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

Nucleosome structure and repair ofN-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 enhancedN-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. Nucleosome structure and repair ofN-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 enhancedN-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. Simple methylating agents, such as methyl methanesulfonate (MMS) 1The abbreviations used are: MMS, methyl methanesulfonate; BER, base excision repair; BLM, bleomycin; DMS, dimethyl sulfate; 3MeA, N 3-methyladenine; 7MeG, N 7-methylguanine; NER, nucleotide excision repair; NMP, N-methylpurine; TS, transcribed strand; NTS, nontranscribed strand; UAS, upstream-activating sequence1The abbreviations used are: MMS, methyl methanesulfonate; BER, base excision repair; BLM, bleomycin; DMS, dimethyl sulfate; 3MeA, N 3-methyladenine; 7MeG, N 7-methylguanine; NER, nucleotide excision repair; NMP, N-methylpurine; TS, transcribed strand; NTS, nontranscribed strand; UAS, upstream-activating sequence and dimethyl sulfate (DMS), produce a variety of damaged bases in DNA of whichN7-methylguanine (7MeG) andN3-methyladenine (3MeA) constitute ∼80 and 10%, respectively (1Singer B. Grunberger D. Molecular Biology of Mutagens and Carcinogens. Plenum Press, NY1983: 55-78Google Scholar, 2Pieper R.O. Nickoloff J.A. Hoekstra M.F. DNA Damage and Repair, Vol. 2: DNA Repair in higher eukaryotes. Humana Press Inc., Totowa, NJ1998: 199-222Google Scholar). These N-methylpurines (NMPs), can be enzymatically removed or they can spontaneously depurinate to produce abasic sites, which may be more mutagenic than NMPs (3Posnick L.M. Samson L.D. Mutat. Res. 1999; 257: 127-143Google Scholar, 4Xiao W. Chow B.L. Hanna M. Doetsch P.W. Mutat. Res. 2001; 487: 137-147Google Scholar). Some DNA lesions that are repaired by nucleotide excision repair (NER) or certain base excision repair (BER) pathways are removed much faster in the transcribed strand (TS) than in the nontranscribed strand (NTS) of an active gene (5Hanawalt P.C. Mutat. Res. 2001; 485: 3-13Google Scholar). In contrast, repair of NMPs does not appear to be coupled to transcription, since the TS and NTS have similar repair rates in the genes analyzed to date (6Scicchitano D.A. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3050-3054Google Scholar, 7Ye N. Holmquist G.P. O'Connor T.R. J. Mol. Biol. 1998; 284: 269-285Google Scholar, 8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar). However, repair rates of NMPs vary dramatically at different sites in both the humanPGK1 gene (7Ye N. Holmquist G.P. O'Connor T.R. J. Mol. Biol. 1998; 284: 269-285Google Scholar) and the yeast minichromosome YRpSO1 (8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar). One reason for this repair heterogeneity is the effect of nearest-neighbor nucleotides (8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar), in that slow repair occurs at NMPs between purines and fast repair occurs at NMPs between pyrimidines. It was proposed that different stabilities of base stacking between adjacent base pairs can affect flipping out of NMPs from the DNA helix during recognition and incision by DNA glycosylase (8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar). In the nucleus of eukaryotic cells, DNA is packaged into a nucleoprotein complex known as chromatin (9Wolffe A.P. Chromatin: Structure and Function. 3rd Ed. Academic Press, London, and New York1999: 7-172Google Scholar). This complex provides the compaction and structural organization of DNA for processes such as replication, transcription, recombination, and repair. The fundamental subunits of chromatin are nucleosome cores, where 147 bp of DNA is wrapped around a histone octamer (10Luger K. Mader A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Google Scholar). DNA between two adjacent nucleosome cores is called linker DNA, which varies in length from about 20 to 90 bp in different organisms and tissues, or between individual nucleosomes in the same cell (11van Holde K.E. Chromatin. Springer-Verlag KG, Berlin1989: 289-354Google Scholar). The effect of nucleosome structure on NMP repair is not understood. In rat liver cells, it was shown that the overall removal of NMPs occurred at a relatively uniform rate in different chromatin fractions (i.e. active chromatin, bulk genome and nuclear matrix) (12Ryan A.J. Billett M.A. O'Connor P.J. Carcinogenesis. 1986; 7: 1497-1503Google Scholar). On the other hand, within the yeast minichromosome YRpSO1, there was a mild correlation between repair rates and nucleosome positioning in regions of the inducible GAL1:URA3 fusion gene, but not in the constitutively expressed HIS3 gene on the same plasmid (8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar). To further address questions on the effect of nucleosome structure on repair of NMPs in intact cells, we examined repair in the divergent yeast GAL1-10 genes, which share a common upstream-activating sequence (UAS). These genes are induced to very high levels of expression in galactose, but are completely repressed in glucose (13Bash R. Lohr D. Prog. Nucleic Acids Res. Mol. Biol. 2001; 65: 197-259Google Scholar). Extensive studies have been done on isolated nuclei or chromatin to map the nucleosome structure in the GAL1-10region and the effects of galactose induction (14Lohr D. Torchia T. Hopper J. J. Biol. Chem. 1987; 262: 15589-15597Google Scholar, 15Fedor M.J. Lue N.F. Kornberg R.D. J. Mol. Biol. 1988; 204: 109-127Google Scholar, 16Fedor M.J. Kornberg R.D. Mol. Cell. Biol. 1989; 9: 1721-1732Google Scholar, 17Cavalli G. Thoma F. EMBO J. 1993; 12: 4603-4613Google Scholar). However, it is possible that subtle changes in nucleosome structure of this region differ in whole cells, and were missed because of the procedure of nuclei and/or chromatin isolation. Thus, to examine the influence of nucleosome structure on repair of NMPs in intact cells, we developed a nucleosome mapping procedure using bleomycin (BLM), which avoids isolation of nuclei or chromatin. This basic glycopeptide-derived antibiotic has been shown to preferentially cleave nucleosome linker DNA in isolated Chinese hamster nuclei (18Kuo M.T. Hsu T.C. Nature. 1978; 271: 83-84Google Scholar), lysophosphatidylcholine-permeabilized human cells (19Sidik K. Smerdon M.J. Cancer Res. 1990; 50: 1613-1619Google Scholar), and whole yeast cells (20Moore C.W. Cancer Res. 1988; 48: 6837-6843Google Scholar). However, BLM has not been used to map nucleosome positions in specific sequences in whole cells, since highly specific cleavage in linker DNA has not been achieved. By using the mild nonionic detergent digitonin to efficiently permeabilize yeast cells, and rich medium to effectively stop BLM cleavage during DNA isolation, we were able to map nucleosome structure at nucleotide resolution in the GAL1-10region in whole yeast cells and directly correlate repair of NMPs with nucleosome structure. DBY747 (MATa ura3-52his3-Δ1 leu2–3 leu2–112 trp1–289) and its isogenic mutant strains JC8901 (mag1Δ::hisG- URA3-hisG), WXY9379 (rad1Δ::LEU2), and WXY9380 (mag1Δ::hisG- URA3-hisG rad1Δ::LEU2) were generously provided by Dr. Wei Xiao (University of Saskatchewan, Canada). Strain Y452 (MATα ura3-52 his3–1 leu2–3 leu2–112) was provided by Dr. Louise Prakash (University of Texas Medical Branch, Galveston, TX). Yeast cells were grown at 30 °C in minimal medium containing 2% glucose or 2% galactose to late-log phase (OD600 ∼1.0). After washing twice with ice-cold 2% glucose (for glucose cultures) or 2% galactose (for galactose cultures), the cells were resuspended in 50 mm NaCl, 2 mm MgCl2, 0.02% glucose or galactose, to give a cell density of 2 × 109cells/ml. Digitonin (Sigma, 10% stock) and Fe(NH4)2(SO4)2 (10 mm, freshly dissolved in H2O) were mixed with the cell suspension to give a final concentration of 0.05% and 50 μm, respectively. BLM (Sigma, 20 units/ml stock) was then added to final concentrations of 0–400 milliunits/ml, and the mixture was incubated at 30 °C for To stop the the cell suspension was mixed with of ice-cold yeast 2% 2% or yeast 2% 2% and by the cells were resuspended in ice-cold 2% glucose or 2% galactose, mixed with of a containing 10% yeast and and by It has been shown that BLM with of cells and can cleave DNA the cells are in the of R.D. 1989; Scholar). Furthermore, BLM can be efficiently removed from cell by R.D. 1989; Scholar). BLM can be efficiently removed by washing the cells with containing yeast and The DNA used in these was a of the 50 mm of a of DNA, of freshly Fe(NH4)2(SO4)2 and of After of at 30 the DNA was from the mixture with the Yeast cells were grown at 30 °C in minimal medium containing 2% glucose or 2% galactose to phase (OD600 and mixed with (Sigma, to give a final concentration of After 2 at cells were twice with ice-cold 2% glucose 2% and resuspended in the same containing to DNA during repair J. Scholar). of a containing 10% yeast and was added to the After different of repair at 30 an was removed and on After BLM or repair of 2 × cells, were mixed with 2 of ice-cold nuclei isolation 50 mm 2 mm NaCl, mm mm and 2 of (Sigma, The were for 30 and on for This procedure was to completely the cells. The were mixed with of 50 mm mm NaCl, 2% 2 mm and incubated at °C for 30 After to the were mixed with of and on The were then at and the was The DNA was with and with After the DNA was dissolved in and at °C The sites of BLM cleavage and NMPs were a region encompassing the common and of the and of these are shown in and on strand of the region were using the procedure S. R. Carcinogenesis. Scholar, S. R. Smerdon M.J. Scholar), with about of DNA was with to the of NMP the DNA was further at the NMP sites by DNA in at 90 °C for 30 and the removed by (8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar). of a which has a to of the to be were mixed with the The in the S. R. Carcinogenesis. Scholar, S. R. Smerdon M.J. Scholar) was to W. Chow B.L. Hanna M. Doetsch P.W. Mutat. Res. 2001; 487: 137-147Google Scholar, P.C. Mutat. Res. 2001; 485: 3-13Google Scholar) of by This of the and between the and of The mixture was to °C for to the DNA and then to an The were to and the other were removed by washing the at the The were using and than S. R. Carcinogenesis. Scholar, S. R. Smerdon M.J. Scholar). The were on and to cleavage of the strand of the GAL1-10 region NTS for and TS for are of for of analyzed a region of the GAL1-10 to were from of using the J. T. Molecular Ed. Scholar). The were using the same procedure as that for the BLM or DNA The BLM cleavage at individual sites is by the on a The of BLM used were relatively to that cleavage by BLM for the of However, these a of the have more than and a of the at a not on a the cleavage occurs to the of the The effect of this increases as a cleave is more from the which at the of a a of nucleotides (i.e. the length on the the at a close to the of a can be as much as of the in a To for the was the in a the (i.e. not by at N in a and the of N sites N in a The of at N is and the of of N is BLM cleavage at specific sites is different (i.e. a then the of at N and those of N on the same is × the of at N and those of N on the same is × × or × the at N on the then × and the at N is × The at in a was by and the to After the was the in different of a were to the same The at a the not with was then using the × The in a containing the were used as a for other that BLM the a of the was by of of in the The in a containing BLM were then by of the The was then to to and to the individual on the S. R. Smerdon M.J. Scholar). for NMPs the same procedure as that for BLM yeast cells were grown in glucose or galactose with digitonin and with The used for digitonin and BLM was which is to BLM cleavage in chromatin these different concentrations of BLM a of and not the cleavage by BLM for the of the chromatin DNA DNA was isolated from the cells and with to the of of were analyzed for strand of The were on DNA and to DNA of the same GAL1-10 region was in with the chromatin can be seen from the in and DNA was at very concentrations of BLM and and cleavage was in the of BLM or in and not pyrimidines to are by BLM 2 in with the and with Proc. Natl. Acad. Sci. U. S. A. 1978; Scholar). to are but to a and to the of Furthermore, sites of cleavage are at pyrimidines to and on of higher concentrations of BLM and are for cleavage of chromatin DNA in cells to the same as that for DNA and Furthermore, there was more cleavage of chromatin DNA in glucose cultures than in galactose cultures, the same concentration of BLM was and not BLM is more in galactose cultures to the induction of BLM which is by the gene J. Biol. Chem. 1997; Scholar). To the of a nucleosome to its DNA, the of for BLM cleavage of DNA to chromatin DNA glucose and galactose cultures) were from of and S. R. Smerdon M.J. Scholar). This was as at a BLM cleavage to the in the gene, 2 on the strand the The at other cleavage sites were then to the at this The are in for both glucose and galactose grown cells, and the in of for these are in B. The BLM cleavage at most sites in the region is similar between chromatin glucose and galactose cultures) and DNA and 2 the region is in both glucose and galactose This is in with D. Nucleic Acids Res. 12: Scholar) and M.J. Kornberg R.D. Mol. Cell. Biol. 1989; 9: 1721-1732Google Scholar) mapping in isolated However, a sites in the region or of BLM cleavage and 2 the of most to this region may modulate BLM In glucose cultures, where the genes are repressed, a region of about bp on of the is from BLM cleavage and and regions by that of these regions a nucleosome with a fixed on The of bp regions and is and the with increasing distance from the by the of in the levels can be in the core regions of nucleosomes and which are adjacent to the UAS, than those of which are from the These results indicate that the variability of nucleosome positions sharply increases with increasing distance from the in intact cells. In galactose cultures, where GAL1-10 genes are the nucleosome core sequences in the GAL1-10 region analyzed are from BLM cleavage nucleosomes are galactose This disruption is most in nucleosomes and which are adjacent to the UAS, to their the of positions in glucose cultures The same yeast cells as those used for nucleosome mapping were used for NMP induction and repair in the GAL1-10 The cells were with for 2 to NMPs After different of repair DNA was with and at NMPs by The were on DNA and to can be seen from the in the of NMPs induced is with is induced to a much with and This of NMP induction is similar to that seen with the yeast minichromosome YRpSO1 (8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar) and to that of other R.O. Nickoloff J.A. Hoekstra M.F. DNA Damage and Repair, Vol. 2: DNA Repair in higher eukaryotes. Humana Press Inc., Totowa, NJ1998: 199-222Google and repair of NMPs in the strand NTS for and TS of the GAL1-10 region for glucose and galactose the to for In galactose cultures, a strong from is seen at in the sequences of or at both of the sites in with on of with a M. Cell. Scholar). an of can be seen in sites of the in the strand on the of there is a between glucose and galactose cultures in NMP at sites in the regions of the two genes and on the of On the other hand, NMP at most sites throughout the region are similar between the two cultures that the of nucleosomes does not affect NMP This with a in that of nucleosomes does not significantly modulate NMP induction by G. Proc. Natl. Acad. Sci. U. S. A. Scholar). The repair rates of NMPs at different sites were dramatically different at can be seen from the in and most NMP sites between pyrimidines or were repaired much faster than NMPs between purines or and those between a and a were repaired at In to the of these the of different of repair in the same of nearest-neighbor nucleotides (i.e. between purines, and between a and a were can be seen from the these sites were for the effect of the nearest-neighbor nucleotides on repair be analyzed by this of nearest-neighbor nucleotides on repair of in the GAL1-10 genes of glucose and galactose cultures, respectively. The in the of the of at the sites that be analyzed in the region between purines pyrimidines and a and a The for the sites between a and a are not to more clearly the of repair rates between the other two of nearest-neighbor In glucose cultures, the correlation between nucleosome positioning and NMP repair can be seen in the two nucleosomes and with the most fixed positions In these slow repair occurs in the nucleosome core DNA and faster repair in nucleosome linker or DNA (i.e. the This correlation sharply in the nucleosomes that are more from the UAS, in with the that the variability of nucleosome positioning sharply increases with distance from the This of correlation can be seen more clearly the individual for of the NMPs are the in However, with the effects of nearest-neighbor nucleotides on NMP repair on the effects of nucleosome the nucleosome effect is and more to in the regions from the Galactose induction causes of repair in both strands of the nucleosome core DNA increases of as much as in the core regions of nucleosomes which are most dramatically this of NMP repair with distance from the with the nucleosome positioning and disruption and It has been shown that are in are to and the effects of these are W. Chow B.L. 1998; Scholar). This that may an for repair of To the of NER, repair of NMPs was analyzed in different regions of isogenic and cells. NMP repair in regions of the gene and the constitutively expressed gene, which the of is shown in can be repair occurred in cells. In contrast, of gene completely repair of repair can be seen the gene is in the cells Repair in other regions the same not in the repair of NMPs in S. have nucleosome structure and repair ofN-methylpurines in whole yeast cells. The nucleosome positions in this with on isolated nuclei or chromatin M.J. Lue N.F. Kornberg R.D. J. Mol. Biol. 1988; 204: 109-127Google Scholar, 16Fedor M.J. Kornberg R.D. Mol. Cell. Biol. 1989; 9: 1721-1732Google Scholar, 17Cavalli G. Thoma F. EMBO J. 1993; 12: 4603-4613Google Scholar). However, in the nucleosomes that fixed positions in the region were in the regions adjacent to the in glucose cultures, and the variability of positions sharply increases with increasing distance from the This with the that the of to a sequence that the of the as a nucleosome positioning M.J. Lue N.F. Kornberg R.D. J. Mol. Biol. 1988; 204: 109-127Google Scholar). In contrast, mapping with isolated nuclei or chromatin of nucleosomes in the GAL1-10 region M.J. Lue N.F. Kornberg R.D. J. Mol. Biol. 1988; 204: 109-127Google Scholar, 16Fedor M.J. Kornberg R.D. Mol. Cell. Biol. 1989; 9: 1721-1732Google Scholar, 17Cavalli G. Thoma F. EMBO J. 1993; 12: 4603-4613Google Scholar). This may a for nucleosome positions in chromatin during nuclei isolation, with more of nucleosomes that are from the in intact cells. This with the NMP repair as as results of nucleotide excision repair of induced and M. J. that nucleosomes in intact cells NMP repair. of in nucleosomes with fixed positions and repair is much in the core DNA sequences than in linker DNA induction of NMP with the regions close to the nucleosome disruption is most being most it is that this is by a between and as strand for repair is in these regions This with nucleosome positioning and disruption and to influence repair of nucleosome affected NMP repair may be in the more nucleosomes in the In to the influence of nucleotides the of may affect NMP repair. repair of NMPs in the region was faster in glucose cultures than in galactose cultures, where the is to the region and These may little or correlation between NMP repair and nucleosome positioning is seen in regions from the In a on NMP repair in the yeast minichromosome YRpSO1 (8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar), we a mild correlation between repair rates and nucleosome positioning in regions of the gene, but not in the HIS3 gene (8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar). these in of nucleosome positions in these genes on the and Mag1-initiated are in to DNA lesions W. Chow B.L. 1998; Scholar), a of these lesions may be repaired by both Furthermore, in with human glycosylase which is the of yeast that with the human of F. M. R. F. G. O'Connor T.R. J. Biol. Chem. Scholar). this the rate of excision from F. M. R. F. G. O'Connor T.R. J. Biol. Chem. Scholar). results with and cells in S. repair of NMPs is primarily by the Mag1-initiated pathway, and that contributes very little to the repair of these lesions. analyzed NMP repair in and of which is a of the S. Prakash Mutat. Res. Scholar). of these a in repair of NMPs not Thus, it is possible that the by and is not but a DNA that be by that a very of repair of NMPs does in cells is This repair may be to the between and the for lesions. of organisms have a strong for In the and can repair of these the for the two D.A. Scholar, M. Sci. Scholar). In NMPs may be repaired primarily the pathway, than a A. Samson J. Scholar). In cells, a may exist that in the of the used DNA glycosylase Nucleic Acids Res. Scholar). However, of the gene in S. cells completely NMP repair not and NMP repair can be seen in cells. This that S. may a strong for as with the yeast minichromosome YRpSO1 (8Li S. Smerdon M.J. J. Biol. Chem. 1999; 274: 12201-12204Google Scholar), there was a correlation between the nucleotides of NMP sites and the repair of NMPs in the yeast These that the same repair is used for NMPs in and minichromosome This is similar to that of and Hanawalt Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. Scholar), repair of in and minichromosome DNA of Dr. Wei Xiao for and and Dr. Louise Prakash for of the Smerdon for and

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