule proliferating cell nuclear antigen (PCNA), making it crucial to elucidate the interaction between PolD and PCNA on a structural level for a full understanding of its functional relevance. We present here the 3D structure of a PolD-PCNA-DNA complex from Thermococcus kodakarensis using single-particle cryo-electron microscopy (EM). RESULTS: Two distinct forms of the PolD-PCNA-DNA complex were identified by 3D classification analysis. Fitting the reported crystal structures of truncated forms of DP1 and DP2 from Pyrococcus abyssi onto our EM map showed the 3D atomic structural model of PolD-PCNA-DNA. In addition to the canonical interaction between PCNA and PolD via PIP (PCNA-interacting protein)-box motif, we found a new contact point consisting of a glutamate residue at position 171 in a ß-hairpin of PCNA, which mediates interactions with DP1 and DP2. The DNA synthesis activity of a mutant PolD with disruption of the E171-mediated PCNA interaction was not stimulated by PCNA in vitro. CONCLUSIONS: Based on our analyses, we propose that glutamate residues at position 171 in each subunit of the PCNA homotrimer ring can function as hooks to lock PolD conformation on PCNA for conversion of its activity. This hook function of the clamp molecule may be conserved in the three domains of life.

A hand-off of DNA between archaeal polymerases allows high-fidelity replication to resume at a discrete intermediate three bases past 8-oxoguanine.

During DNA replication, the presence of 8-oxoguanine (8-oxoG) lesions in the template strand cause the high-fidelity (HiFi) DNA polymerase (Pol) to stall. An early response to 8-oxoG lesions involves 'on-the-fly' translesion synthesis (TLS), in which a specialized TLS Pol is recruited and replaces the stalled HiFi Pol for lesion bypass. The length of TLS must be long enough for effective bypass, but it must also be regulated to minimize replication errors by the TLS Pol. The exact position where the TLS Pol ends and the HiFi Pol resumes (i.e. the length of the TLS patch) has not been described. We use steady-state and pre-steady-state kinetic assays to characterize lesion bypass intermediates formed by different archaeal polymerase holoenzyme complexes that include PCNA123 and RFC. After bypass of 8-oxoG by TLS PolY, products accumulate at the template position three base pairs beyond the lesion. PolY is catalytically poor for subsequent extension from this +3 position beyond 8-oxoG, but this inefficiency is overcome by rapid extension of HiFi PolB1. The reciprocation of Pol activities at this intermediate indicates a defined position where TLS Pol extension is limited and where the DNA substrate is handed back to the HiFi Pol after bypass of 8-oxoG.

The N-terminal domain of Staphylothermus marinus McrB shares structural homology with PUA-like RNA binding proteins.

McrBC is a conserved modification-dependent restriction system that in Escherichia coli specifically targets foreign DNA containing methylated cytosines. Crystallographic data show that the N-terminal domain of Escherichia coli McrB binds substrates via a base flipping mechanism. This region is poorly conserved among the plethora of McrB homologs, suggesting that other species may use alternative binding strategies and/or recognize different targets. Here we present the crystal structure of the N-terminal domain from Stayphlothermus marinus McrB (Sm3-180) at 1.92 Å, which adopts a PUA-like EVE fold that is closely related to the YTH and ASCH RNA binding domains. Unlike most PUA-like domains, Sm3-180 binds DNA and can associate with different modified substrates. We find the canonical 'aromatic cage' binding pocket that confers specificity for methylated bases in other EVE/YTH domains is degenerate and occluded in Sm3-180, which may contribute to its promiscuity in target recognition. Further structural comparison between different PUA-like domains identifies motifs and conformational variations that correlate with the preference for binding either DNA or RNA. Together these data have important implications for PUA-like domain specificity and suggest a broader biological versatility for the McrBC family than previously described.

Role of RadA and DNA Polymerases in Recombination-Associated DNA Synthesis in Hyperthermophilic Archaea.

Among the three domains of life, the process of homologous recombination (HR) plays a central role in the repair of double-strand DNA breaks and the restart of stalled replication forks. Curiously, main protein actors involved in the HR process appear to be essential for hyperthermophilic Archaea raising interesting questions about the role of HR in replication and repair strategies of those Archaea living in extreme conditions. One key actor of this process is the recombinase RadA, which allows the homologous strand search and provides a DNA substrate required for following DNA synthesis and restoring genetic information. DNA polymerase operation after the strand exchange step is unclear in Archaea. Working with Pyrococcus abyssi proteins, here we show that both DNA polymerases, family-B polymerase (PolB) and family-D polymerase (PolD), can take charge of processing the RadA-mediated recombination intermediates. Our results also indicate that PolD is far less efficient, as compared with PolB, to extend the invaded DNA at the displacement-loop (D-loop) substrate. These observations coincide with previous genetic analyses obtained on Thermococcus species showing that PolB is mainly involved in DNA repair without being essential probably because PolD could take over combined with additional partners.

Mechanistic insights into Lhr helicase function in DNA repair.

The DNA helicase Large helicase-related (Lhr) is present throughout archaea, including in the Asgard and Nanoarchaea, and has homologues in bacteria and eukaryotes. It is thought to function in DNA repair but in a context that is not known. Our data show that archaeal Lhr preferentially targets DNA replication fork structures. In a genetic assay, expression of archaeal Lhr gave a phenotype identical to the replication-coupled DNA repair enzymes Hel308 and RecQ. Purified archaeal Lhr preferentially unwound model forked DNA substrates compared with DNA duplexes, flaps and Holliday junctions, and unwound them with directionality. Single-molecule FRET measurements showed that binding of Lhr to a DNA fork causes ATP-independent distortion and base-pair melting at, or close to, the fork branchpoint. ATP-dependent directional translocation of Lhr resulted in fork DNA unwinding through the 'parental' DNA strands. Interaction of Lhr with replication forks in vivo and in vitro suggests that it contributes to DNA repair at stalled or broken DNA replication.

Archaeal Chromatin Proteins Cren7 and Sul7d Compact DNA by Bending and Bridging.

Archaeal chromatin proteins Cren7 and Sul7d from Sulfolobus are DNA benders. To better understand their architectural roles in chromosomal DNA organization, we analyzed DNA compaction by Cren7 and Sis7d, a Sul7d family member, from Sulfolobus islandicus at the single-molecule (SM) level by total single-molecule internal reflection fluorescence microscopy (SM-TIRFM) and atomic force microscopy (AFM). We show that both Cren7 and Sis7d were able to compact singly tethered λ DNA into a highly condensed structure in a three-step process and that Cren7 was over an order of magnitude more efficient than Sis7d in DNA compaction. The two proteins were similar in DNA bending kinetics but different in DNA condensation patterns. At saturating concentrations, Sis7d formed randomly distributed clusters whereas Cren7 generated a single and highly condensed core on plasmid DNA. This observation is consistent with the greater ability of Cren7 than of Sis7d to bridge DNA. Our results offer significant insights into the mechanism and kinetics of chromosomal DNA organization in Crenarchaea.IMPORTANCE A long-standing question is how chromosomal DNA is packaged in Crenarchaeota, a major group of archaea, which synthesize large amounts of unique small DNA-binding proteins but in general contain no archaeal histones. In the present work, we tested our hypothesis that the two well-studied crenarchaeal chromatin proteins Cren7 and Sul7d compact DNA by both DNA bending and bridging. We show that the two proteins are capable of compacting DNA, albeit with different efficiencies and in different manners, at the single molecule level. We demonstrate for the first time that the two proteins, which have long been regarded as DNA binders and benders, are able to mediate DNA bridging, and this previously unknown property of the proteins allows DNA to be packaged into highly condensed structures. Therefore, our results provide significant insights into the mechanism and kinetics of chromosomal DNA organization in Crenarchaeota.

Modes of action of the archaeal Mre11/Rad50 DNA-repair complex revealed by fast-scan atomic force microscopy.

Mre11 and Rad50 (M/R) proteins are part of an evolutionarily conserved macromolecular apparatus that maintains genomic integrity through repair pathways. Prior structural studies have revealed that this apparatus is extremely dynamic, displaying flexibility in the long coiled-coil regions of Rad50, a member of the structural maintenance of chromosome (SMC) superfamily of ATPases. However, many details of the mechanics of M/R chromosomal manipulation during DNA-repair events remain unclear. Here, we investigate the properties of the thermostable M/R complex from the archaeon Sulfolobus acidocaldarius using atomic force microscopy (AFM) to understand how this macromolecular machinery orchestrates DNA repair. While previous studies have observed canonical interactions between the globular domains of M/R and DNA, we observe transient interactions between DNA substrates and the Rad50 coiled coils. Fast-scan AFM videos (at 1-2 frames per second) of M/R complexes reveal that these interactions result in manipulation and translocation of the DNA substrates. Our study also shows dramatic and unprecedented ATP-dependent DNA unwinding events by the M/R complex, which extend hundreds of base pairs in length. Supported by molecular dynamic simulations, we propose a model for M/R recognition at DNA breaks in which the Rad50 coiled coils aid movement along DNA substrates until a DNA end is encountered, after which the DNA unwinding activity potentiates the downstream homologous recombination (HR)-mediated DNA repair.

Optimizing ultracentrifugation conditions for DNA-based stable isotope probing (DNA-SIP).

DNA-SIP (DNA-based stable isotope probing) is increasingly being employed in soil microbial ecology to identify those microbes assimilating the 13C/15N labelled substrate. Isopycnic gradient centrifugation is the primary experimental process for conducting DNA-SIP. However, diverse centrifugal conditions have been used in various recent studies. In order to get the optimum conditions of centrifugation for DNA-SIP, centrifugation time (36, 42, 48, 60 h), speed (45,000, 55,000 rpm) and the initial buoyant density (1.69, 1.71, 1.725 g ml-1), as were used extensively in related studies, were tested in this experiment with the Vti 65.2 rotor. DNA with either 13C-labelling or unlabelled was extracted from a paddy soil pre-incubated with either 13C-labelled or natural abundance glucose. After ultracentrifugation, the gene abundance of bacterial 16S rRNA, fungal 18S rRNA, bacterial and archaeal amoA within the fractioned DNA was detected. The results showed that centrifugation for 48 h was enough for the DNA to reach stabilization in the CsCl solution. The initial density of the mixed solution was best adjusted to 1.71 g ml-1 to ensure that most of the genes were concentrated on the middle fractions of the density gradient. Increasing the centrifugation speed would increase the density gradient of fractions; therefore, 45,000 rpm (184,000 g) was recommended so as to obtain the more widespread pattern of DNA in the centrifugal tube. We hope these findings will assist future researchers to conduct optimum ultracentrifugation for DNA-SIP.

Casposase structure and the mechanistic link between DNA transposition and spacer acquisition by CRISPR-Cas.

Key to CRISPR-Cas adaptive immunity is maintaining an ongoing record of invading nucleic acids, a process carried out by the Cas1-Cas2 complex that integrates short segments of foreign genetic material (spacers) into the CRISPR locus. It is hypothesized that Cas1 evolved from casposases, a novel class of transposases. We show here that the Methanosarcina mazei casposase can integrate varied forms of the casposon end in vitro, and recapitulates several properties of CRISPR-Cas integrases including site-specificity. The X-ray structure of the casposase bound to DNA representing the product of integration reveals a tetramer with target DNA bound snugly between two dimers in which single-stranded casposon end binding resembles that of spacer 3'-overhangs. The differences between transposase and CRISPR-Cas integrase are largely architectural, and it appears that evolutionary change involved changes in protein-protein interactions to favor Cas2 binding over tetramerization; this in turn led to preferred integration of single spacers over two transposon ends.

An alternative pathway for repair of deaminated bases in DNA triggered by archaeal NucS endonuclease.

Recent studies show that NucS endonucleases participate in mismatch repair in several archaea and bacteria. However, the function of archaeal NucS endonucleases has not been completely clarified. Here, we describe a NucS endonuclease from the hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans (Tga NucS) that can cleave uracil (U)- and hypoxanthine (I)-containing dsDNA at 80 °C. Biochemical evidence shows that the cleavage sites of the enzyme are at the second phosphodiester on the 5'- site of U or I, and at the third phosphodiester on the 5'-site of the opposite base of U or I, creating a double strand break with a 4-nt 5'-overhang.The ends of the cleaved product of Tga NucS are ligatable, possessing 5'-phosphate and 3'-hydroxyl termini, which can be utilized by DNA repair proteins or enzymes. Tga NucS displays a preference for U/G- and I/T-containing dsDNA over other pairs with U or I, suggesting that the enzyme is responsible for repair of U and I in DNA that arise from deamination. Biochemical characterization of cleaving U- and I-containing DNA by Tga NucS was also investigated. The DNA-binding results show that the enzyme exhibits a higher affinity for normal, U- and I-containing dsDNA than for normal, U- and I-containing ssDNA. Therefore, we present an alternative pathway for repair of deaminated bases in DNA triggered by archaeal NucS endonuclease in hyperthermophilic archaea.