Summary auto-generated
This study investigates how adenovirus type 2 (Ad2) chromatin structure changes during lytic infection by mapping nuclease-sensitive sites. Researchers infected human HEp-2 cells with Ad2 and used micrococcal nuclease, DNase I, and endogenous nuclease to digest chromatin at early (5 hours) and late (20 hours) post-infection timepoints. They mapped cleavage sites within a specific genomic region using a terminal labeling method. Results revealed that early chromatin displayed hypersensitive sites at map positions 16.0 and 14.3, near the IVa2 promoter and upstream of the major late promoter. By late infection, these sites shifted to positions 13.5 and 13.0. The repositioning of nuclease-sensitive sites correlated with the switch from early to late gene transcription and reflected profound changes in viral chromatin structure, where early histone-based chromatin transformed into late core-protein-containing structures. All three nucleases revealed similar digestion patterns, suggesting the identified sites reflect underlying chromatin organization rather than nuclease sequence preferences.
Key findings
- Early Ad2 chromatin (5 h post-infection) contains hypersensitive nuclease cleavage sites at map positions 16.0 and 14.3, located at or near the IVa2 promoter
- Late Ad2 chromatin (20 h post-infection) shows hypersensitive sites shifted to positions 13.5 and 13.0, with reduced sensitivity around the early promoter regions
- The shift in nuclease-sensitive sites parallels the early-to-late switch in viral transcription patterns and the structural transformation from histone-based to core-protein-based chromatin organization
- Micrococcal nuclease, DNase I, and endogenous nuclease produced similar digestion patterns, indicating site specificity is determined by chromatin structure rather than nuclease type
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Abstract
We have investigated the sensitivity of adenovirus type 2 naked DNA and chromatin at 5 h and 20 h after infection to digestion by DNase I, micrococcal nuclease and endogenous nuclease between map coordinates 11.3 and 18.0 (SmaI-F fragment) using a terminal labelling method. Infected cell nuclei were gently digested with nucleases, DNA was extracted and digested to completion with SmaI and the fragments shorter than the SmaI-F fragment mapped by hybridization with a 708 base pair probe co-terminal with the SmaI-F fragment. Early chromatin contained hypersensitive sites at 16.0 and 14.3. These sites became minor cleavage sites in late chromatin and new hypersensitive sites appeared at 13.5 and 13.0. The change in the location of the hypersensitive sites in the course of infection correlated with the early to late switch in the transcription pattern in this region and the early to late change in the overall structure of adenovirus chromatin.