Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes. XII. Evidence for enhanced photolysis enzyme-substrate complexes by a 2-photon reaction.

Yeast photoreactivating enzyme (PRE), preilluminated with wavelengths ranging from the near-UV to the red spectral region, forms with 254 nm-irradiated transforming DNA of Haemophilus influenzae enzyme-substrate complexes that are more efficiently photorepaired than complexes formed from non-preilluminated PRE. The action spectrum for this "preillumination effect", previously shown to have a maximum in the near-UV region, has another maximum near 577 nm. In complexes formed from non-preilluminated PRE the repair probability per incident photon is only about 25% of that in complexes formed from preilluminated PRE, if low-intensity photoreactivating light is applied continuously or as a sequence of flashes. However, photoreactivating light in the form of a single, high-intensity flash of 1 msec duration raises the repair probability to greater than 50%. Two light flashes, discharged with a delay of slightly more than a millisecond, may already achieve less photorepair than the same energy given as a single flash. These results are explained by the assumption that the great majority of PRE molecules in a non-preilluminated preparation have reduced activity (of the order of 1/4 of maximal activity). These less reactive molecules form enzyme-substrate complexes ("non-activated complexes") in which the repair probability per incident photon is considerably increased if 2 or more photons are absorbed within a time period of the order of milliseconds. This phenomenon, tentatively termed "2-photon photolysis" does not occur in "activated complexes" (i.e. those formed form preilluminated enzyme). The data are compatible with suggestion that the first absorption leads to a metastable excited state of the complex, during which the repair probability is increased by absorption of another photon. The generally observed heterogeneity of the photolytic response of enzyme-substrate complexes can be partly explained by heterogeneity of PRE molecules regarding their activity. In particular, uncontrolled exposure of enzyme to almost any kind of room light before its experimental use can enhance the heterogeneity.     

Analysis of photoernzymatic repair of UV lesions in DNA by single light flashes. XI. Light-induced activation of the yeast photoreactivating enzyme

Photoreactivating enzyme (PRE) from yeast (as semi-crude extract, or in highly purified form) shows increased activity if its is illuminated with near UV or short wavelength visible light prior to its use for photoenzymatic repair of UV-induced pyrimidine dimers in transforming DNA in vitro. This effect results from an alternation in PRE molecules changing those with low activity in the light-dependent step of the reaction to a higher activity. Light-induced activation of PRE preparations is slowly lost by dark storage for several hours to 1 day (faster at 23°C than at 5°C), but can be recovered repeatedly by renewed preillumination. The action spectrum for these preillumination effects generally resembles that for the photoenzymatic repair reaction itself, having its maximum in the same 355–385 nm region as the latter, but light of somewhat longer wavelengths (546 nm) is still effective. Preilluminated PRE is also more stable to thermal inactivation (65°C) than untreated enzyme.     

In vivo studies of repair of 2-aminopurine in Escherichia coli.

The repair of the base analog 2-aminopurine has been studied in vivo by using a temperature-sensitive mutant of the cloned mutH gene of Escherichia coli. Our results suggest that the lethal event in killing of dam mutants by 2-aminopurine does not result simply from incorporation of 2-aminopurine into the DNA and its subsequent repair. Furthermore, a 10-fold increase in the level of 2-aminopurine incorporated into the DNA of a dam mutH double mutant has little effect on the mutation frequency of this strain. An alternative mechanism for the mutagenicity of 2-aminopurine in E. coli is proposed.     

Stimulation of DNA repair and increased light output in response to UV irradiation in Escherichia coli expressing lux genes.

It has previously been suggested that the evolutionary drive of bacterial bioluminescence is a mechanism of DNA repair. By assessing the UV sensitivity of Escherichia coli, it is shown that the survival of UV-irradiated E. coli constitutively expressing luxABCDE in the dark is significantly better than either a strain with no lux gene expression or the same strain expressing only luciferase (luxAB) genes. This shows that UV resistance is dependent on light output, and not merely on luciferase production. Also, bacterial survival was found to be dependent on the conditions following UV irradiation, as bioluminescence-mediated repair was not as efficient as repair in visible light. Moreover, photon emission revealed a dose-dependent increase in light output per cell after UV exposure, suggesting that increased lux gene expression correlates with UV-induced DNA damage. This phenomenon has been previously documented in organisms where the lux genes are under their natural luxR regulation but has not previously been demonstrated under the regulation of a constitutive promoter.     

Behavior of bacteriophage Mu DNA upon infecton of Escherichia coli cells.

The question whether the ends of bacteriophage Mu DNA are fused to form a ring in host cells is critical to the understanding of the mechanism of integrative recombination between Mu DNA and host DNA. We have examined the fate of ³²P-labeled Mu DNA, after infection of sensitive and immune (lysogenic) cells, by sedimentation in sucrose gradients, ethidium bromide/CsCl density centrifugation and by electrophoresis of parental Mu DNA and its fragments in agarose gels. We find that the parental Mu DNA cannot be detected as covalently closed circles at any stage during the Mu life cycle. An interesting form of Mu DNA can be seen after superinfection of immune cells. This form sediments about twice as fast as the mature phage DNA marker in neutral sucrose gradients but yields linear molecules upon phenol extraction. Upon infection of sensitive cells, most of the parental DNA associates with a large complex, presumably containing the host chromosome. When Mu-sensitive cells are infected with unlabeled Mu particles and Mu DNA examined at different times after infection by fractionation in 0.3% agarose gels and hybridization with ³²P-labeled Mu DNA, Mu sequences are found to appear with the bulk host DNA as the phage lytic cycle progresses. However, no distinct replicative or integrative intermediate of Mu, that behaves differently from linear Mu DNA and is separate from the host DNA, can be detected.     

Escherichia coli Fpg protein and UvrABC endonuclease repair DNA damage induced by methylene blue plus visible light in vivo and in vitro.

pBR322 plasmid DNA was treated with methylene blue plus visible light (MB-light) and tested for transformation efficiency in Escherichia coli mutants defective in either formamidopyrimidine-DNA glycosylase (Fpg protein) and/or UvrABC endonuclease. The survival of pBR322 DNA treated with MB-light was not significantly reduced when transformed into either fpg-1 or uvrA single mutants compared with that in the wild-type strain. In contrast, the survival of MB-light-treated pBR322 DNA was greatly reduced in the fpg-1 uvrA double mutant. The synergistic effect of these two mutations was not observed in transformation experiments using pBR322 DNA treated with methyl methanesulfonate, UV light at 254 nm, or ionizing radiation. In vitro experiments showed that MB-light-treated pBR322 DNA is a substrate for the Fpg protein and UvrABC endonuclease. The number of sites sensitive to cleavage by either Fpg protein or UvrABC endonuclease was 10-fold greater than the number of apurinic-apyrimidinic sites indicated as Nfo protein (endonuclease IR)-sensitive sites. Seven Fpg protein-sensitive sites per PBR322 molecule were required to produce a lethal hit when transformed into the uvrA fpg-1 mutant. These results suggest that MB-light induces DNA base modifications which are lethal and that these modifications are repaired by Fpg protein and UvrABC endonuclease in vivo and in vitro. Therefore, one of the physiological functions of Fpg protein might be to repair DNA base damage induced by photosensitizers and light.     

Replication of bacteriophage M13 DNA in plasmolysed Escherichia coli cells

Plasmolysed M13 infected E. coli cells utilize deoxynucleoside triphosphates to synthesize phage-specific DNA in an ATP-dependent, nalidixic acid sensitive, semi-conservative replication process. Whereas the major fraction of the reaction product consists of replicative form I molecules (RF) labeled asymmetrically in the viral strand, a minor fraction of the label is found in mature viral single strands. We therefore conclude that the system is capable of initiating second rounds of replication, for which ring closure seems to be a precondition.