Conservation and diversification of genes by mismatch correction and SOS induction.

Regulation of genetic stability is discussed in terms of interactions between constitutive and inducible DNA repair processes with specific emphasis on the results of our experimental studies of mismatch correction and SOS induction in Escherichia coli.

The effect of growth conditions on inducible, recA-dependent resistance to X rays in Escherichia coli.

Escherichia coli cells grown to logarithmic phase in, and plated on, rich medium (yeast extract-nutrient broth) were more resistant to X rays, ultraviolet (uv) radiation, and methyl methanesulfonate (MMS) than cells grown in, and plated on, minimal medium. We have called this enhanced survival capability medium-dependent resistance (MDR). The magnitude of MDR observed after oxic X irradiation was greater than that observed after anoxic X irradiation, uv irradiation, or MMS treatment. MDR was not observed in stationary-phase cells with X or uv radiation. MDR was associated with an increased ability to repair X-ray-induced DNA single-strand breaks, and with reduced X-ray-induced DNA degradation and protein synthesis retardation. Postirradiation protein synthesis was concluded to be critical in allowing the high X-ray survival associated with MDR, because of the large radiosensitization caused by a postirradiation growth medium shift down or treatment with rifampicin (RIF), recA protein must be at least one of the proteins whose synthesis is critical to MDR, as judged by the absence of MDR or a RIF effect in X-irradiated recA and lexA mutants. The results with X-irradiated temperature-conditional recA cells suggest that it is only after cells have been damaged that the recA gene plays a role in MDR.

The effects of mutations in the polA and recA genes on mutagenesis by nitrosoguanidine in Salmonella typhimurium.

Results of semi-quantitative plate tests indicated that polA and recA mutants of Salmonella typhimurium strain LT2 trpB1 might be significantly less mutable by nitrosoguanidine (MNNG) than were their repair-proficient parents strains. Quantitative data obtained in treat-and-plate experiments showed that this was not the case, at least for low doses of MNNG, and also that the recA strain was significantly more mutable at low doses than its Rec+ parent. On the basis of these results it is suggested that cells of S. typhimurium may possess a recA+-dependent repair pathway capable of error-free removal of MNNG-induced pre-mutational lesions from their DNA.

Clonally derived cell lines with altered DNA methylation as models for error correction and mutation in tumours.

We have investigated the relative importance of DNA methylation-directed repair of replication errors to the production of mutation/gene rearrangements in mammalian cells with hypomethylated genomes. Using a series of sibling (Chinese hamster derived) cell clones with stably altered DNA methylation levels, we found that genomic hypomethylation was frequently associated with an elevated spontaneous mutation rate in the hypoxanthine phosphoribosyl transferase (HPRT) gene and with increased sensitivity to 2-aminopurine, a known inducer of DNA base mismatches. However, while there are some alterations in sites of DNA methylation around the HPRT gene, there was no systematic direction of change to differentiate between cells with normal or with elevated mutation rates. Further, the nature of gene alterations giving rise to the HPRT mutations suggested that methylation-directed mismatch repair is of little if any significance in influencing error avoidance in mammalian cells with hypomethylated genomes and that increased mutation associated with DNA hypomethylation is more likely to be due to increased genomic instability through activation of such factors as transposable elements.

2-Aminopurine as a probe for mismatch repair in mammalian cells and its relationship to DNA methylation.

We have used a series of clonally related mammalian cell lines with different levels of DNA methylation and the known inducer of DNA mismatch repair in Escherichia coli, 2-aminopurine (2AP), to test for the presence of methylation-directed DNA repair in mammalian cells. While a number of these hypomethylated clones showed increased sensitivity to 2AP, another similarly hypomethylated clone was resistant. DNA replication and both the immediate and delayed classes of DNA methylation were strongly inhibited and to similar extents in both sensitive and resistant clones. The inclusion of deoxycytidine reversed most of the 2AP-induced inhibition of replication and methylation without reducing 2AP toxicity. While 2AP-induced DNA mismatches are repaired by a methylation-directed process in E. coli, this analogue has major secondary effects in mammalian cells unrelated to its toxicity. Genomic methylation levels are also not a determinant of resistance or sensitivity to this base analogue in mammalian cells.

The majority of methylated deoxycytidines in human DNA are not in the CpG dinucleotide.

The only natural postsynthetic modification known to occur in mammalian DNA is the methylation in the 5 position of deoxycytidines. Of the four 5'-CpN-3' dinucleotides (ie. CpG, CpC, CpA, and CpT), the dinucleotide which contains the highest proportion of deoxycytidines methylated is CpG, with 40 to 80% methylation in different mammalian genomes. It has also been shown that CpA, CpT, and CpC are methylated as well but to a much lower extent. Here we report the result of a full nearest neighbour analysis (together with quantitation of methylation levels in the 4 CpN dinucleotides) for DNA from human spleen. Using the values we have calculated the overall frequencies for all the methylated dinucleotides in the human genome. Because of the relative underrepresentation (by 7 to 10 fold) of the CpG dinucleotide, only 45.5% of total mC was present in mCpG, with 54.5% in mCpA, mCpT plus mCpC. These calculations have implications for studies into the function and significance of DNA methylation in mammalian cells.

Human cellular sequences detectable with adenovirus probes. I. Evidence for novel repeat sequences and a possible E1a-like cellular "gene".

Previous studies suggesting homology between human cellular DNA and the DNAs from adenovirus types 2 and 5 are extended in the present paper. A clone (ChAdh), isolated from a human genomic DNA library using an adenovirus probe, hybridized to discrete regions of adenovirus 2 DNA, including part of the transforming genes E1a and E1b, as well as to repeated sequences within human DNA. The E1a and E1b genes both hybridize to the same 300 base pair Sau3AI fragment within ChAdh although there is no obvious homology between E1a and E1b. The Ad 2 E1a gene was also used as a probe to screen other cellular DNAs to determine whether repeated sequences detectable with Ad2 DNA probes were conserved over long evolutionary periods. Hybridization was detected to the genomes of man, rat, mouse and fruit fly, but not to those of yeast and bacteria. In addition to a "smear" hybridization, discrete fragments were detected in both rodent and fruit fly DNAs. The experiments reported suggest the existence of two different types of cellular sequences detected by Ad 2 DNA: (1) repeated sequences conserved in a variety of eukaryote genomes and (2) a possible unique sequence detected with an E1a probe different from that responsible for hybridization to repeated sequences. This unique sequence was detected as an EcoRI fragment in mouse DNA and had a molecular size of about 8.8 kb.

DNA sequences of and complementation by the tnpR genes of Tn21, Tn501 and Tn1721.

DNA sequences that encode the tnpR genes and internal resolution (res) sites of transposons Tn21 and Tn501, and the res site and the start of the tnpR gene of Tn1721 have been determined. There is considerable homology between all three sequences. The homology between Tn21 and Tn501 extends further than that between Tn1721 and Tn501 (or Tn21), but in the homologous regions, Tn1721 is 93% homologous with Tn501, while Tn21 is only 72-73% homologous. The tnpR genes of Tn21 and Tn501 encode proteins of 186 amino acids which show homology with the tnpR gene product of Tn3 and with other enzymes that carry out site-specific recombination. However, in all three transposons, and in contrast to Tn3, the tnpR gene is transcribed towards tnpA gene, and the res site is upstream of both. The res site of Tn3 shows no obvious homology with the res regions of these three transposons. Just upstream of the tnpR gene and within the region that displays common homology between the three elements, there is a 50 bp deletion in Tn21, compared to the other two elements. A TnpR- derivative of Tn21 was complemented by Tn21, Tn501 and Tn1721, but not by Tn3.

RglB facilitated cloning of highly methylated eukaryotic DNA: the human L1 transposon, plant DNA, and DNA methylated in vitro with human DNA methyltransferase.

In vitro methylation of Bluescribe plasmid DNA (pBS) with human placental DNA methyltransferase to 6% 5-methylcytosine (mC) reduced transformation efficiencies in rglB+ host strains C600 and DS410 by almost 2 orders of magnitude. By contrast, the rglB- derivative of DS410 showed no reduction in transformation efficiency with methylation while the rglB- derivative of C600 was partially tolerant to methylation. Further, we show that the 1.8 kilobase (kb) and 1.2 kb KpnI fragments derived from the human L1 repeat have respectively 18.3% and 2.3% mC in vivo. Using these hyper- and hypo-methylated genomic segments ligated into the pBS plasmid, transformants with the highly methylated 1.8 kb L1 insert were recovered at 17 to 40 fold higher frequency with the rglB- host strains than with the rglB+ hosts. In addition, recombinant phage (lambda 2001) containing inserts of plant genomic DNA with 26.7% mC (from Petunia hybrida) when plated on rglB- hosts gave titres up to 222 times higher than on the rglB+ strains.