DNA Binding and Cleavage by the Fowlpox Virus
Resolvase |
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Authors: | Matthew J Culyba Young Hwang Nana Minkah and Frederic D Bushman |
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Institution: | Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 |
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Abstract: | The first steps of poxvirus DNA synthesis yield concatemeric arrays of
covalently linked genomes. The virus-encoded Holliday junction resolvase is
required to process concatemers into unit-length genomes for packaging.
Previous studies of the vaccinia virus resolvase have been problematic due to
poor protein solubility. We found that fowlpox virus resolvase was much more
tractable. Fowlpox resolvase formed complexes with a variety of branched DNA
substrates, but not linear DNA, and had the highest affinity for a Holliday
junction substrate, illustrating a previously unappreciated affinity for
Holliday junctions over other substrates. The cleavage activity was monitored
in fixed time assays, showing that, as with vaccinia resolvase, the fowlpox
enzyme could cleave a wide array of branched DNA substrates. Single turnover
kinetic analysis revealed the Holliday junction substrate was cleaved 90-fold
faster than a splayed duplex substrate containing a single to double strand
transition. Multiple turnover kinetic analysis, however, showed that the
cleavage step was not limiting for the full reaction cycle. Cleavage by
resolvase was also tightly coupled at symmetrical positions across the
junction, and coupling required the complete Holliday junction structure.
Last, we found that cleavage of an extruded cruciform yielded a product, which
after treatment with ligase, had the properties expected for covalently closed
DNA hairpin ends, as is seen for poxvirus genome monomers. These findings
provide a tractable poxvirus resolvase usable for the development of small
molecule inhibitors.Poxvirus DNA replication is proposed to proceed by a “rolling
hairpin” mechanism to yield linear concatemers, in which genomes are
arranged in mostly head-to-head and tail-to tail orientation
(, step 1)
(1). The terminal sequences at
each junction form an inverted repeat, which can be extruded to form a
cruciform structure (step 2)
(2). Cleavage of the resulting
Holliday junctions on each end frees the monomer genome from the concatemer
(step 3). The nicks left behind after resolution of the Holliday junction can
then be ligated, yielding the hairpin DNA ends characteristic of poxviruses
(step 4).Open in a separate windowRole of poxvirus resolvase during viral replication. Black
lines indicate single DNA strands. Half-arrows indicate repeated
sequences. Small arrows indicate resolvase cleavage sites.
1) Poxvirus genome replication yields concatemers; 2)
inverted repeat sequences at concatemer junctions extrude to form cruciform
structures; 3) Holliday junction cleavage by resolvase at cruciform
structures yields unit-length genomes with preserved hairpin ends; 4)
ligase seals nicks to yield mature genome monomers.The vaccinia virus resolvase gene, A22R, was first recognized in
bioinformatic surveys to encode a member of the RNase H superfamily of
polynucleotide phosphotransfer enzymes
(3). These enzymes catalyze
attack of a hydroxyl group on a phosphodiester bond, thereby supporting a
variety of nuclease or DNA joining reactions. Garcia et al.
(3) purified recombinant
vaccinia resolvase and showed that it displayed cleaving activity on model
Holliday junctions. They also generated a conditional A22R
recombinant vaccinia virus and showed that in the absence of A22R
expression, vaccinia failed to replicate and concatemer junctions accumulated,
indicating that A22 resolvase indeed is required for concatemer resolution
in vivo (4).
Subsequent studies by Garcia et al.
(5) and Culyba et al.
(6) showed that vaccinia
resolvase had little sequence specificity, and that cleavage yielded a
3′-hydroxyl group suitable for subsequent DNA ligation. Culyba et
al. (7) also showed that
several further branched DNA molecules could be cleaved by vaccinia resolvase,
establishing that the enzyme could potentially process a variety of branched
DNA forms expected to arise during recombination or replication, suggesting
possible additional roles for poxvirus resolvase.Progress in studying poxvirus resolvase has been limited by the poor
solubility of the purified vaccinia protein. For example, in Garcia et
al. (5), the vaccinia
resolvase was fused to maltose-binding protein to improve solubility, but
consequently the properties of the maltose-binding protein portion of the
fusion must be considered in interpreting the results. Pilot studies from our
laboratory showed that the insolubility and low activity of the vaccinia virus
resolvase precluded its use in high-throughput screens for inhibitors (data
not shown).In an effort to identify a more tractable poxvirus resolvase protein, we
attempted to clone four other poxvirus resolvase genes and purify the gene
products after overexpression in bacteria. We found that the fowlpox resolvase
was much more soluble and active than the others tested. Analysis of cleavage
revealed that a wide range of branched DNA forms were substrates, paralleling
results with vaccinia resolvase and establishing that these activities are a
conserved property of poxvirus resolvases. Binding analysis on these same DNA
forms also revealed a strict specificity for branched DNA, with the highest
affinity binding for the Holliday junction, suggesting that DNA binding
specificity is the major discriminatory mechanism for DNA cleavage activity.
Kinetic analysis was feasible with fowlpox resolvase, allowing us to show that
the first-order rate constant for strand cleavage under single turnover
conditions is 90-fold greater for a Holliday junction substrate than for a
splayed duplex substrate. However, this rate constant was not limiting for the
Holliday junction under multiple turnover conditions, where the rate of strand
cleavage is 1.9-fold slower for the Holliday junction than for the splayed
duplex. Last, we show that fowlpox resolvase cleavage at Holliday junctions is
coupled, so that nicking on one strand also promoted nicking on the strand
located across the junction from it. These studies indicate that fowlpox
resolvase is well suited to in vitro analysis and suggests approaches
to high-throughput screening for resolvase inhibitors. |
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