Analysis of stress-induced duplex destabilization (SIDD) properties of replication origins, genes and intergenes in the fission yeast, Schizosaccharomyces pombe

Abstract

Background: Replication and transcription, the two key functions of DNA, require unwinding of the DNA double helix. It has been shown that replication origins in the budding yeast, Saccharomyces cerevisiae contain an easily unwound stretch of DNA. We have used a recently developed method for determining the locations and degrees of stress-induced duplex destabilization (SIDD) for all the reported replication origins in the genome of the fission yeast, Schizosaccharomyces pombe.

Results: We have found that the origins are more susceptible to SIDD as compared to the non-origin intergenic regions (NOIRs) and genes. SIDD analysis of many known origins in other eukaryotes suggests that SIDD is a common property of replication origins. Interestingly, the previously shown deletion-dependent changes in the activities of the origins of the ura4 origin region on chromosome 3 are paralleled by changes in SIDD properties, suggesting SIDD's role in origin activity. SIDD profiling following in silico deletions of some origins suggests that many of the closely spaced S. pombe origins could be clusters of two or three weak origins, similar to the ura4 origin region.

Conclusion: SIDD appears to be a highly conserved, functionally important property of replication origins in S. pombe and other organisms. The distinctly low SIDD scores of origins and the long range effects of genetic alterations on SIDD properties provide a unique predictive potential to the SIDD analysis. This could be used in exploring different aspects of structural and functional organization of origins including interactions between closely spaced origins.

Figure 1

Variability patterns of SIDD profiles in sliding and fixed windows. (A-C) Variability of SIDD profiles of Ori1098 in sliding windows. Note that the deep SIDD valley corresponding to the origin (grey bar) in the center of a 5-kb window (A) becomes shallow on sliding 1 kb from left to right (B) and shallower on sliding 1kb from right to left (C). (D-F) The locations of the SIDD valleys show little or no alteration although their depths change to different extents between 5-kb and 10-kb windows centered on an origin.

Figure 2

Representative patterns of the SIDD profiles of different genomic entities. (A-G) origins, (H) a NOIR and, (I) a gene. All these origins except (D) and (G) are 2D-gel proven. The locations of the origins/AT islands are marked with grey bars. Note in (G) the presence of a deep SIDD valley not overlapping but close to Ori1003, one of the four origins found to be highly stable in this study.

Figure 3

Origins are more susceptible to SIDD than NOIRs and genes. (A) Distribution patterns of the lowest G(x) values of 564 origins (ChIP-chip origins and AT-islands) [25,28] (black dots), 440 weak origins [37] (grey dots), 434 NOIRs (magenta dots) and 360 genes (cyan dots). (B) The graph of AT% vs lowest G(x) values of the four analyzed entities, ChIP-chip origins and ATislands (black dots), weak origins (grey dots), NOIRs (magenta dots) and genes (cyan dots) in the 5-kb context.

Figure 4

Alterations in the origin activity are paralleled by SIDD changes in the ura4 origin region. Relationship between the extent of SIDD and origin activity in the ura4 origin region in (A) wild-type, and, (BD) ARS deletion S. pombe strains [18]. The map of the concerned region, locations of ARS elements, restriction sites (Av=AvrII, B=BamHI, Bg=BglII, Bs=BstBI, C=ClaI RI=EcoRI, RV=EcoRV, H=HinDIII, N=NruI, Ns=NsiI,S=SmaI) and the 2D autoradiograms of different fragments shown on the top of each section have been reproduced from [18] with permission. The arrows mark the bubble arc signal, indicative of the origin activity of the detected fragment. The lowest G(x) value for each ARS element is shown in kcal/mol near the valley.

Figure 5

Other closely spaced stress-destabilized intergenes show SIDD interactions akin to the ura4 origin region. SIDD profiles of two ChIP-chip origins, Ori1092 (rip1) and Ori1047 (ars1), to check the interactions between the closely spaced stress destabilized intergenes following in silico deletions. In case of Ori1092, the intergenes were deleted in silico and replaced with ura4 gene somewhat mimicking the experiments done in [18]. The locations of the origins (dark grey bar), other SIDD valleys (light grey bar) and genes (top) are shown (S. pombe GeneDB). The lowest G(x) values are mentioned below the valleys.