TY - JOUR
T1 - Characterization of the Holliday Junction Resolving Enzyme Encoded by the Bacillus subtilis Bacteriophage SPP1
AU - Zecchi, Lisa
AU - Lo Piano, Ambra
AU - Suzuki, Yuki
AU - Cañas, Cristina
AU - Takeyasu, Kunio
AU - Ayora, Silvia
N1 - Funding Information:
We thank Juan C. Alonso for providing us with strains and phages, for his interest in the project, and for his critical reading of the manuscript. David Sherratt providing us with the plasmid and strain to produce the χ-structures. Three anonymous referees provided helpful comments, which improved the manuscript, and Melissa Stauffer edited the text. A.L.P. received a contract from Community of Madrid, L.Z. was a recipient of a EU Leonardo grant, and Y.S. was a recipient of the JSPS (Japan Society for the Promotion of Science) research fellowship.
PY - 2012/10/31
Y1 - 2012/10/31
N2 - 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 PE2 and PE3 promoters. Resequencing the region corresponding to the last two hypothetical genes transcribed from the PE2 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.
AB - 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 PE2 and PE3 promoters. Resequencing the region corresponding to the last two hypothetical genes transcribed from the PE2 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.
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U2 - 10.1371/journal.pone.0048440
DO - 10.1371/journal.pone.0048440
M3 - Article
C2 - 23119018
AN - SCOPUS:84868312980
SN - 1932-6203
VL - 7
JO - PLoS One
JF - PLoS One
IS - 10
M1 - e48440
ER -