G-quadruplexes may determine the landscape of recombination in HSV-1

Figures

• 

  Fig. 1. Genome-wide distribution of recombination breakpoints mirrors G4 densities in HSV-1. A nucleotide window of 100 bp was slid across the genome of HSV-1 strain 17 (Genbank accession number: NC_001806) at a step size of 1 bp (i.e a total of 152162 windows were analyzed for a genome that is 152,163 bp long). The G4 density and the number of breakpoints in each window were computed; the fold difference over their respective genomic averages was calculated window-wise and smoothened line graphs are plotted. The green line at 1 represents the normalized genome-wide average and demarcates the regions of local enrichment (above the green line) and depletion (below the green line). The blue and red lines superimpose each other genome-wide, indicating a spatial association between recombination breakpoints and G4-motifs in the HSV-1 genome

• 

  Fig. 2. Flanking sites (500 bp on both sides) of recombination breakpoints are enriched for G4-motifs. a In majority of the recombinant strains analyzed, the average G4 density in the 1-kb bins centered on the recombination breakpoints is higher than that in the rest of the genome, indicating specific enrichment of G4-motifs near recombination breakpoints. b The violin plot summarizes the data from all 40 strains

• 

  Fig. 3. CD spectroscopic analysis of G4-motifs in flanking regions of breakpoints. The spectral signatures of the 8 oligomers chosen from the flanking sites of recombination breakpoints indicate the formation of G4 structures. Oligomers 1,3,4,5, and 7 form G4 s with parallel topology, while oligomers 2,6,8 form G4 s with hybrid topology. (The sequence of the oligomers 1–8 are provided in Additional file 1: Table S2)

• 

  Fig. 4. NMR spectroscopic analysis of G4-motifs in flanking regions of breakpoints. The 8 oligomers chosen from the flanking sites of recombination breakpoints form G4 structures in vitro as indicated by the appearance of peaks in the downfield range 10.5–12 ppm in 1H NMR. (The sequence of the oligomers 1–8 are provided in Additional file 1: Table S2)

• 

  Fig. 5. The proximity of recombination breakpoints to G4-motifs is non-random. The median distance of actual recombination breakpoints to their nearest G4-motifs in the genome of the recombinants is significantly lesser than that of the randomly selected breakpoint positions, indicating that, the occurrence of recombination breakpoints near G4-motifs is not stochastic

• 

  Fig. 6. G-quadruplexes are sites of recombination in HSV-1. a The proportion of actual breakpoints and randomly selected breakpoints contained within G4-motifs was determined strain-wise and is consolidated in the violin plot. The median proportion of actual breakpoints within G4 motifs was significantly higher than that of randomized breakpoints within G4 motifs, suggesting that G4 motifs in the HSV-1 genome are enriched for breakpoints. b When the recombination breakpoint occurs within G4-motif in the parental strains, the G4-motif is often conserved (and not lost) in the process of recombination (i.e. the recombinant strain has a G4-motif in the same region). ‘+’ indicates presence of G4-motif at the breakpoint ‘-‘indicates absence of G4-motif at the breakpoint

• 

  Fig. 7. Understanding G4-clusters and unique features of breakpoints within G4-clusters. a Understanding G4-clusters: The complete square denotes all G4-motifs. The axes apply only to the blue portions of the square and take only integer values. The subset of all singlet G4-motifs (denoted by ‘S’), characterized by exactly 4 ‘G’ triplets (and hence only 1 independent G4), is represented by the grey portion in the top-left region. The blue portions represent G4-clusters. The red line is a bivariate characteristics curve of G4-clusters, the two variables being, number of G triplets and size. Given the number of ‘G’ triplets, the size of G4 cluster can be determined by extrapolation and vice versa. The white ‘˩’ shaped region merely demarcates the unit order and higher order G4 clusters. b Summary of the unique features of breakpoints contained within G4-motifs depicted as a multilevel nested cartoon: The colored bars indicate a unique aspect among the breakpoints present in G4-motifs. Bars are zoomed in at the subsequent level to indicate the features nested within them. The proportion of breakpoints containing the indicated feature is mentioned in each bar

• 

  Fig. 8. G4-motifs at the boundary of the UL segment are hotspots of recombination. G4-motifs at the boundary of UL are approximately 46-fold more enriched for recombination breakpoints as compared to other G4-motifs, making them the most common G4-motifs to contain a breakpoint in HSV-1 recombination. *The proportion of G4-motifs containing a breakpoint is significantly higher among the UL boundary G4-motifs than among other G4-motifs (P < 0.0001; Chi-squared test)