DNA Polymerase Mutants Research Articles

The behavior of temperature-sensitive T4 DNA polymerase mutants in temperature shift experiments

Temperature-sensitive mutants of gene 43 of bacteriophage T4 produce either temperature-stable or temperature-labile DNA polymerases. Molecules produced under restrictive conditions can be activated in the presence of chloramphenicol by exposure to a permissive temperature to yield proteins which behave similarly at low temperature to those enzymes actually synthesized at the low temperature.

Increased frequency of deletions in DNA polymerase mutants of Escherichia coli.

Mutants of E. coli, deficient in DNA polymerase activity in vitro, spontaneously generate chromosomal deletions at a high frequency. Such deletions may arise from the inability of these strains to repair single-strand breaks in DNA efficiently.

A possible function of DNA polymerase in chromosome replication

The Escherichia coli DNA polymerase mutant isolated by de Lucia and Cairns is impaired in its ability to convert low molecular weight, newly synthesized DNA into high molecular weight material. The mutation affects the joining of the low molecular weight strands and not their synthesis, but the strands are eventually incorporated into high molecular weight DNA. The in vitro DNA ligase activity of the mutant is normal. It is proposed both DNA polymerase and DNA ligase are required to close the breaks in newly synthesized DNA in vivo , and either the low levels of polymerase activity or interference with DNA ligase by an altered DNA polymerase reduce the rate of closing in the mutant.

Enzymic joining of polynucleotides: VIII. Structure of hybrids of parental T4 DNA molecules

The structures of the “joint” (hydrogen-bonded) and “recombinant” (covalently-linked) hybrids of parental phage DNA's formed after infection of Escherichia coli strain BB with T4 amber mutants defective in the DNA polymerase or in the DNA polymerase and polynucleotide ligase genes were examined. The mixture of joint and recombinant DNA isolated after infection with DNA polymerase mutants consisted of molecules whose approximate size was one-fourth that of the parental T4 DNA and contained an average of two single-strand interruptions per molecule. The joint molecules isolated after infection with mutants defective in both the DNA polymerase and ligase were also one-fourth the size of the parental DNA; however, these contained an average of six single-strand interruptions per molecule. In both instances the interruptions were found to be gaps with mean lengths of 300 to 400 nucleotide residues.

Spontaneous Mutation: Genetic Control of Mutation Rates in Bacteriophage T4

The following two articles are concerned with the genetic control of mutation rate. The first describes the anti-mutagenic effect of some DNA polymerase mutations on phage T4 mutation rates, and discusses the implications they hold for the mechanism of action of DNA polymerase. The second compares spontaneous mutation rates in different species, and considers the biological factors which may determine these rates.

Problems in protein biosynthesis.

Outline of the steps in protein synthesis. Nature of the genetic code. The use of synthetic oligo- and polynucleotides in deciphering the code. Structure of the code: relatedness of synonym codons. The wobble hypothesis. Chain initiation and N-formyl-methionine. Chain termination and nonsense codons. Mistakes in translation: ambiguity in vitro. Suppressor mutations resulting in ambiguity. Limitations in the universality of the code. Attempts to determine the particular codons used by a species. Mechanisms of suppression, caused by (a) abnormal aminoacyl-tRNA, (b) ribosomal malfunction. Effect of streptomycin. The problem of "reading" a nucleic acid template. Different ribosomal mutants and DNA polymerase mutants might cause different mistakes. The possibility of involvement of allosteric proteins in template reading.

Mutagenic DNA polymerase

An experiment will be described which shows that DNA polymerase helps select the base in DNA replication. After elucidating the structure of DNA (1953a), Watson and Crick proposed a template mechanism for the replication of DNA (1953b). This hypothesis invokes the same hydrogen bonds which hold the finished DNA strands together as the agents for the selection of precursor nucleotides to complement those of the parental DNA strand. The enzyme for this polymerization process was subsequently discovered, Kornberg (1960), and was not previously known to play a selective role. Nor was it implicated in mutagenesis. In another selective polymerization directed by a nucleic acid, protein synthesis, the ribosomes-which may be considered as enzymes-affect the specificity of the process, Gorini and Kataja (1964). This suggested that the enzymes involved in nucleic acid synthesis might also affect the specificity. Since enzymes may change their substrate specificity when their gene is mutated, Hotchkiss and Evans (1960), a mutant DNA polymerase might therefore cause errors in DNA synthesis. These errors can be detected as an increase in the frequency of mutations. When a mutant DNA polymerase was discovered a direct test became possible. This discovery was made by Epstein and Edgar and their colleagues (1963) who mapped the genes of coliphage T4. They have found about 70 genes, of which several are involved in DNA replication. One of these, gene 43, (Edgar et al , 1964) has been identified as a structural gene of DNA polymerase by de Waard et al (1965) . Dr. Edgar generously gave us thirteen temperature sensitive mutants of this gene. We have studied the effect of two ts alleles of this gene on mutagenesis.