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DNA from lambdagt-lambdaB bacteriophage was cleaved with EcoRI endonuclease and fragments from EcoRI-digested E. coli DNA were inserted. This DNA was used to infect E. coli, and phages containing the gene for DNA ligase were isolated by genetic selection. Two different hybrids were found with the same E. coli segment inserted in opposite orientations. Both hybrids produced similar levels of ligase as measured in crude extracts of infected cells.

Strand breaks accumulated in the DNA of a temperature-sensitive DNA ligase mutant of Escherichia coli growing at the restrictive temperature, as detected by zone sedimentation through alkaline sucrose density gradients. The rate of strand breakage was increased by concomitant thymine starvation. Rifampicin and chloramphenicol inhibited the accumulation of strand breaks in the DNA. There was a correlation between the accumulation of strand breaks in the DNA and lethality, suggesting that such breaks are the basis for lethality at the restrictive temperature.

The recA1 mutation was transduced into the tag-2 mutant of E. coli, thus making a strain deficient in the induction of SOS repair as well as in the constitutive repair of 3-alkylated adenines in DNA. The double mutant recA tag is more sensitive to methyl methanesulfonate exposure than either single mutant, indicating that recA and tag mutations block different pathways in repair of alkylation damage. The double mutant is more deficient in host cell reactivation of alkylated phages than the tag single mutant. However, alkylation induction of the double mutant with N-methyl-N'-nitro-N-nitrosoguanidine resulted in killing adaptation of the cells to methyl methanesulfonate and restored the host cell reactivation capacity for alkylated lambda phage to wild-type levels. These adaptive responses can be ascribed to the induction of 3-methyladenine DNA glycosylase II which is shown by enzyme analysis to proceed normally in the recA mutant background. The results imply that the induction of the alkA gene encoding 3-methyladenine DNA glycosylase II is independent of SOS induction.

The ada gene of Escherichia coli K12, the regulatory gene for the adaptive response of bacteria to alkylating agents, was cloned in multicopy plasmids. O6-Methylguanine-DNA methyltransferase and 3-methyladenine-DNA glycosylase II, which are known to be inducible as part of the adaptive response, were produced in ada- cells bearing ada+ plasmids, even without treatment with alkylating agents. When such cells had been treated with methyl methanesulfonate, even higher levels of the enzyme activities were produced. Maxicell experiments revealed that the ada gene codes for a polypeptide with a molecular weight of 38 000. We constructed a hybrid plasmid carrying an ada'-lacZ' fused gene, with the proper control region for ada expression. beta-Galactosidase synthesis from the fused gene was strongly induced only when cells were treated with low doses of methylating agents, but was weakly induced with relatively high doses of ethylating agents. The induction was autogenously regulated by the ada gene product, in a positive manner.

When bacteriophage lambda DNA replication is blocked by mutation in phage genes O or P, the efficiency of lysogenization drops to a very low value unless high multiplicities of infecting phage are used. Our results show that even at high multiplicity, lambda O or P mutants cannot efficiently lysogenize some hosts that are defective in either DNA polymerase I or DNA ligase. Covalent closure of infecting DNA molecules, a preliminary step for insertion according to Campbell's model and an obvious candidate for this lysogenization defect, appears to occur normally under our conditions. In addition, prophage excision as measured by the frequency of curing O- and P- lysogens seemed normal when tested in the poll- strain. These results suggest that the Escherichia coli enzymes DNA polymerase I and ligase, and phage proteins O and P, are able to provide some complementary activity whose function is required specifically for prophage integration.

The biosynthesis of early enzymes of DNA-ligase, RNA-ligase, DNA-polymerase, polynucleotide kinase, exonuclease A induced by bacteriophage T4amN82 was studied. The maximal activity of DNA-ligase was observed at the 60th min after the infection, while that of the other enzymes was revealed at the 90th min and reached 4, 45, 529, 120 and 78 units per mg of protein for DNA-ligase, RNA-ligase, polynucleotide kinase, DNA-polymerase and exonuclease A, respectively. Bacteriophage T4amN82 induced the maximal biosynthesis of the tested enzymes, when E. coli B-23 cells were grown in medium I containing trypton bacto ("Merck", West Germany) and a yeast extract ("Difco", USA). Similar events were observed when E. coli B-23 cells infected with phage T4amN82 were grown in a medium (II) with casein hydrolysate and yeast extract. Ultrasonication used for the disruption of E. coli B-23 cells infected with bacteriophage T4 had no effect on the enzyme activities.

Genetic analyses of an Escherichia coli K-12 mutant possessing the amber mutation lig-321 were carried out. This mutant is defective in deoxyribonucleic acid (DNA) ligase and conditionally lethal. We constructed strains harboring an F'lig+ or F'lig-321 plasmid. Genetic complementation analyses were done by using these plasmids and by constructing a lig-4/F'lig-321 merodiploid. It was shown that lig-321 does not complement lig-4, unless the former is suppressed by an amber suppressor. The same was found to be the case between lig-321 and lig-ts7. Transductional mapping of lig-321, by a four-factor cross, revealed that lig-321 is very closely linked to lig-4. The frequency of recombinants between the two alleles was not unreasonable for assuming that they arose by intragenic recombination. The lig-4 and lig-ts7 alleles are known to reside in the structural gene for DNA ligase, in which lig-321 may also be located.

Bacteriophage T7 bearing amber mutations in both gene 1.3 (T7 DNA ligase) and gene 3 (T7 endonuclease I) are viable when grown in suppressor-negative, ligase-negative hosts. This is evidenced by a high plating efficiency and a large burst size compared to the single mutants. These findings may be explained by a limited destruction of cellular DNA by the double mutant.