RESUMO
The loading of oligomeric helicases onto replication origins marks an essential step in replisome assembly. In cells, dedicated AAA+ ATPases regulate loading, however, the mechanism by which these factors recruit and deposit helicases has remained unclear. To better understand this process, we determined the structure of the ATPase region of the bacterial helicase loader DnaC from Aquifex aeolicus to 2.7 A resolution. The structure shows that DnaC is a close paralog of the bacterial replication initiator, DnaA, and unexpectedly shares an ability to form a helical assembly similar to that of ATP-bound DnaA. Complementation and ssDNA-binding assays validate the importance of homomeric DnaC interactions, while pull-down experiments show that the DnaC and DnaA AAA+ domains interact in a nucleotide-dependent manner. These findings implicate DnaC as a molecular adaptor that uses ATP-activated DnaA as a docking site for regulating the recruitment and correct spatial deposition of the DnaB helicase onto origins.
Assuntos
Proteínas de Bactérias/química , DNA Helicases/fisiologia , Replicação do DNA , Proteínas de Ligação a DNA/química , DnaB Helicases/química , Proteínas de Escherichia coli/química , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Bactérias/enzimologia , Cristalografia por Raios X/métodos , DNA Helicases/metabolismo , DNA de Cadeia Simples/química , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
The initiation of DNA replication requires the melting of chromosomal origins to provide a template for replisomal polymerases. In bacteria, the DnaA initiator plays a key role in this process, forming a large nucleoprotein complex that opens DNA through a complex and poorly understood mechanism. Using structure-guided mutagenesis, biochemical, and genetic approaches, we establish an unexpected link between the duplex DNA-binding domain of DnaA and the ability of the protein to both self-assemble and engage single-stranded DNA in an ATP-dependent manner. Intersubunit cross-talk between this domain and the DnaA ATPase region regulates this link and is required for both origin unwinding in vitro and initiator function in vivo. These findings indicate that DnaA utilizes at least two different oligomeric conformations for engaging single- and double-stranded DNA, and that these states play distinct roles in controlling the progression of initiation.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Replicação do DNA , DNA Bacteriano/biossíntese , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Modelos Moleculares , Mutagênese , Mutação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Especificidade por SubstratoRESUMO
In bacteria, the initiation of replication is controlled by DnaA, a member of the ATPases associated with various cellular activities (AAA+) protein superfamily. ATP binding allows DnaA to transition from a monomeric state into a large oligomeric complex that remodels replication origins, triggers duplex melting and facilitates replisome assembly. The crystal structure of AMP-PCP-bound DnaA reveals a right-handed superhelix defined by specific protein-ATP interactions. The observed quaternary structure of DnaA, along with topology footprint assays, indicates that a right-handed DNA wrap is formed around the initiation nucleoprotein complex. This model clarifies how DnaA engages and unwinds bacterial origins and suggests that additional, regulatory AAA+ proteins engage DnaA at filament ends. Eukaryotic and archaeal initiators also have the structural elements that promote open-helix formation, indicating that a spiral, open-ring AAA+ assembly forms the core element of initiators in all domains of life.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Bactérias/enzimologia , Proteínas de Bactérias/genética , Sequência Conservada , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/genética , Modelos Moleculares , Conformação Proteica , Origem de ReplicaçãoRESUMO
BACKGROUND: Despite an increase in the content of palliative medicine curricula in medical schools, students are rarely exposed to end-of-life (EOL) care through real-patient experiences during their preclinical education. OBJECTIVE: To evaluate the utility and impact of exposure to EOL care for first year medical students (MS-1s) through a hospice volunteer experience. METHODS: Patients and Families First (PFF), a hospice volunteer training program in EOL care, was piloted on three cohorts of MS-1s as an elective. Fifty-five students received 3 hours of volunteer training, and were then required to conduct at least two consecutive hospice visits on assigned patients to obtain course credit. Students' reflective essays on their experiences were analyzed using qualitative methodology and salient themes were extracted by two investigators independently and then collaboratively. RESULTS: The following five themes were identified from students' reflective essays: perceptions regarding hospice patients; reactions regarding self; normalcy of EOL care at home; impact of witnessing death and dying; and suggestions for improving EOL care education for medical students. CONCLUSION: Hospice volunteering during preclinical years may provide valuable experiential training for MS-1s in caring for seriously ill patients and their families by fostering personal reflection and empathic skills, thereby providing a foundation for future patient encounters during clinical training.
Assuntos
Atitude Frente a Morte , Educação de Graduação em Medicina/métodos , Cuidados Paliativos na Terminalidade da Vida/psicologia , Estudantes de Medicina/psicologia , Doente Terminal/psicologia , Voluntários/psicologia , Feminino , Humanos , Masculino , Meio-Oeste dos Estados Unidos , Pesquisa Qualitativa , Autoavaliação (Psicologia) , Assistência Terminal/métodosRESUMO
In all organisms, multi-subunit replicases are responsible for the accurate duplication of genetic material during cellular division. Initiator proteins control the onset of DNA replication and direct the assembly of replisomal components through a series of precisely timed protein-DNA and protein-protein interactions. Recent structural studies of the bacterial protein DnaA have helped to clarify the molecular mechanisms underlying initiator function, and suggest that key structural features of cellular initiators are universally conserved. Moreover, it appears that bacteria use a diverse range of regulatory strategies dedicated to tightly controlling replication initiation; in many cases, these mechanisms are intricately connected to the activities of DnaA at the origin of replication. This Review presents an overview of both the mechanism and regulation of bacterial DNA replication initiation, with emphasis on the features that are similar in eukaryotic and archaeal systems.