Nucleosome Structure and Repair of N-Methylpurines in the GAL1-10 Genes of Saccharomyces cerevisiae

Abstract

Nucleosome structure and repair of N-methylpurines were analyzed at nucleotide resolution in the divergent GAL1-10 genes of intact yeast cells, encompassing their common upstream-activating sequence. In glucose cultures where genes are repressed, nucleosomes with fixed positions exist in regions adjacent to the upstream-activating sequence, and the variability of nucleosome positioning sharply increases with increasing distance from this sequence. Galactose induction causes nucleosome disruption throughout the region analyzed, with those nucleosomes close to the upstream-activating sequence being most striking. In glucose cultures, a strong correlation between N-methylpurine repair and nucleosome positioning was seen in nucleosomes with fixed positions, where slow and fast repair occurred in nucleosome core and linker DNA, respectively. Galactose induction enhanced N-methylpurine repair in both strands of nucleosome core DNA, being most dramatic in the clearly disrupted, fixed nucleosomes. Furthermore, N-methylpurines are repaired primarily by the Mag1-initiated base excision repair pathway, and nucleotide excision repair contributes little to repair of these lesions. Finally, N-methylpurine repair is significantly affected by nearest-neighbor nucleotides, where fast and slow repair occurred in sites between pyrimidines and purines, respectively. These results indicate that nucleosome positioning and DNA sequence significantly modulate Mag1-initiated base excision repair in intact yeast cells.

Dissecting Transcription-coupled and Global Genomic Repair in the Chromatin of Yeast GAL1-10 Genes

“…In glucose cultures, nucleosomes with fixed positions exist in regions adjacent to the UAS, and the variability of nucleosome positioning sharply increases with increasing distance from this sequence. In agreement with the past reports using isolated nuclei or chromatin (24 -27), galactose induction causes nucleosome disruption throughout the region analyzed in permeabilized whole yeast cells, with the disruption of those nucleosomes close to the UAS being the most striking (28).…”

“…The effect of this "hidden signal" increases as a CPD site is more distant from the labeled 3Ј end. This hidden signal was corrected as described previously (28).…”

“…Repair incubation, DNA isolation, and CPD mapping were done as described (3,12,28,36,37). Briefly, genomic DNA was isolated following UV irradiation and different times of repair incubation, using glass beads to break the cells (3).…”

“…Extensive studies have been done on isolated nuclei or chromatin to map the nucleosome structure in the GAL1-10 region (24 -27). Using bleomycin as a DNA cleaving agent, we recently mapped nucleosome positioning in the GAL1-10 genes in permeabilized whole yeast cells (28). In glucose cultures, nucleosomes with fixed positions exist in regions adjacent to the UAS, and the variability of nucleosome positioning sharply increases with increasing distance from this sequence.…”

Recruitment and Dissociation of Nonhomologous End Joining Proteins at a DNA Double-Strand Break in Saccharomyces cerevisiae

“…A GAL1 cut site (GAL1-cs) was placed within a positioned nucleosome that is bound when the promoter is inactive. This nucleosome becomes undetectable when GAL1 is activated by galactose (Reagan and Majors 1998;Li and Smerdon 2002), suggesting a means of regulating cut-site accessibility. The sequence and length of the replaced region are otherwise unimportant for GAL1 function (Reagan and Majors 1998).…”