RESUMO
Hef is an archaeal member of the DNA repair endonuclease XPF (XPF)/Crossover junction endonuclease MUS81 (MUS81)/Fanconi anemia, complementation group M (FANCM) protein family that in eukaryotes participates in the restart of stalled DNA replication forks. To investigate the physiological roles of Hef in maintaining genome stability in living archaeal cells, we studied the localization of Hef-green fluorescent protein fusions by fluorescence microscopy. Our studies revealed that Haloferax volcanii Hef proteins formed specific localization foci under regular growth conditions, the number of which specifically increased in response to replication arrest. Purification of the full-length Hef protein from its native host revealed that it forms a stable homodimer in solution, with a peculiar elongated configuration. Altogether our data indicate that the shape of Hef, significant physicochemical constraints and/or interactions with DNA limit the apparent cytosolic diffusion of halophilic DNA replication/repair complexes, and demonstrate that Hef proteins are dynamically recruited to archaeal eukaryotic-like chromatin to counteract DNA replication stress. We suggest that the evolutionary conserved function of Hef/FANCM proteins is to enhance replication fork stability by directly interacting with collapsed replication forks.
Assuntos
Proteínas Arqueais/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Afidicolina/farmacologia , Proteínas Arqueais/análise , Proteínas Arqueais/genética , Tamanho Celular/efeitos dos fármacos , Dano ao DNA , DNA Helicases/análise , DNA Helicases/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/análise , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Fluorescência , Corantes Fluorescentes/análise , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Haloferax volcanii/citologia , Haloferax volcanii/metabolismo , Resolvases de Junção Holliday/fisiologia , Multimerização Proteica , Proteínas Recombinantes de Fusão/análiseRESUMO
We show that Pyrococcus abyssi PAB2263 (dubbed NucS (nuclease for ss DNA) is a novel archaeal endonuclease that interacts with the replication clamp PCNA. Structural determination of P. abyssi NucS revealed a two-domain dumbbell-like structure that in overall does not resemble any known protein structure. Biochemical and structural studies indicate that NucS orthologues use a non-catalytic ssDNA-binding domain to regulate the cleavage activity at another site, thus resulting into the specific cleavage at double-stranded DNA (dsDNA)/ssDNA junctions on branched DNA substrates. Both 3' and 5' extremities of the ssDNA can be cleaved at the nuclease channel that is too narrow to accommodate duplex DNA. Altogether, our data suggest that NucS proteins constitute a new family of structure-specific DNA endonucleases that are widely distributed in archaea and in bacteria, including Mycobacterium tuberculosis.
Assuntos
DNA Arqueal/metabolismo , DNA de Cadeia Simples/metabolismo , Endonucleases/química , Endonucleases/metabolismo , Pyrococcus abyssi/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , DNA Arqueal/química , DNA de Cadeia Simples/química , Endonucleases/genética , Endonucleases/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mycobacterium tuberculosis/enzimologia , Conformação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Branched DNA structures that occur during DNA repair and recombination must be efficiently processed by structure-specific endonucleases in order to avoid cell death. In the present paper, we summarize our screen for new interaction partners for the archaeal replication clamp that led to the functional characterization of a novel endonuclease family, dubbed NucS. Structural analyses of Pyrococcus abyssi NucS revealed an unexpected binding site for ssDNA (single-stranded DNA) that directs, together with the replication clamp, the nuclease activity of this protein towards ssDNA-dsDNA (double-stranded DNA) junctions. Our studies suggest that understanding the detailed architecture and dynamic behaviour of the NucS (nuclease specific for ssDNA)-PCNA (proliferating-cell nuclear antigen) complex with DNA will be crucial for identification of its physiologically relevant activities.
Assuntos
DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Endodesoxirribonucleases/metabolismo , Conformação de Ácido Nucleico , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Replicação do DNA , Endodesoxirribonucleases/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Antígeno Nuclear de Célula em Proliferação/metabolismo , Conformação Proteica , Pyrococcus abyssi/genética , Pyrococcus abyssi/metabolismo , Ribonucleases/química , Ribonucleases/metabolismo , Alinhamento de SequênciaRESUMO
During DNA replication and repair, many proteins bind to and dissociate in a highly specific and ordered manner from proliferating cell nuclear antigen (PCNA). We describe a combined approach of in silico searches at the genome level and combinatorial peptide synthesis to investigate the binding properties of hundreds of short PCNA-interacting peptides (PIP-peptides) to archaeal and eukaryal PCNAs. Biological relevance of our combined approach was demonstrated by identification an inactive complex of Pyrococcus abyssi ribonuclease HII with PCNA. Furthermore we show that PIP-peptides interact with PCNA largely in a sequence independent manner. Our experimental approach also identified many so far unidentified PCNA interacting peptides in a number of human proteins.
Assuntos
Peptídeos/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteoma , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , DNA Ligases/genética , DNA Ligases/metabolismo , Reparo do DNA , Endonucleases Flap/genética , Endonucleases Flap/metabolismo , Humanos , Dados de Sequência Molecular , Peptídeos/genética , Antígeno Nuclear de Célula em Proliferação/genética , Pyrococcus abyssi/enzimologia , Ribonuclease H/genética , Ribonuclease H/metabolismo , Alinhamento de SequênciaRESUMO
Nucleases are required to process and repair DNA damage in living cells. One of the best studied nucleases is the RecB protein, which functions in Escherichia coli as a component of the RecBCD enzyme complex that amends double-strand breaks in DNA. Although archaea do not contain the RecBCD complex, a RecB-like nuclease from Pyrococcus abyssi has been cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 81.5, b = 159.8, c = 100.8 A. Self-rotation function and native Patterson map calculations revealed that there is a dimer in the asymmetric unit with its local twofold axis running parallel to the crystallographic twofold screw axis. The crystals diffracted to about 2 A and a complete native data set was collected to 2.65 A resolution.
Assuntos
Exonucleases/química , Pyrococcus abyssi/enzimologia , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Conformação ProteicaRESUMO
As DNA repair enzymes are essential for preserving genome integrity, understanding their substrate interaction dynamics and the regulation of their catalytic mechanisms is crucial. Using single-molecule imaging, we investigated the association and dissociation kinetics of the bipolar endonuclease NucS from Pyrococcus abyssi (Pab) on 5' and 3'-flap structures under various experimental conditions. We show that association of the PabNucS with ssDNA flaps is largely controlled by diffusion in the NucS-DNA energy landscape and does not require a free 5' or 3' extremity. On the other hand, NucS dissociation is independent of the flap length and thus independent of sliding on the single-stranded portion of the flapped DNA substrates. Our kinetic measurements have revealed previously unnoticed asymmetry in dissociation kinetics from these substrates that is markedly modulated by the replication clamp PCNA. We propose that the replication clamp PCNA enhances the cleavage specificity of NucS proteins by accelerating NucS loading at the ssDNA/dsDNA junctions and by minimizing the nuclease interaction time with its DNA substrate. Our data are also consistent with marked reorganization of ssDNA and nuclease domains occurring during NucS catalysis, and indicate that NucS binds its substrate directly at the ssDNA-dsDNA junction and then threads the ssDNA extremity into the catalytic site. The powerful techniques used here for probing the dynamics of DNA-enzyme binding at the single-molecule have provided new insight regarding substrate specificity of NucS nucleases.
Assuntos
Proteínas Arqueais/metabolismo , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Endonucleases Flap/metabolismo , Proteínas Arqueais/química , Domínio Catalítico , Replicação do DNA , Endonucleases Flap/química , Cinética , Manganês/química , Fotodegradação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Pyrococcus abyssi/enzimologia , Especificidade por Substrato , Temperatura , ViscosidadeRESUMO
The genome of Pyrococcus abyssi contains two open reading frames encoding proteins which had been previously predicted to be DNA ligases, Pab2002 and Pab1020. We show that while the former is indeed a DNA ligase, Pab1020 had no effect on the substrate deoxyoligo-ribonucleotides tested. Instead, Pab1020 catalyzes the nucleotidylation of oligo-ribonucleotides in an ATP-dependent reaction, suggesting that it is an RNA ligase. We have solved the structure of Pab1020 in complex with the ATP analog AMPPNP by single-wavelength anomalous dispersion (SAD), elucidating a structure with high structural similarity to the catalytic domains of two RNA ligases from the bacteriophage T4. Additional carboxy-terminal domains are also present, and one of these mediates contacts with a second protomer, which is related by noncrystallographic symmetry, generating a homodimeric structure. These C-terminal domains are terminated by short domain swaps which themselves end within 5 A of the active sites of the partner molecules. Additionally, we show that the protein is indeed capable of circularizing RNA molecules in an ATP-dependent reaction. These structural and biochemical results provide an insight into the potential physiological roles of Pab1020.