Characterization of the Holliday Junction Resolving Enzyme Encoded by the <em>Bacillus subtilis</em> Bacteriophage SPP1

<div><p>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 (<em>ori</em>L), and when replication progresses, the fork might stall by the binding of the origin binding protein G<em>38</em>P to the late replication origin (<em>ori</em>R<em>).</em> 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 <em>P</em>_E2 and <em>P</em>_E3 promoters. Resequencing the region corresponding to the last two hypothetical genes transcribed from the <em>P</em>_E2 operon (genes <em>44</em> and <em>45</em>) showed that they are in fact a single gene, re-annotated here as gene <em>44</em>, that encodes a single polypeptide, named gene <em>44</em> product (G<em>44</em>P, 27.5 kDa). G<em>44</em>P 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 G<em>44</em>P, which is a dimer in solution, binds with high affinity but without sequence specificity to several double-stranded DNA recombination intermediates. G<em>44</em>P preferentially cleaves Holliday junctions, but also, with lower efficiency, replicated D-loops. It also partially complemented the loss of RecU resolvase activity in <em>B. subtilis</em> cells. These <em>in vitro</em> and <em>in vivo</em> data suggest a role for G<em>44</em>P in replication restart during the transition to concatemeric viral replication.</p> </div

G<i>44</i>P-mediated cleavage of replicated D-loops.

<p>Different D-loop variants resembling several recombination intermediates were end-labeled at the 5′end of oligonucleotide 19-M (the invading strand, in A) or of oligonucleotide 17-M (the displaced strand, in B) and were incubated with 10 nM G<i>44</i>P in buffer B containing 10 mM MgCl₂ for 30 min at 37°C. Reaction products were analyzed using 20% denaturing PAGE and revealed by autoradiography. Drawings indicate the different substrates analyzed (D-loops A to F, and control HJ and ssDNA). Asterisks indicate the [γ³²P]-ATP labeling of oligonucleotides at the 5′end. In the HJ substrate, an arrow indicates the major cleavage site. As markers, the G+A sequencing ladder obtained for the corresponding labeled oligonucleotide and the 41-nt and 21-nt primers for the corresponding sequence were loaded.</p

G<i>44</i>P binding to different DNA substrates.

<p>EMSAs showing binding of G<i>44</i>P to the indicated [γ³²P]-labeled DNA substrates: (A) HJ-J3, (B) D-loop DL-D, (C) 80-bp dsDNA, and (D) 80-nt ssDNA. DNA (0.2 nM) was incubated with increasing amounts of G<i>44</i>P as indicated in buffer B containing 1 mM EDTA for 20 min at 37°C. The three types of complexes formed are denoted by I, II, and III.</p

Determination of the cleavage ability of G<i>44</i>P on static and mobile HJs.

<p>(A and C) A fixed HJ (HJ-23M, in A) or a mobile HJ containing a 13-bp homologous core (HJ-jbm6, in C) [γ³²P]-labeled at the indicated strand was incubated with 10 nM G<i>44</i>P in buffer B containing 10 mM MgCl₂ for 30 min at 37°C. Reaction products were analyzed using 15% denaturing PAGE in the presence (+) or absence (-) of protein. “m” indicates the G+A sequencing ladder obtained for the corresponding labeled oligonucleotide. To serve as additional molecular weight markers, and denoted by C, 41-nt, 21-nt, 23-nt and 18-nt primers were loaded. In lane 13 of panel A, a degraded 17-M oligonucleotide was loaded. (B and D) The cleavage sites detected are indicated by arrows in the core of the two HJ sequences.</p

Visualization of G<i>44</i>P bound to HJ structures by AFM.

<p>(A) Representative AFM images of the χ-structure DNA alone and (B) after incubation with G<i>44</i>P (10 nM) in the presence of 1 mM EDTA. G<i>44</i>P was observed bound to the junction as well as to the dsDNA arms. Arrows indicate protein-DNA complexes. Scale bar  = 100 nm.</p

Identification of the correct G<i>44</i>P protein.

<p>(A) Part of the gene <i>44</i> carrying the sequence error. The arrow indicates the position of the missing guanine. Under the nucleotide sequence, the three possible reading frames are listed. The change of frame caused by the insertion of the guanine is underlined in red. The corrected sequence is shown in the bottom row. (B) Alignment with the ClustalW2 program of <i>Eco</i>RusA with the corrected sequence of G<i>44</i>P. In the G<i>44</i>P sequence the residues corresponding to former G<i>45</i>P are shown in bold. The region highlighted in part A is underlined in red, and the three putative starting methinonine residues are highlighted in red. The <i>Eco</i>RusA residues essential for catalysis are highlighted by red stars, and the region suggested to be involved in DNA binding and sequence specific cleavage is highlighted by a blue line. (C) G<i>44</i>P accumulation in the cell after SPP1 infection. <i>B. subtilis</i> cells were infected at an m.o.i of 10 and aliquots were taken every 5 min (from 0 [lane 2] to 25 [lane 7] min after infection). Crude extracts were prepared. Proteins (30 µg total protein) were separated in 15% SDS-PAGE and immunoblotted. As a control, 10, 20 and 40 ng of purified G<i>44</i>P were loaded (lanes 8-10). G<i>44</i>P was detected in the blots using anti-G<i>44</i>P rat polyclonal antisera. C: Crude extracts of non-infected cells. The running positions of the molecular weight markers are shown on the left.</p

Purified G<i>44</i>P is a dimer in solution.

<p>(A) Electrophoresis of the purified protein on a 15% SDS-polyacrylamide gel. Lane 1: molecular weight marker; lanes 2-4: increasing concentrations of G<i>44</i>P (1–4 µg). (B) Estimation of the molecular mass of G<i>44</i>P by gel filtration chromatography on a Superdex 75 column. A standard curve of K_av versus log₁₀ of molecular mass of protein standards was determined. The K_av of G<i>44</i>P was 0.05, corresponding to a molecular mass of 54 kDa.</p

Putative roles of G<i>44</i>P in the generation of concatemeric linear SPP1 DNA after replication fork stalling.

<p> (A) The generation of a 3′-tailed dsDNA molecule. 1, G<i>38</i>P (grey ovals) bound to <i>ori</i>R may hinder the progression of the replisome (white ovals), causing its subsequent disassembly. 2, The fork reverses and a Holliday junction (HJ) is formed. 3, The G<i>44</i>P HJ resolvase (scissors) may cleave this substrate and a one-ended double strand break is formed. 4, The G<i>34</i>.<i>1</i>P 5′-3′ exonuclease (pacman) processes the end. 5, The G<i>35</i>P recombinase (red ovals) forms filaments on the generated 3′-tailed duplex DNA. The 3′-ends of the strands are shown by arrows. From this substrate two alternatives were proposed to generate SPP1concatemeric DNA (B and B’). (B) Generation of concatemeric DNA by a sigma-like mechanism. 1, G<i>35</i>P promotes strand invasion on a supercoiled SPP1 molecule. 2, The replisome is recruited and the invaded strand primes DNA synthesis with subsequent dislodging of G<i>35</i>P. 3, G<i>44</i>P may cleave the strands of the replicated D-loop. 4 and 5, DNA synthesis followed by DNA ligation would generate the proper substrate for concatemeric DNA synthesis by a semiconservative mechanism. (B’) Bubble migration model for the generation of concatemeric DNA. 1′ and 2′, These steps are common in both avenues. 3′ and 4′, The replication bubble migrates and the newly synthesized strands are extruded, so that by this mechanism a concatemer is formed by conservative DNA synthesis, without an obvious need for a D-loop resolvase. In B and B’ the template DNA is drawn in black and newly synthesized DNA in yellow. The steps where G<i>44</i>P could participate are indicated by the scissors.</p