I was appointed to my first faculty position as an Instructor at Rutgers Medical School (as it was known then) in Piscataway, NJ, in 1971. I had come to join N. Ronald Morris who had been researching DNA methylation in eukaryotes. It seemed worthwhile studying the problem in Escherichia coli which, unlike eukaryotes, had both 6-methyladenine (6-meA) and 5-methylcytosine (5-meC) in its DNA. The approach would be a standard one - isolate mutants lacking methylated bases and deduce their functions by examining their properties.
The assay we used to isolate methylation-deficient mutants was based on two prior observations. First, DNA isolated from E. coli
grown in the presence of ethionine, a methionine analog, was found to be deficient in methylation because it was a substrate for the transfer of methyl groups from S-adenosyl-methionine (SAM) to such DNA in crude extracts prepared from wild-type cells grown without ethionine [7
]. DNA isolated from untreated E. coli
was not a substrate because the DNA was fully methylated. Second, Herb Boyer’s lab had located the gene (near his
) for cytosine methylation on the E. coli
K-12 map by using this assay on recombinants obtained from crosses between K-12 and B which does not have methylated cytosine in its DNA [8
]. These findings suggested a way to detect mutants deficient in methylation - they would incorporate methyl groups into their DNA while wild-type cells would not. Accordingly, I treated my wild-type cells with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and combined the survivors in groups of ten. DNA was isolated from the pool and assayed for methyl group transfer from tritiated SAM into DNA. If a positive signal was obtained, the pool was split in two for re-testing and finally to individuals. This brute force screen led to the isolation of 14 DNA and 10 RNA methylation mutants from about 1500 survivors. The RNA mutants were easily identified by testing radioactivity in the alkali-treated nucleic acid supernatant fraction designed to remove RNA.
The 14 DNA methylation mutants were grown with tritiated methionine and the amount of 6-meA and 5-meC quantified. This led to the identification of three mutants lacking 6-meA and 11 lacking 5-meC [9
]. Seven tRNA methylation mutants were also recovered and in collaboration with Dieter Soll’s lab at Yale were shown to be deficient in ribothymine (5 isolates), 7-methylguanine and 2-thio-5-methylaminoethyluracil [10
]. At this point it was necessary to identify the genes involved by mapping the mutations and this was done first by conjugational crosses and then by transductional crosses [11
]. I tested recombinant classes using the assay above, which was successful but laborious. The 6-meA and 5-meC mutants were designated dam
(DNA adenine methylase) and dcm
(DNA cytosine methylase), respectively, although in recent years I’ve been using methyltransferase instead of methylase. I had toyed with mad
(methyladenine deficient) as the designation for the gene but dam
won out. The Dam and Dcm methyltransferases methylate -GATC- and -CC(A/T)GG- sequences, respectively, of which there are 19,120 and 12,045, respectively, in the E. coli
chromosome. Stan Hattman’s lab had also isolated dcm
mutants at the same time by looking for E. coli
mutants that would not protect phage lambda from the restriction system encoded by plasmid N3 [12
]. For many years this was the only phenotype associated with dcm
mutants. From this point on I will deal only with the dam
mutants since these had not been previously isolated, and I had concentrated my efforts on them.
The mutations in a clean genetic background were tested for a variety of phenotypic traits. I had included in the isolation protocol the possibility that the mutations conferred a temperature-sensitive phenotype but none of the mutants were temperature-sensitive (Ts) for growth. This was somewhat disappointing but K. Brooks Low, then a junior faculty member in Therapeutic Radiology at Yale, consoled me by pointing out that recA
mutants were not conditionally lethal but were still interesting to study. Microscopic observation showed that the dam
cells were not uniform in size confirming that for a given optical density in broth cultures the viable count was always lower for the dam
mutant than the wild type. In my previous work with dnaB
mutants I had also observed this when the cells were grown at the non-permissive temperature and this led me to look at the DNA sedimentation profile in alkaline sucrose gradients. There were single-strand breaks in the chromosomal DNA of the dam
cells and these were amplified in dam polA
(Ts) and dam lig
(Ts) strains. These latter strains were inviable at the non-permissive temperature as were dam
mutations in combination with recA
mutant alleles. It was clear that the dam
mutants were defective in some kind of DNA repair but the best that could be done at the time was to exclude nucleotide excision repair since the uvr
genes had no effect on dam
]. During the mapping of the dam
gene, I noticed that my control plates for the dam
mutants often had colonies on them while those of the wild type did not. The mutator phenotype of the dam
mutants was quickly confirmed.
These results were published as my three-year appointment at Rutgers Medical School was coming to an end, and I was busy trying to find a new position. My wife was seven months pregnant when in June 1974 she drove our Volvo and I drove the U-Haul truck to Worcester, Massachusetts, where I was to take up a faculty position in the newly-formed University of Massachusetts Medical School. I had expected to be there for only a few years before continuing our nomadic existence but I have remained there ever since.
In my assistant professor position at UMass Medical School, I isolated more dam
mutants by various means and all had the same range of phenotypes as those previously isolated. In order to confirm that these were associated with the dam
mutation and not something else, advantage was taken of the inviability of dam recA
mutants to isolate true revertants. These had none of the phenotypes associated with the dam
strains. In addition to the true revertants there were also suppressed revertants which had mutations in the mutS
genes (see below). These did not have the phenotypes associated with dam
except for the mutator phenotype which was much stronger [14
]. The interesting result was that suppressor mutations of the dam
mutator phenotype were in mutator genes which had a stronger mutator phenotype.
In 1974 the SOS hypothesis had not yet been formulated, and it was a few years later that we showed the dam mutants to be sub-induced for the SOS response. Of all the SOS genes only expression of the recA, ruvA and ruvB genes was necessary for dam cell survival. It was also shown subsequently that double-strand breaks were present in the DNA of dam bacteria. The evidence made it clear that mismatch repair is responsible for the formation of DNA breaks, that DNA ligase is required for repair of single-strand interruptions, and that homologous recombination is essential for double-strand break repair. What is still not known is how the double-strand breaks are formed, and shows two possibilities. First, a replication fork encountering a gap or nick in duplex DNA will collapse () but can be repaired by homologous recombination. It is not known what fraction of collapsed forks is mended. Second, the presence of a nick on each strand at a GATC sequence is equivalent to a double-strand break () which requires a sister chromosome as a template for recombinational repair.
Fig. 1 Formation of double-strand breaks in dam bacteria. (A) The encounter of a replication fork with a strand discontinuity due to mismatch repair (MMR) results in fork collapse and the formation of a double-stranded end. This end is a substrate for the RecBCD (more ...)