01-12-2012, 02:34 PM
Characterization of Sulfolobus islandicus rod-shaped virus 2 gp19,
a single-strand specific endonuclease
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Abstract
The hyperthermophilic Sulfolobus islandicus
rod-shaped virus 2 (SIRV2) encodes a 25-kDa protein
(SIRV2gp19) annotated as a hypothetical protein with
sequence homology to the RecB nuclease superfamily.
Even though SIRV2gp19 homologs are conserved
throughout the rudivirus family and presumably play a role
in the viral life cycle, SIRV2gp19 has not been functionally
characterized. To define the minimal requirements for
activity, SIRV2gp19 was purified and tested under varying
conditions. SIRV2gp19 is a single-strand specific endonuclease
that requires Mg2? for activity and is inactive on
double-stranded DNA. A conserved aspartic acid in RecB
nuclease superfamily Motif II (D89) is also essential for
SIRV2gp19 activity and mutation to alanine (D89A)
abolishes activity. Therefore, the SIRV2gp19 cleavage
mechanism is similar to previously described RecB
nucleases. Finally, SIRV2gp19 single-stranded DNA endonuclease
activity could play a role in host chromosome
degradation during SIRV2 lytic infection.
Introduction
Sulfolobus islandicus rod-shaped virus 2 (SIRV2) infects
the archeaon Sulfolobus islandicus at both extreme temperature
(70–80C) and acidity (pH 3) and has been the
focus of structural, genomic, and transcriptional studies
(Blum et al. 2001; Kessler et al. 2004; Peng et al. 2001;
Prangishvili et al. 2006a). The SIRV2 genome has been
completely sequenced; however, the majority of open
reading frames (ORFs) are annotated as hypothetical proteins
that share little sequence homology to other proteins
in GenBank (Blum et al. 2001; Prangishvili et al. 2006b).
Thus, a key step in understanding SIRV2 biology will be
assigning biochemical and biological functions to hypothetical
SIRV2 proteins.
Results
SIRV2gp19 substrate specificity and requirements
for cleavage activity
The similarity to the RecB nuclease superfamily led us to
test SIRV2gp19 nuclease activity on a panel of DNA
substrates to determine substrate specificity. Circular
double-stranded M13mp18 DNA and linear double-stranded
phiX174/HaeIII DNA were not substrates for SIRV2gp19
cleavage (Fig. 1a, b). SIRV2gp19 cleaved circular singlestranded
M13mp18 DNA into smaller disperse fragments,
appearing as a smear rather than discrete fragments, suggesting
that SIRV2gp19 has minimal sequence or structure
specificity (Fig. 1c).
Discussion
SIRV2gp19, previously annotated as a hypothetical RecB
nuclease, is a single-stranded specific DNA endonuclease.
Mutagenesis of conserved SIRV2gp19 Motif II aspartate to
alanine (D89A) abolishes nuclease activity, presumably by
disrupting Mg2? coordination in the active site. As little
sequence or structure specificity was observed with the
SIRV2gp19 nuclease, it could act with other components to
direct nuclease activity like the related archaeal cas4
nuclease involved in CRISPR processing (Haft et al. 2005;
Jansen et al. 2002). Alternatively, SIRV2gp19 could act as
a broad spectrum nuclease, presumably to degrade DNA.
Broad degradative function has been observed in the
metabolism of other bacteriophages, e.g., bacteriophage T4
(Parson and Snustad 1975). Bacteriophage T4 degrades
host chromosomal DNA by a combination of several
nucleases (Fig. 3).