RESUMO
Complex viruses that encode their own initiation proteins and subvert the host's elongation apparatus have provided valuable insights into DNA replication. Using purified bacteriophage SPP1 and Bacillus subtilis proteins, we have reconstituted a rolling circle replication system that recapitulates genetically defined protein requirements. Eleven proteins are required: phage-encoded helicase (G40P), helicase loader (G39P), origin binding protein (G38P) and G36P single-stranded DNA-binding protein (SSB); and host-encoded PolC and DnaE polymerases, processivity factor (ß(2)), clamp loader (τ-δ-δ') and primase (DnaG). This study revealed a new role for the SPP1 origin binding protein. In the presence of SSB, it is required for initiation on replication forks that lack origin sequences, mimicking the activity of the PriA replication restart protein in bacteria. The SPP1 replisome is supported by both host and viral SSBs, but phage SSB is unable to support B. subtilis replication, likely owing to its inability to stimulate the PolC holoenzyme in the B. subtilis context. Moreover, phage SSB inhibits host replication, defining a new mechanism by which bacterial replication could be regulated by a viral factor.
Assuntos
Fagos Bacilares/genética , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Replicação do DNA , Proteínas Virais/metabolismo , Proteínas de Transporte/metabolismo , DNA/metabolismo , DNA Helicases/metabolismo , DNA Polimerase III/metabolismo , DNA Primase/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas não Estruturais Virais/metabolismoRESUMO
Recombination-dependent DNA replication, which is a central component of viral replication restart, is poorly understood in Firmicutes bacteriophages. Phage SPP1 initiates unidirectional theta DNA replication from a discrete replication origin (oriL), and when replication progresses, the fork might stall by the binding of the origin binding protein G38P to the late replication origin (oriR). Replication restart is dependent on viral recombination proteins to synthesize a linear head-to-tail concatemer, which is the substrate for viral DNA packaging. To identify new functions involved in this process, uncharacterized genes from phage SPP1 were analyzed. Immediately after infection, SPP1 transcribes a number of genes involved in recombination and replication from P(E2) and P(E3) promoters. Resequencing the region corresponding to the last two hypothetical genes transcribed from the P(E2) operon (genes 44 and 45) showed that they are in fact a single gene, re-annotated here as gene 44, that encodes a single polypeptide, named gene 44 product (G44P, 27.5 kDa). G44P shares a low but significant degree of identity in its C-terminal region with virus-encoded RusA-like resolvases. The data presented here demonstrate that G44P, which is a dimer in solution, binds with high affinity but without sequence specificity to several double-stranded DNA recombination intermediates. G44P preferentially cleaves Holliday junctions, but also, with lower efficiency, replicated D-loops. It also partially complemented the loss of RecU resolvase activity in B. subtilis cells. These in vitro and in vivo data suggest a role for G44P in replication restart during the transition to concatemeric viral replication.
Assuntos
Fagos Bacilares/enzimologia , Fagos Bacilares/genética , DNA Bacteriano/metabolismo , DNA Cruciforme/metabolismo , Resolvases de Junção Holliday/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/genética , Bacillus subtilis/virologia , Sequência de Bases , Replicação do DNA , DNA Bacteriano/química , DNA Cruciforme/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genoma Viral , Resolvases de Junção Holliday/química , Resolvases de Junção Holliday/genética , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Recombinação Genética , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
The initiation of viral double stranded (ds) DNA replication involves proteins that recruit and load the replisome at the replication origin (ori). Any block in replication fork progression or a programmed barrier may act as a factor for ori-independent remodelling and assembly of a new replisome at the stalled fork. Then replication initiation becomes dependent on recombination proteins, a process called recombination-dependent replication (RDR). RDR, which is recognized as being important for replication restart and stability in all living organisms, plays an essential role in the replication cycle of many dsDNA viruses. The SPP1 virus, which infects Bacillus subtilis cells, serves as a paradigm to understand the links between replication and recombination in circular dsDNA viruses. SPP1-encoded initiator and replisome assembly proteins control the onset of viral replication and direct the recruitment of host-encoded replisomal components at viral oriL. SPP1 uses replication fork reactivation to switch from ori-dependent θ-type (circle-to-circle) replication to σ-type RDR. Replication fork arrest leads to a double strand break that is processed by viral-encoded factors to generate a D-loop into which a new replisome is assembled, leading to σ-type viral replication. SPP1 RDR proteins are compared with similar proteins encoded by other viruses and their possible in vivo roles are discussed.
