Alexei I. Slesarev

The complete genome of hyperthermophile Methanopyrus kandleri AV19 and monophyly of archaeal methanogens

We have determined the complete 1,694,969-nt sequence of the GC-rich genome of Methanopyrus kandleri by using a whole direct genome sequencing approach. This approach is based on unlinking of genomic DNA with the ThermoFidelase version of M. kandleri topoisomerase V and cycle sequencing directed by 2′-modified oligonucleotides (Fimers). Sequencing redundancy (3.3×) was sufficient to assemble the genome with less than one error per 40 kb. Using a combination of sequence database searches and coding potential prediction, 1,692 protein-coding genes and 39 genes for structural RNAs were identified. M. kandleri proteins show an unusually high content of negatively charged amino acids, which might be an adaptation to the high intracellular salinity. Previous phylogenetic analysis of 16S RNA suggested that M. kandleri belonged to a very deep branch, close to the root of the archaeal tree. However, genome comparisons indicate that, in both trees constructed using concatenated alignments of ribosomal proteins and trees based on gene content, M. kandleri consistently groups with other archaeal methanogens. M. kandleri shares the set of genes implicated in methanogenesis and, in part, its operon organization with Methanococcus jannaschii and Methanothermobacter thermoautotrophicum. These findings indicate that archaeal methanogens are monophyletic. A distinctive feature of M. kandleri is the paucity of proteins involved in signaling and regulation of gene expression. Also, M. kandleri appears to have fewer genes acquired via lateral transfer than other archaea. These features might reflect the extreme habitat of this organism.

Helix–hairpin–helix motifs confer salt resistance and processivity on chimeric DNA polymerases

Helix–hairpin–helix (HhH) is a widespread motif involved in sequence-nonspecific DNA binding. The majority of HhH motifs function as DNA-binding modules with typical occurrence of one HhH motif or one or two (HhH)2 domains in proteins. We recently identified 24 HhH motifs in DNA topoisomerase V (Topo V). Although these motifs are dispensable for the topoisomerase activity of Topo V, their removal narrows the salt concentration range for topoisomerase activity tenfold. Here, we demonstrate the utility of Topo Vs HhH motifs for modulating DNA-binding properties of the Stoffel fragment of TaqDNA polymerase and Pfu DNA polymerase. Different HhH cassettes fused with either NH2 terminus or COOH terminus of DNA polymerases broaden the salt concentration range of the polymerase activity significantly (up to 0.5 M NaCl or 1.8 M potassium glutamate). We found that anions play a major role in the inhibition of DNA polymerase activity. The resistance of initial extension rates and the processivity of chimeric polymerases to salts depend on the structure of added HhH motifs. Regardless of the type of the construct, the thermal stability of chimeric Taq polymerases increases under the optimal ionic conditions, as compared with that of TaqDNA polymerase or its Stoffel fragment. Our approach to raise the salt tolerance, processivity, and thermostability of Taq and Pfu DNA polymerases may be applied to all pol1- and polB-type polymerases, as well as to other DNA processing enzymes.

Topoisomerase V relaxes supercoiled DNA by a constrained swiveling mechanism

Topoisomerase V is a type I topoisomerase without structural or sequence similarities to other topoisomerases. Although it belongs to the type I subfamily of topoisomerases, it is unrelated to either type IA or IB enzymes. We used real-time single-molecule micromechanical experiments to show that topoisomerase V relaxes DNA via events that release multiple DNA turns, employing a constrained swiveling mechanism similar to that for type IB enzymes. Relaxation is powered by the torque in the supercoiled DNA and is constrained by friction between the protein and the DNA. Although all type IB enzymes share a common structure and mechanism and type IA and type II enzymes show marked structural and functional similarities, topoisomerase V represents a different type of topoisomerase that relaxes DNA in a similar overall manner as type IB molecules but by using a completely different structural and mechanistic framework.

An Ancestral Nuclear Protein Assembly: Crystal Structure of the 'Methanopyrus kandleri' Histone

Eukaryotic histone proteins condense DNA into compact structures called nucleosomes. Nucleosomes were viewed as a distinguishing feature of eukaryotes prior to ideication of histone orthologs in methanogens. Although evolutionarily distinct from methanogens, the methane‐producing hyperthermophile Methanopyrus kandleri produces a novel, 154‐residue histone (HMk). Amino acid sequence comparisons show that HMk differs from both methanogenic and eukaryotic histones, in that it contains two histone‐fold ms within a single chain. The two HMk histone‐fold ms, N and C terminal, are 28% identical in amino acid sequence to each other and ∼21% identical in amino acid sequence to other histone proteins. Here we present the 1.37‐Å‐resolution crystal structure of HMk and report that the HMk monomer structure is homologous to the eukaryotic histone heterodimers. In the crystal, HMk forms a dimer homologous to [H3–H4]2 in the eukaryotic nucleosome. Based on the spatial similarities to structural ms found in the eukaryotic nucleosome that are important for DNA‐binding, we infer that the Methanopyrus histone binds DNA in a manner similar to the eukaryotic histone tetramer [H3–H4]2.

Structure of the N-terminal fragment of topoisomerase V reveals a new family of topoisomerases

Topoisomerases are involved in controlling and maintaining the topology of DNA and are present in all kingdoms of life. Unlike all other types of topoisomerases, similar type IB enzymes have only been identified in bacteria and eukarya. The only putative type IB topoisomerase in archaea is represented by Methanopyrus kandleri topoisomerase V. Despite several common functional characteristics, topoisomerase V shows no sequence similarity to other members of the same type. The structure of the 61 kDa N‐terminal fragment of topoisomerase V reveals no structural similarity to other topoisomerases. Furthermore, the structure of the active site region is different, suggesting no conservation in the cleavage and religation mechanism. Additionally, the active site is buried, indicating the need of a conformational change for activity. The presence of a topoisomerase in archaea with a unique structure suggests the evolution of a separate mechanism to alter DNA.

Genome Comparison and Proteomic Characterization of Thermus thermophilus Bacteriophages P23-45 and P74-26 : Siphoviruses with Triplex-forming Sequences and the Longest Known Tails

The genomes of two closely related lytic Thermus thermophilus siphoviruses with exceptionally long (approximately 800 nm) tails, bacteriophages P23-45 and P74-26, were sequenced completely. The P23-45 genome consists of 84,201 bp with 117 putative open reading frames (ORFs), and the P74-26 genome has 83,319 bp and 116 putative ORFs. The two genomes are 92% identical with 113 ORFs shared. Only 25% of phage gene product functions can be predicted from similarities to proteins and protein domains with known functions. The structural genes of P23-45, most of which have no similarity to sequences from public databases, were identified by mass spectrometric analysis of virions. An unusual feature of the P23-45 and P74-26 genomes is the presence, in their largest intergenic regions, of long polypurine-polypyrimidine (R-Y) sequences with mirror repeat symmetry. Such sequences, abundant in eukaryotic genomes but rare in prokaryotes, are known to form stable triple helices that block replication and transcription and induce genetic instability. Comparative analysis of the two phage genomes shows that the area around the triplex-forming elements is enriched in mutational variations. In vitro, phage R-Y sequences form triplexes and block DNA synthesis by Taq DNA polymerase in orientation-dependent manner, suggesting that they may play a regulatory role during P23-45 and P74-26 development.

A type IB topoisomerase with DNA repair activities

Previously we have characterized type IB DNA topoisomerase V (topo V) in the hyperthermophile Methanopyrus kandleri. The enzyme has a powerful topoisomerase activity and is abundant in M. kandleri. Here we report two characterizations of topo V. First, we found that its N-terminal domain has sequence homology with both eukaryotic type IB topoisomerases and the integrase family of tyrosine recombinases. The C-terminal part of the sequence includes 12 repeats, each repeat consisting of two similar but distinct helix-hairpin-helix motifs; the same arrangement is seen in recombination protein RuvA and mammalian DNA polymerase β. Second, on the basis of sequence homology between topo V and polymerase β, we predict and demonstrate that topo V possesses apurinic/apyrimidinic (AP) site-processing activities that are important in base excision DNA repair: (i) it incises the phosphodiester backbone at the AP site, and (ii) at the AP endonuclease cleaved AP site, it removes the 5′ 2-deoxyribose 5-phosphate moiety so that a single-nucleotide gap with a 3′-hydroxyl and 5′-phosphate can be filled by a DNA polymerase. Topo V is thus the prototype for a new subfamily of type IB topoisomerases and is the first example of a topoisomerase with associated DNA repair activities.

Cooperation between Catalytic and DNA Binding Domains Enhances Thermostability and Supports DNA Synthesis at Higher Temperatures by Thermostable DNA Polymerases

We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases [Pavlov, A. R., et al. (2002) Proc. Natl. Acad. Sci. U.S.A.99, 13510-13515]. The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various sequence-nonspecific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting helix-hairpin-helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of Topo V HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105 °C by maintaining processivity of DNA synthesis at high temperatures. We found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding of templates to DNA polymerases.

[15] Topoisomerase V from Methanopyrus kandleri

Publisher Summary Topoisomerases are enzymes that alter the topological state of nucleic acids by generating transient breaks in the sugar-phosphate backbone of DNA. In order to maintain the integrity of the genetic material during this process, topoisomerases form covalent bonds with DNA termini created by their actions. This covalent linkage is a hallmark of all topoisomerases. The first, and so far the only, prokaryotic type IB topoisomerase, named topoisomerase V, was discovered in Methanopyrus kandleri , a hyperthermophilic methanogen, which grows in temperatures up to 110 ° 15 and represents a separate lineage distinct from other methanogens. It has been shown that topoisomerase V is a single polypeptide with a molecular mass of about 110 kDa. The enzyme relaxes positive and negative supercoils with a rate similar to or even higher than other topoisomerases, does not require divalent cations for activity, and binds to the 3ʹ end of the cleaved DNA strand. The chapter describes the purification and characterization of DNA topoisomerase V from Methanopyrus kandleri cells. Furthermore, current applications of topoisomerase V in DNA technology are also presented, including discussion of those features of the enzyme that are of interest for DNA sequencing.