RESUMO
The minichromosome maintenance (MCM) helicase is the presumptive replicative helicase in archaea and eukaryotes. The archaeal homomultimeric MCM has a two-tier structure. One tier contains the AAA+ motor domains of the proteins, and these are the minimal functional helicase domains. The second tier is formed by the N-terminal domains. These domains are not essential for MCM helicase activity but act to enhance the processivity of the helicase. We reveal that a conserved loop facilitates communication between processivity and motor tiers. Interestingly, this allostery seems to be mediated by interactions between, rather than within, individual protomers in the MCM ring.
Assuntos
Sequência Conservada , DNA Helicases/química , DNA Helicases/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Sulfolobus solfataricus/enzimologia , Regulação Alostérica , Sequência de Aminoácidos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Nucleotídeos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Deleção de Sequência , Relação Estrutura-AtividadeRESUMO
The dinoflagellate chloroplast genome is fragmented into a number of plasmid-like minicircles, mostly containing one or more genes, and with a conserved core. We show here that, in addition to the transcripts of similar sizes to individual genes that have been reported previously, there are larger transcripts beginning and ending close to the core region. These may give rise to the smaller transcripts by processing. We also show that previously reported ORFs (open reading frames) are represented by transcripts that are significantly more abundant than those for non-coding regions, indicating that the ORFs are indeed functional. We show that 'empty' minicircles are also transcribed. We propose a model for linkage of DNA replication and transcription in dinoflagellate chloroplasts.
Assuntos
DNA de Cloroplastos/genética , Dinoflagellida/genética , Plastídeos/genética , Transcrição Gênica , Animais , Northern Blotting , Modelos Genéticos , Plasmídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The mini-chromosome maintenance (MCM) complex is the principal candidate for the replicative helicase of archaea and eukaryotes. Here, we describe a functional dissection of the roles of the three principal structural modules of the homomultimeric MCM of the hyperthermophilic archaeon Sulfolobus solfataricus. Our results include the first analysis of the central AAA+ domain in isolation. This domain possesses ATPase and helicase activity, defining this as the minimal helicase domain. Reconstitution experiments show that the helicase activity of the AAA+ domain can be stimulated by addition of the isolated N-terminal half in trans. Addition of the N-terminus influences both the processivity of the helicase and the choice of substrate that can be melted by the ATPase domain. The degenerate helix-turn-helix domain at the C-terminus of MCM exerts a negative effect on the helicase activity of the complex. These results provide the first evidence for extensive regulatory inter-domain communication within the MCM complex.
Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Sulfolobus solfataricus/enzimologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas Arqueais/genética , DNA/metabolismo , DNA Helicases/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Estrutura Terciária de Proteína , Deleção de Sequência , Especificidade por SubstratoRESUMO
The archaeal DNA replication machinery bears striking similarity to that of eukaryotes and is clearly distinct from the bacterial apparatus. In recent years, considerable advances have been made in understanding the biochemistry of the archaeal replication proteins. Furthermore, a number of structures have now been obtained for individual components and higher-order assemblies of archaeal replication factors, yielding important insights into the mechanisms of DNA replication in both archaea and eukaryotes.
Assuntos
Archaea/genética , Replicação do DNA , Genoma Arqueal , Archaea/enzimologia , Proteínas de Transporte/metabolismo , DNA Arqueal/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Regulação da Expressão Gênica em Archaea/fisiologiaRESUMO
In eukaryotes, the GINS complex is essential for DNA replication and has been implicated as having a role at the replication fork. This complex consists of four paralogous GINS subunits, Psf1, Psf2, Psf3 and Sld5. Here, we identify an archaeal GINS homologue as a direct interaction partner of the MCM helicase. The core archaeal GINS complex contains two subunits that are poorly conserved homologues of the eukaryotic GINS subunits, in complex with a protein containing a domain homologous to the DNA-binding domain of bacterial RecJ. Interaction studies show that archaeal GINS interacts directly with the heterodimeric core primase. Our data suggest that GINS is important in coordinating the architecture of the replication fork and provide a mechanism to couple progression of the MCM helicase on the leading strand with priming events on the lagging strand.