By using a microscopic approach, field inversion single-cell gel electrophoresis, we show that preformed single-strand discontinuities are present in the chromatin of resting and proliferating mammalian and yeast cells. These single-strand breaks are primarily nicks positioned at approximately 50-kbp intervals throughout the entire genome that could be efficiently labeled in situ by DNA polymerase I holoenzyme but not by Klenow fragment and terminal transferase unless after ribonucleolytic treatments. The RNA molecules involved appear to comprise R-loops, recognized by the S9.6 RNA/DNA hybrid-specific antibody. By using the breakpoint cluster region of the Mixed Lineage Leukemia (MLL) gene as a model, we have found that the number of manifest nicks detected by FISH performed after field inversion single-cell gel electrophoresis depends on epigenetic context, but the difference between germ-line and translocated MLL alleles is abolished by protease treatment. Our data imply that the double-stranded genomic DNA is composed of contiguous rather than continuous single strands and reveal an aspect of higher-order chromatin organization with ribonucleoprotein-associated persistent nicks defining approximately 50-kbp domains.