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.
Highlights
Simple methylating agents, such as methyl methanesulfonate (MMS)1 and dimethyl sulfate (DMS), produce a variety of damaged bases in DNA of which N7-methylguanine (7MeG) and N3-methyladenine (3MeA) constitute ϳ80 and 10%, respectively [1, 2]
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
Nucleosome Structure in the GAL1-10 Genes—Wild type (Y452) yeast cells were grown in glucose or galactose medium, permeabilized with digitonin and treated with BLM
Summary
Yeast Strains—DBY747 (MATa ura his3-⌬1 leu leu112 trp1–289) and its isogenic mutant strains JC8901 (mag1⌬::hisGURA3-hisG), WXY9379 (rad1⌬::LEU2), and WXY9380 (mag1⌬::hisGURA3-hisG rad1⌬::LEU2) were generously provided by Dr Wei Xiao (University of Saskatchewan, Canada). Cells were resuspended in ice-cold 2% glucose or 2% galactose, mixed with 1:10 volume of a stock solution containing 10% yeast extract and 20% peptone, and pelleted by centrifugation. After 2 min incubation at room temperature, cells were washed twice with ice-cold 2% glucose (or 2% galactose) and resuspended in the same solutions containing 100 mM hydroxyurea, to prevent DNA replication during repair incubation [22]. The doses of BLM used were relatively low, to ensure that cleavage by BLM followed single-hit kinetics for the majority of fragments analyzed Even under these conditions, a small portion of the fragments still have more than one cleavage, and a small portion of the signal at a site will not show up on a gel if the cleavage occurs upstream (relative to the labeled end) of the site. Quantitation for NMPs followed the same procedure as that for BLM cleavage
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