RESUMO
To date, methanogens are the only group within the archaea where firing DNA replication origins have not been demonstrated in vivo. In the present study we show that a previously identified cluster of ORB (origin recognition box) sequences do indeed function as an origin of replication in vivo in the archaeon Methanothermobacter thermautotrophicus. Although the consensus sequence of ORBs in M. thermautotrophicus is somewhat conserved when compared with ORB sequences in other archaea, the Cdc6-1 protein from M. thermautotrophicus (termed MthCdc6-1) displays sequence-specific binding that is selective for the MthORB sequence and does not recognize ORBs from other archaeal species. Stabilization of in vitro MthORB DNA binding by MthCdc6-1 requires additional conserved sequences 3' to those originally described for M. thermautotrophicus. By testing synthetic sequences bearing mutations in the MthORB consensus sequence, we show that Cdc6/ORB binding is critically dependent on the presence of an invariant guanine found in all archaeal ORB sequences. Mutation of a universally conserved arginine residue in the recognition helix of the winged helix domain of archaeal Cdc6-1 shows that specific origin sequence recognition is dependent on the interaction of this arginine residue with the invariant guanine. Recognition of a mutated origin sequence can be achieved by mutation of the conserved arginine residue to a lysine or glutamine residue. Thus despite a number of differences in protein and DNA sequences between species, the mechanism of origin recognition and binding appears to be conserved throughout the archaea.
Assuntos
Archaea/genética , Complexo de Reconhecimento de Origem/metabolismo , Sequência de Aminoácidos , Archaea/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Cinética , Dados de Sequência MolecularRESUMO
The genome of Methanothermobacter thermautotrophicus, as a hitherto unique case, is apparently devoid of genes coding for general uracil DNA glycosylases, the universal mediators of base excision repair following hydrolytic deamination of DNA cytosine residues. We have now identified protein Mth212, a member of the ExoIII family of nucleases, as a possible initiator of DNA uracil repair in this organism. This enzyme, in addition to bearing all the enzymological hallmarks of an ExoIII homologue, is a DNA uridine endonuclease (U-endo) that nicks double-stranded DNA at the 5'-side of a 2'-d-uridine residue, irrespective of the nature of the opposing nucleotide. This type of activity has not been described before; it is absent from the ExoIII homologues of Escherichia coli, Homo sapiens and Methanosarcina mazei, all of which are equipped with uracil DNA repair glycosylases. The U-endo activity of Mth212 is served by the same catalytic center as its AP-endo activity.
Assuntos
Proteínas Arqueais/metabolismo , Endodesoxirribonucleases/metabolismo , Methanobacteriaceae/enzimologia , Uridina/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Catálise , Extratos Celulares/química , Clonagem Molecular , DNA/química , DNA/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/química , Genes Arqueais , Methanobacteriaceae/genética , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
A spontaneous mutant of Methanothermobacter thermautotrophicus resistant to the Na+/H+ antiporter inhibitor amiloride was isolated. The Na+/H+ exchanger activity in the mutant cells was remarkably decreased in comparison with wild-type cells. Methanogenesis rates in the mutant strain were higher than wild-type cells and resistant to the inhibitory effect of 2 mM amiloride. In contrast, methanogenesis in wild-type cells was completely inhibited by the same amiloride concentration. ATP synthesis driven by methanogenic electron transport or by an electrogenic potassium efflux in the presence of sodium ions was significantly enhanced in the mutant cells. ATP synthesis driven by potassium diffusion potential was profoundly inhibited in wild-type cells by the presence of uncoupler 3,3',4',5- tetrachlorosalicylanilide and sodium ions, whereas c. 50% inhibition was observed in the mutant cells under the same conditions.
Assuntos
Amilorida/farmacologia , Farmacorresistência Bacteriana , Methanobacteriaceae/classificação , Mutação , Bloqueadores dos Canais de Sódio/farmacologia , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Metano/metabolismo , Methanobacteriaceae/efeitos dos fármacos , Methanobacteriaceae/genética , Methanobacteriaceae/isolamento & purificação , FenótipoRESUMO
The effect of Ca2+ ions on methanogenesis and growth of Methanothermobacter thermautotrophicus was investigated. The calcium chelator ethylene glycol bis(2-aminoethylether)-N,N,N',N'-tetra-acetic acid, calcium ionophore A23187 and ruthenium red all inhibited growth of this strain. Methane formation was strongly dependent on the external Ca2+ concentration in a resting cell suspension. In addition, methanogenesis of Ca2+ preloaded cells was stimulated by 400%. Inhibitor studies revealed that Co2+ and Ni2+, inorganic antagonists of Ca2+ transport, strongly inhibited methanogenesis in these cells. Interestingly, our findings imply that one of the enzymes of methanogenesis might catalyse a Ca2+ -dependent step and allow a direct activation of methanogenesis by Ca2+ ions.
Assuntos
Cálcio/fisiologia , Metano/biossíntese , Methanobacteriaceae/metabolismo , Trifosfato de Adenosina/biossíntese , Transporte Biológico/fisiologia , Cálcio/antagonistas & inibidores , Bloqueadores dos Canais de Cálcio/farmacologia , Meios de Cultura , Ácido Egtázico/farmacologia , Methanobacteriaceae/efeitos dos fármacos , Methanobacteriaceae/crescimento & desenvolvimento , Rutênio Vermelho/farmacologiaRESUMO
We have identified a novel structure-specific nuclease in highly fractionated extracts of the thermophilic archaeon Methanothermobacter thermautotrophicus (Mth). The 71 kDa protein product of open reading frame mth1090 is a nuclease with ATPase activity, which we call Nar71 (Nuclease-ATPase in Repair, 71 kDa). The nar71 gene is located in a gene neighbourhood proposed by genomics to encode a novel DNA repair system conserved in thermophiles. The biochemical characterization of Nar71 presented here is the first analysis from within this neighbourhood, and it supports the insight from genomics. Nuclease activity of Nar71 is specific for 3' flaps and flayed duplexes, targeting single-stranded DNA (ssDNA) regions. This activity requires Mg2+ or Mn2+ and is greatly reduced in ATP. In ATP, Nar71 displaces ssDNA, also with high specificity for 3' flap and flayed duplex DNA. Strand displacement is weak compared with nuclease activity, but in ATPS it is abolished, suggesting that Nar71 couples ATP hydrolysis to DNA strand separation. ATPase assays confirmed that Nar71 is stimulated by ssDNA, though not double-stranded DNA. Mutation of Lys-117 in Nar71 abolished ATPase and nuclease activity, and we describe a separation-of-function mutant (K68A) that has lost ATPase activity but retains nuclease activity. A model of possible Nar71 function in DNA repair is presented.
Assuntos
Adenosina Trifosfatases/metabolismo , Reparo do DNA , Desoxirribonucleases/metabolismo , Methanobacteriaceae/enzimologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Catálise , DNA de Cadeia Simples/metabolismo , Desoxirribonucleases/química , Desoxirribonucleases/genética , Methanobacteriaceae/genética , Dados de Sequência Molecular , Fases de Leitura AbertaRESUMO
Flow cytometry and epifluorescence microscopy results for the euryarchaeon Methanothermobacter thermautotrophicus were consistent with filaments containing multiple cells. Filaments of one to four cells contained two to eight nucleoids. Single chromosome-containing cells were not observed. Filaments containing multiple genome copies displayed synchronous DNA replication initiation. Chromosome segregation occurred during replication or rapidly after replication termination.