The bacterial alkyltransferase-like (eATL) protein protects mammalian cells against methylating agent-induced toxicity

In both pro- and eukaryotes, the mutagenic and toxic DNA adduct O⁶-methylguanine (O⁶MeG) is subject to repair by alkyltransferase proteins via methyl group transfer. In addition, in prokaryotes, there are proteins with sequence homology to alkyltransferases, collectively designated as alkyltransferase-like (ATL) proteins, which bind to O⁶-alkylguanine adducts and mediate resistance to alkylating agents. Whether such proteins might enable similar protection in higher eukaryotes is unknown. Here we expressed the ATL protein of Escherichia coli (eATL) in mammalian cells and addressed the question whether it is able to protect them against the cytotoxic effects of alkylating agents. The Chinese hamster cell line CHO-9, the nucleotide excision repair (NER) deficient derivative 43-3B and the DNA mismatch repair (MMR) impaired derivative Tk22-C1 were transfected with eATL cloned in an expression plasmid and the sensitivity to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was determined in reproductive survival, DNA double-strand break (DSB) and apoptosis assays. The results indicate that eATL expression is tolerated in mammalian cells and conferes protection against killing by MNNG in both wild-type and 43-3B cells, but not in the MMR-impaired cell line. The protection effect was dependent on the expression level of eATL and was completely ablated in cells co-expressing the human O⁶-methylguanine-DNA methyltransferase (MGMT). eATL did not protect against cytotoxicity induced by the chloroethylating agent lomustine, suggesting that O⁶-chloroethylguanine adducts are not target of eATL. To investigate the mechanism of protection, we determined O⁶MeG levels in DNA after MNNG treatment and found that eATL did not cause removal of the adduct. However, eATL expression resulted in a significantly lower level of DSBs in MNNG-treated cells, and this was concomitant with attenuation of G2 blockage and a lower level of apoptosis. The results suggest that eATL confers protection against methylating agents by masking O⁶MeG/thymine mispaired adducts, preventing them from becoming a substrate for mismatch repair-mediated DSB formation and cell death.     

Synthesis and characterisation of novel chromogenic substrates for human pancreatic alpha-amylase

Derivatives of maltose and maltotriose were chemically synthesised as substrates for human pancreatic alpha-amylases and subjected to kinetic analysis. Rates measured were shown to reflect both hydrolysis and transglycosylation reactions. 4-O-Methylated derivatives of these substrates underwent only hydrolysis, thereby simplifying kinetic analyses. These modified substrates may be used for the detection and kinetic analysis of alpha-amylases, and are useful in rapidly screening for novel alpha-amylase inhibitors and for subsequent kinetic characterisation.     

In vivo effect of DNA repair on the transition frequency produced from a single O6-methyl- or O6-n-butyl-guanine in a T:G base pair

Summary We have previously reported some effects of DNA repair on the transition frequencies produced by an O⁶-methyl-guanine (MeG) or an O⁶-n-butyl-guanine (BuG) paired with C at the first position of the third codon in gene G of bacteriophage X174 form I'DNA (Chambers et al. 1985). We now report experiments in which the transition is produced from T:MeG or T:BuG, instead of C:MeG or C:BuG, located at this site. The site-modified DNAs were transfected into cells with normal DNA repair as well as into cells with repair defects (uvrA, uvrB, uvrC, recA, uvrArecA). The lysates were screened for phage carrying the expected transition using a characteristic change in phenotype. The data demonstrate that the transition frequency from T:BuG is low (0.3% of total phage progeny) in cells with normal repair (Escherichia coli AB1157) and increases 7-fold in uvrA cells (E. coli AB1886). A similar increase is seen in uvrB and uvrC cells (AB1885, AB1884). These data, like our previous data, indicate BuG is repaired primarily by excision. In contranst to this, the transition frequency from T:MeG is high (5±2%) in cells with normal repair. After induction of alkyl transfer repair in E. coli AB1157, the transition frequency goes up 5-fold. Compared with cells with normal repair, the transition frequency goes up 2-fold in uvrA, uvrB and uvrC cells; it goes up 1.5-fold in recA cells (E. coli AB2463). The data reinforce our earlier conclusion that MeG is repaired primarily by alkyl transfer, but the ABC excinuclease as well as RecA protein inhibit this repair process. Using the BuG data reported here and in our previous paper, we calculate that BuG pairs with a thymine residue 0.5%–0.62% of the time during replication in vivo, and that BuG markedly inhibits replication of the strand that contains it. Because of the complication introduced by alkyl transfer repair, similar calculations for MeG cannot be made from the current data.Abbreviations MeG and BuG O⁶-methyl-or O⁶-n-butyl-guanine moiety in X DNA (in each case, the plus strand nucleotide is specified first) - form I'DNA relaxed, covalently closed, circular, double-stranded DNA - Wt wild-type phenotype - Am amber phenotype - pfu plaque forming units - MNNG N-methyl-N'-nitro-N-nitrosoguanidine X mutants are named by designating the gene, the type of mutation (e.g. ms=missense), the codon number, the mutant codon and the new amino acid (where pertinent) in that order (e.g. XGam3) carries an amber in the third codon of gene G, and should not be confused with the classical am3 mutant used in the older literature to designate what is now known to be XEam7     

Differential methylation of mitochondrial and nuclear DNA in cultured mouse, hamster and virus-transformed hamster cells. In vivo and in vitro methylation

Information has been lacking as to whether mitochondrial DNA of animal cells is methylated. The methylation patterns of mitochondrial and nuclear DNAs of several mammalian cell lines have therefore been compared by four methods: (1) in vivo transfer of the methyl group from [methyl-³H]methionine; (2) in vivo incorporation of [³²P]orthophosphate and a combination of (1) and (2); (3) in vivo incorporation of [³H]deoxycytidine; (4) in vitro methylation of DNAs with ³H-labeled S-adenosylmethionine as methyl donor and DNA methylase preparations from L cell nuclei. The cell lines were mouse L cells, $\text{BHK21}\text{C13}$, $\text{C13}\text{B4}$ (baby hamster kidney cells transformed by the Bryan strain of Rouse sarcoma virus), and PyY (BHK cells transformed by polyoma virus). DNA bases were separated chromatographically, using 5-methylcytosine, 6-methylaminopurine and, in some cases, 7-methylguanine as markers.Mitochondrial DNA was found to be significantly less methylated than nuclear DNA with respect to 5-methylcytosine in all cell types studied and by all methods used. The relative advantages and disadvantages of each method have been discussed. The level of 5-methylcytosine in mitochondrial DNA as compared with that in nuclear DNA was estimated as one-fourth to one-fourteenth in various cell lines. The estimated 5-methylcytosine content per circular mitochondrial DNA molecule (mol. wt 10 × 10⁶) was about 12 methylcytosine residues for L cells and 24, 30 and 36 methylcytosine residues for BHK, B4 and PyY cells, respectively. Relative to cytosine residues, the estimate was one 5-methylcytosine per 500 cytosine residues of mitochondrial DNA and one 5-methylcytosine per 36 cytosine residues of nuclear DNA from L-cells. The values for methylcytosine of mitochondrial DNA are presumed to be maximal. PyY cells as compared with other cells had the highest methylcytosine content of both mitochondrial and nuclear DNA as estimated by method (3). No methylation of nuclear DNA was observed in confluent L cells.Evidence for the presence of DNA methylase activity associated with mitochondrial fractions was obtained. This activity could be distinguished from other cellular DNA methylase activity by differential response to mercaptoethanol. Radioactivity from ³H-labeled S-adenosylmethionine was found only in 5-methyl-cytosine of DNA.     

Selectivity of the excision of alkylation products in a xeroderma pigmentosum-derived lymphoblastoid line.

Lymphoblastoid cell derived from a complementation group C xeroderma patient were unable to remove 06-methyl guanine residues formed in DNA by treatment of cells with low concentration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The xeroderma cells were competent in their ability to excise 3-methyl adenine adducts. MNNG treatment induced excision repair in the xeroderma line and in addition the treatment resulted in the presence of numerous single-strand breaks in the DNA. The single gene, UV-excision-defective mutants of Escherichia coli, uvrA and uvrB, are able to excise MNNG-induced 06-methyl guanine adducts indicating that excision of this compound is not due to operation of UV endonuclease system.