Base Excision Repair of N-Methylpurines in a Yeast Minichromosome

Abstract

Base excision repair of dimethyl sulfate induced Nmethylpurines (NMPs) was measured in a yeast minichromosome that has a galactose-inducible GAL1: URA3 fusion gene, a constitutively expressed HIS3 gene, and varied regions of chromatin structure. Removal rates of NMPs varied dramatically (>20-fold) at different sites along three selected fragments encompassing a total length of 1775 base pairs. Repair of NMPs was not coupled to transcription, because the transcribed strands of HIS3 and induced GAL1:URA3 were not repaired faster than the nontranscribed strands. However, the repair rate of NMPs was significantly affected by the nearest neighbor nucleotides. Slow repair occurred at NMPs between purines, especially guanines, whereas fast repair occurred at NMPs between pyrimidines. NMPs between a purine and pyrimidine were repaired at moderate rates. Moreover, a rough correlation between nucleosome positions and repair rates exists in some but not all regions that were analyzed.Subtle alterations in DNA molecules, such as oxidized or alkylated bases, are repaired by the base excision repair (BER) 1 pathway (reviewed in Refs. 1 and 2). BER is initiated by specific DNA glycosylases that release the damaged base. There are two families of alkylated base DNA glycosylases (3). Although their primary sequences and three-dimensional structures are unrelated, they may have common themes in recognition of alkylated bases and catalysis of glycosylic bond cleavage (3). Crystal structures of the human AAG protein (3) and the Escherichia coli AlkA protein (4, 5) suggest that the glycosylases flip the target base out of the DNA duplex into the active site cleft of the enzymes. The "flipping out" may occur in a later step during recognition and/or incision of damaged bases (6).Alkylation damage to DNA occurs frequently in nature and can be introduced either by alkylating agents present in the environment or by erroneous alkylation induced by the natural methyl donor S-adenosyl-methione (7). Simple methylating agents such as dimethyl sulfate (DMS) produce a variety of damaged bases in DNA of which N-methylpurines (N 7 -methylguanine and N 3 -methyladenine) or NMPs constitute approximately 90% of the alterations (8). In Saccharomyces cerevisiae, the repair of NMPs occurs mainly through the BER pathway, although nucleotide excision repair (NER) may play a role if the BER pathway is abolished (9). Initiation of this pathway in S. cerevisiae is by the action of the small (34 kDa) protein MAG DNA glycosylase (10).Although the repair of thymine glycols is coupled to transcription in both yeast and mammalian cells (11,12), a link between transcription and BER of other lesions has not been well established. It was found that the alkali-labile sites (representing NMPs) induced by methylnitrosourea are preferentially repaired within the active insulin gene of rat insulinoma cells (13). However, no preferential repair of these lesions induced by DMS was found in the actively transcribed DHFR gene of Chinese hamster ovary cells (14 -16) or in t...

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

“…compare 0 h lanes with G and GA lanes). This pattern of NMP induction is similar to that seen with the yeast minichromosome YRpSO1 (8) and to that of other past reports (2).…”

“…Briefly, about 2-3 g of genomic DNA was digested with restriction endonuclease(s) to release the fragments of interest. For NMP mapping, the restricted DNA was further cleaved at the NMP sites by incubating DNA in 1 M piperidine at 90°C for 30 min, and the piperidine removed by evaporation (8). Excess copies of a biotinylated oligonucleotide, which has a portion complementary to one end of the fragment to be labeled, were mixed with the sample.…”

“…active chromatin, bulk genome and nuclear matrix) (12). 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 (8).…”

“…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 (6 -8). However, repair rates of NMPs vary dramatically at different sites in both the human PGK1 gene (7) and the yeast minichromosome YRpSO1 (8). One reason for this repair heterogeneity is the effect of nearest-neighbor nucleotides (8), in that slow repair occurs at NMPs between purines and fast repair occurs at NMPs between pyrimidines.…”

“…One reason for this repair heterogeneity is the effect of nearest-neighbor nucleotides (8), 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 (8).…”