Recombinant DNA technology, in which artificially "cut and pasted" DNA
is introduced into living cells, contributed extensively to the rapid development of molecular biology over the past 5 decades ...since the latter half of the 20
century. Although the original technology required special experiences and skills, the development of polymerase chain reaction (PCR) has greatly eased
genetic manipulation for various experimental methods. The current development of a simple genome-editing technique using CRISPR-Cas9 gave great impetus to molecular biology. Genome editing is a major technique for elucidating the functions of many unknown genes. Genetic manipulation technologies rely on enzymes that act on DNA. It involves artificially synthesizing, cleaving, and ligating DNA strands by making good use of DNA-related enzymes present in organisms to maintain their life activities. In this review, I focus on key enzymes involved in the development of genetic manipulation technologies.
The recent discovery of the CRISPR-Cas-mediated acquired immunity system highlights the fact that our knowledge of phage/virus defense mechanisms encoded in bacterial and archaeal genomes is far from ...complete. Indeed, new prokaryotic immune systems are now continually being discovered. A recent report described a novel glycosylase that recognizes α-glycosyl-hydroxymethyl cytosin (α-Glu-hmC), a modified base observed in the T4 phage genome, where it produces an abasic site, thereby inhibiting the phage propagation.
Highlights • Hef may function in stalled fork repair by association of its IDR with multiple partners. • EndoQ cleaves the phosphodiester bond 5′ from dI, dU, dX, and the AP site in DNA strand. • ...PprA plays a principal role in CNDEJ by forming a larger nucleoprotein complex. • PprI cleaves DdrO, a radiation-inducible repressor of RDR regulon.
Origin of DNA replication is an enigma because the replicative DNA polymerases (DNAPs) are not homologous among the three domains of life, Bacteria, Archaea, and Eukarya. The homology between the ...archaeal replicative DNAP (PolD) and the large subunits of the universal RNA polymerase (RNAP) responsible for transcription suggests a parsimonious evolutionary scenario. Under this model, RNAPs and replicative DNAPs evolved from a common ancestor that functioned as an RNA-dependent RNA polymerase in the RNA-protein world that predated the advent of DNA replication. The replicative DNAP of the Last Universal Cellular Ancestor (LUCA) would be the ancestor of the archaeal PolD.
DNA base deamination occurs spontaneously under physiological conditions and is promoted by high temperature. Therefore, hyperthermophiles are expected to have efficient repair systems of the ...deaminated bases in their genomes. Endonuclease Q (EndoQ) was originally identified from the hyperthermophlic archaeon, Pyrococcus furiosus, as a hypoxanthine-specific endonuclease recently. Further biochemical analyses revealed that EndoQ also recognizes uracil, xanthine, and the AP site in DNA, and is probably involved in a specific repair process for damaged bases. Initial phylogenetic analysis showed that an EndoQ homolog is found only in the Thermococcales and some of the methanogens in Archaea, and is not present in most members of the domains Bacteria and Eukarya. A better understanding of the distribution of the EndoQ-mediated repair system is, therefore, of evolutionary interest. We showed here that an EndoQ-like polypeptide from Bacillus pumilus, belonging to the bacterial domain, is functional and has similar properties with the archaeal EndoQs.
The common mismatch repair system processed by MutS and MutL and their homologs was identified in Bacteria and Eukarya. However, no evidence of a functional MutS/L homolog has been reported for ...archaeal organisms, and it is not known whether the mismatch repair system is conserved in Archaea. Here, we describe an endonuclease that cleaves double-stranded DNA containing a mismatched base pair, from the hyperthermophilic archaeon Pyrococcus furiosus The corresponding gene revealed that the activity originates from PF0012, and we named this enzyme Endonuclease MS (EndoMS) as the mismatch-specific Endonuclease. The sequence similarity suggested that EndoMS is the ortholog of NucS isolated from Pyrococcus abyssi, published previously. Biochemical characterizations of the EndoMS homolog from Thermococcus kodakarensis clearly showed that EndoMS specifically cleaves both strands of double-stranded DNA into 5'-protruding forms, with the mismatched base pair in the central position. EndoMS cleaves G/T, G/G, T/T, T/C and A/G mismatches, with a more preference for G/T, G/G and T/T, but has very little or no effect on C/C, A/C and A/A mismatches. The discovery of this endonuclease suggests the existence of a novel mismatch repair process, initiated by the double-strand break generated by the EndoMS endonuclease, in Archaea and some Bacteria.
Summary
Viruses infecting hyperthermophilic archaea of the phylum Crenarchaeota display enormous morphological and genetic diversity, and are classified into 12 families. Eight of these families ...include only one or two species, indicating sparse sampling of the crenarchaeal virus diversity. In an attempt to expand the crenarchaeal virome, we explored virus diversity in the acidic, hot spring Umi Jigoku in Beppu, Japan. Environmental samples were used to establish enrichment cultures under conditions favouring virus replication. The host diversity in the enrichment cultures was restricted to members of the order Sulfolobales. Metagenomic sequencing of the viral communities yielded seven complete or near‐complete double‐stranded DNA virus genomes. Six of these genomes could be attributed to polyhedral and filamentous viruses that were observed by electron microscopy in the enrichment cultures. Two icosahedral viruses represented species in the family Portogloboviridae. Among the filamentous viruses, two were identified as new species in the families Rudiviridae and Lipothrixviridae, whereas two other formed a group seemingly distinct from the known virus genera. No particle morphotype could be unequivocally assigned to the seventh viral genome, which apparently represents a new virus type. Our results suggest that filamentous viruses are globally distributed and are prevalent virus types in extreme geothermal environments.
Translation elongation factor EF1A delivers aminoacyl-tRNA to the ribosome in a GTP-bound form, and is released from the ribosome in a GDP-bound form. This association/dissociation cycle proceeds ...efficiently via a marked conformational change in EF1A. EF1A function is dependent on the ribosomal "stalk" protein of the ribosomal large subunit, although the precise mechanism of action of the stalk on EF1A remains unclear. Here, we clarify the binding mode of archaeal stalk aP1 to GTP-bound aEF1A associated with aPelota. Intriguingly, the C-terminal domain (CTD) of aP1 binds to aEF1A•GTP with a similar affinity to aEF1A•GDP. We have also determined the crystal structure of the aP1-CTD•aEF1A•GTP•aPelota complex at 3.0 Å resolution. The structure shows that aP1-CTD binds to a space between domains 1 and 3 of aEF1A. Biochemical analyses show that this binding is crucial for protein synthesis. Comparison of the structures of aP1-CTD•aEF1A•GTP and aP1-CTD•aEF1A•GDP demonstrates that the binding mode of aP1 changes markedly upon a conformational switch between the GTP- and GDP-bound forms of aEF1A. Taking into account biochemical data, we infer that aP1 employs its structural flexibility to bind to aEF1A before and after GTP hydrolysis for efficient protein synthesis.
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