title: Characterization of the Holliday junction resolving enzyme encoded by the Bacillus subtilis bacteriophage SPP1.
abstract: 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.
authors.affiliation: Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Madrid, Spain.
authors.forename: Lisa
authors.initials: L
authors.lastname: Zecchi
authors.forename: Ambra
authors.initials: A
authors.lastname: Lo Piano
authors.forename: Yuki
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Cristina
authors.initials: C
authors.lastname: Cañas
authors.forename: Kunio
authors.initials: K
authors.lastname: Takeyasu
authors.forename: Silvia
authors.initials: S
authors.lastname: Ayora
mesh_terms: D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000201:enzymology* / Q000235:genetics*; D001412:Bacillus subtilis / Q000235:genetics / Q000821:virology; D001483:Base Sequence; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D045566:DNA, Cruciform / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016679:Genome, Viral; D045565:Holliday Junction Resolvases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D055503:Protein Multimerization; D011995:Recombination, Genetic; D016415:Sequence Alignment; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D045566:DNA, Cruciform; D004268:DNA-Binding Proteins; D014764:Viral Proteins; D045565:Holliday Junction Resolvases