PMC

A model of SIRV2gp19 host chromosome during SIRV2 infection. Bacteriophage T4 or SIRV2 infection triggers an enzymatic cascade that degrades of the host chromosome (Bize et al. ; Parson and Snustad ). a T4 endonuclease II creates nicks in dCMP regions of the host chromosome. T4 46/47 exonuclease removes mononucleotides at gaps to create single-stranded DNA regions. Then T4 endonuclease IV cleaves these single-stranded gaps to fragment DNA. b If SIRV2 degrades its host chromosome by a similar mechanism as bacteriophage T4, then a predicted nuclease could first create single-stranded gaps in the host chromosome. This nuclease has not been identified. Then SIRV2gp19, a single-stranded endonuclease, could cleave single-stranded gaps and fragment the host chromosome

SIRV2gp19 cleaves single-stranded DNA. MBP-SIRV2gp19 nuclease activity was measured by incubating MBP-SIRV2gp19 dilutions (lanes 1–5 1, 0.5, 0.25, 0.125, or 0.0625 pmol) with 1 μg of a circular double-stranded M13mp18 RF I DNA, b linear double-stranded phiX/HaeIII DNA, or c circular single-stranded M13mp18 DNA in 1× ThermoPol Buffer for 1 h at 55°C. SIRV2gp19/D89A was assayed for nuclease activity as described above with substrates d circular double-stranded pBR322 DNA, e linear double-stranded phiX/HaeIII DNA, or f circular single-stranded M13mp18 DNA. As a control, DNA was incubated in the absence of MBP-SIRV2gp19 or MBP-SIRV2/D89A (−). Reaction products were separated by 0.7% agarose gel electrophoresis. The NEB 1 kb DNA ladder (M) as a reference

SIRV2gp19 is a single-strand specific endonuclease. Nuclease activity was characterized using a synthetic single-stranded oligonucleotide labeled on the a 5′ or b 3′ end with a fluorescent FAM-label for detection. MBP-SIRV2gp19 (0.2 μM) was incubated with a 5′ or b 3′ FAM-labeled oligonucleotide (1 μM) in NEBuffer 4 at 55°C. E. coli exonuclease I (exo I) (0.05 μM) was incubated with a 5′ or b 3′ FAM-labeled oligonucleotide (1 μM) in exonuclease I reaction buffer at 37°C. Mung Bean Nuclease (0.3 μM) was incubated with a 5′ or b 3′ FAM-labeled oligonucleotide (1 μM) in Mung Bean Nuclease buffer at 30°C. Reaction aliquots were sampled at the indicated times and reaction was halted by the addition of EDTA (10 mM) in formamide. Reaction products were separated by 15% denaturing PAGE and fluorescence detected by a GE Typhoon scanner