Formation of a thymine photoproduct in transforming DNA by near ultraviolet irradiation.

Irradiation at 334 and 365 nm of a highly purified preparation of thymine-labeled transforming DNA from Haemophilus influenzae produced a photo product containing label from thymine but different from the cyclobutane dimer. The photoproduct is soluble in water and in ethanol and Rf values in a number of solvents are presented. The photoproduct has properties similar in a number of respects to those of the spore photoproduct, 5-thyminyl-5,6-dihydrothymine. The near ultraviolet photoproduct is more likely to affect the oxygen independent inactivation of transforming DNA rather than its mutagenesis, as judged by the quantitative relationship between amount of photboproduct and inactivation and mutagenesis.     

Inactivation of transforming DNA by ultraviolet light. I. Near-UV action spectrum for marker inactivation

The storage effect is defined as an increase of mutational damage after cessation of treatment. It differs from other kinds of delayed effect (replication errors, replicating instabilities) in not requiring replication of DNA. In Neurospora ad3A 38701 inos 37401 diepoxybutane (DEB) yields a storage effect for adenine reversions, but none for inositol reversions. The storage effect takes place in treated washed spores that are sedimented in a centrifuge tube, but not in spores that are agitated in water. Under the latter conditions, response to storage is gradually lost. The storage effect can be imitated by administering very small amounts of DEB to cells that had been previously treated with a moderately high dose (booster effect). During post-treatment shaking in water, responses to booster and to storage conditions disappear together; response to booster disappears at the same rate in spores that are sedimented in centrifuge tubes. No mutagenic action could be detected in eluates from heavily treated cells. We have concluded that treatment with DEB sensitizes the conidia to further small doses of DEB whether these are administered extraneously as booster or present intracellularly during storage. Sensitization is lost in the course of a few hours in shaken as well as in sedimented spores. Thus, while the storage effect is due to traces of mutagen, its gradual disappearance after treatment is not due to loss of these traces.Correlated with the ability to yield a storage effect, and probably part of the storage effect, is the response to temperature between treatment and plating. Conidia that can give a storage effect yield fewer mutations when spread on cold agar than when inplated into warm agar or heat-shocked before spreading; the excess of mutation under the latter two conditions forms part of the final storage effect. The true base line for calculation of the storage effect is therefore mutation frequency among spread spores.For DEB as mutagen, response to storage by the adenine locus and lack of response by the inositol locus are correlated with the responses of these two loci to dose of DEB and to combination treatment of DEB with UV, or DEB with nitrous acid (NA). This makes it possible to fit all observations into the picture of a general hypothesis on the cellular effects of DEB. Because of the differential response of the two loci, storage and plating procedures offer two additional means for manipulating specificity in this system.     

Altered dynamics of DNA bases adjacent to a mismatch: a cue for mismatch recognition by MutS

The structural deviations as well as the alteration in the dynamics of DNA at mismatch sites are considered to have a crucial role in mismatch recognition followed by its repair utilizing mismatch repair family proteins. To compare the dynamics at a mismatch and a non-mismatch site, we incorporated 2-aminopurine, a fluorescent analogue of adenine next to a G.T mismatch, a C.C mismatch, or an unpaired T, and at several other non-mismatch positions. Rotational diffusion of 2-aminopurine at these locations, monitored by time-resolved fluorescence anisotropy, showed distinct differences in the dynamics. This alteration in the motional dynamics is largely confined to the normally matched base-pairs that are immediately adjacent to a mismatch/ unpaired base and could be used by MutS as a cue for mismatch-specific recognition. Interestingly, the enhanced dynamics associated with base-pairs adjacent to a mismatch are significantly restricted upon MutS binding, perhaps “resetting” the cues for downstream events that follow MutS binding. Recognition of such details of motional dynamics of DNA for the first time in the current study enabled us to propose a model that integrates the details of mismatch recognition by MutS as revealed by the high-resolution crystal structure with that of observed base dynamics, and unveils a minimal composite read-out involving the base mismatch and its adjacent normal base-pairs.     

Similar ultraviolet action spectra for the protection by glycerol of transforming DNA against single-strand breaks and inactivation of biological activity

An action spectrum for the protection of purified DNA by glycerol against the induction of single-strand breaks in the DNA by ultraviolet (uv) light is described. Protection was not observed below 300 nm, was maximal between 334 and 365 nm, and decreased at 405 nm. This spectrum closely matched the spectrum for the protection by glycerol against the inactivation of biological transforming activity by near uv, described previously. Also, deviations from the reciprocity rule are similar for inactivation of transforming activity and for induction of DNA breaks by 365-nm radiation. That is, the deviations for the two end points are quantitatively the same, such that high fluence rates are less effective than low fluence rates.     

DNA mismatch repair mediates protection from mutagenesis induced by short-wave ultraviolet light

To investigate involvement of DNA mismatch repair in the response to short-wave ultraviolet (UVC) light, we compared UVC-induced mutant frequencies and mutational spectra at the Hprt gene between wild type and mismatch-repair-deficient mouse embryonic stem (ES) cells. Whereas mismatch repair gene status did not significantly affect survival of these cells after UVC irradiation, UVC induced substantially more mutations in ES cells that lack the MutSalpha mismatch-recognizing heterodimer than in wild type ES cells. The global UVC-induced mutational spectra at Hprt and the distribution of most spectral mutational hotspots were found to be similar in mismatch-repair-deficient and wild type cells. However, at one predominant spectral hot spot for mutagenesis in wild type cells, the UVC-induced mutation frequency was not affected by the mismatch repair status. Together these data reveal a major role of mismatch repair in controlling mutagenesis induced by UVC light and may suggest the sequence context-dependent direct mismatch repair of misincorporations opposite UVC-induced pyrimidine dimers.     

Acridone-tagged DNA as a new probe for DNA detection by fluorescence resonance energy transfer and for mismatch DNA recognition

Acridone is highly fluorescent and stable against photodegradation, oxidation, and heat. It is also a small molecule with no charge, making it a promising fluorescent agent for use in a DNA probe. Thus, we have prepared 5'-terminal acridone-labeled DNAs by post-modification, and have examined their photophysical properties and their use as donors for a fluorescence resonance energy transfer (FRET) system in combination with a 3'-terminal dabcyl-tagged DNA as an acceptor, which can detect the target DNA by emission-quenching caused by FRET. The FRET with an acridone and dabcyl pair has been found to complement that with fluorescence and dabcyl and other fluorescence-quencher pairs. Significant amounts of quenching of the acridone emissions by guanine in the DNA were observed when guanine was close to acridone, which can be applied as a quencher-free probe for the detection of special sequence of DNA. The DNA bearing acridone at the C5 position of inner thymidine could discriminate the opposite T-T base mismatch, although enhancement of discrimination ability is needed for the practical use of SNP typing.     

Specific inactivation of heterospecific transforming DNA by a factor derived from Streptococcus sanguis lysates.

Summary A heat-sensitive factor obtained from lysates of competent Streptococcus sanguis cells reacts specifically with native DNA of heterospecific (S. pneumoniae or calf thymus) origin. In vitro it does not alter the double or single strand length of the DNA, nor does it affect uptake of the DNA by competent S. sanguis or S. pneumoniae cells in DNase I-resistant form. Following uptake, however, DNA previously exposed to the factor loses over 90% of its biological activity. Reaction of heterospecific DNA with the factor is cometitive, suggesting a competition for binding to the factor. Heating treated DNA prior to its reaction with recipient cells, apparently by irreversibly dissociating the factor, restores to the DNA its original potential transforming activity. Specific activity of the factor can be increased in cells grown under certain conditions; this increase is blocked by erythromycin.     

Sensitivity of carcinogen-treated DNA to inactivation by ultraviolet radiation

The DNA of bacteriophage SPO2c12 was treated with methylmethane sulfonate (MMS), beta-propiolactone (BPL), 2-anthramine (AA) or benzo[a]pyrene (BP) and then exposed to 254-nm radiation. Competent Bacillus subtilis host cells were transfected with DNA subjected to the carcinogen-UV treatment or with DNA treated with carcinogen only. Survival curves were obtained for loss of plaque-forming ability as a function of UV dose. The UV sensitivity of DNA treated with MMS, BPL or AA was not significantly different from that of untreated DNA. The results indicate that in competent B. subtilis the pathways for repair of alkylating agent damage and for repair of UV damage are probably different.     

DNA repair: models for damage and mismatch recognition

Maintaining the integrity of the genome is critical for the survival of any organism. To achieve this, many families of enzymatic repair systems which recognize and repair DNA damage have evolved. Perhaps most intriguing about the workings of these repair systems is the actual damage recognition process. What are the chemical characteristics which are common to sites of nucleic acid damage that DNA repair proteins may exploit in targeting sites? Importantly, thermodynamic and kinetic principles, as much as structural factors, make damage sites distinct from the native DNA bases, and indeed, in many cases, these are the features which are believed to be exploited by repair enzymes. Current proposals for damage recognition may not fulfill all of the demands required of enzymatic repair systems given the sheer size of many genomes, and the efficiency with which the genome is screened for damage. Here we discuss current models for how DNA damage recognition may occur and the chemical characteristics, shared by damaged DNA sites, of which repair proteins may take advantage. These include recognition based upon the thermodynamic and kinetic instabilities associated with aberrant sites. Additionally, we describe how small changes in base pair structure can alter also the unique electronic properties of the DNA base pair pi-stack. Further, we describe photophysical, electrochemical, and biochemical experiments in which mismatches and other local perturbations in structure are detected using DNA-mediated charge transport. Finally, we speculate as to how this DNA electron transfer chemistry might be exploited by repair enzymes in order to scan the genome for sites of damage.     

Inactivation of transforming DNA by ultraviolet light. II. Protection by histidine against near-UV irradiation: action spectrum

Histadine, at concentrations greater than 0.075 M, quenches the inhibition of the transforming activity of DNA by near UV. This effect does not occur at wavelengths below about 300 nm and is maximal at about 380 nm. Since histidine at this concentration has no effect upon the inactivation by far UV, this observation shows that inactivation by near UV is clearly different, and suggests an indirect photosensitization mechanism. The histidine protection spectrum coincides closely with the near-UV DNA inactivation spectrum derived from the shoulder in the near UV.     

Structures of mismatched base pairs in DNA and their recognition by the Escherichia coli mismatch repair system.

The Escherichia coli mismatch repair system does not recognize and/or repair all mismatched base pairs with equal efficiency: whereas transition mismatches (G X T and A X C) are well repaired, the repair of some transversion mismatches (e.g. A X G or C X T) appears to depend on their position in heteroduplex DNA of phage lambda. Undecamers were synthesized and annealed to form heteroduplexes with a single base-pair mismatch in the centre and with the five base pairs flanking each side corresponding to either repaired or unrepaired heteroduplexes of lambda DNA. Nuclear magnetic resonance (n.m.r.) studies show that a G X A mismatch gives rise to an equilibrium between fully helical and a looped-out structure. In the unrepaired G X A mismatch duplex the latter predominates, while the helical structure is predominant in the case of repaired G X A and G X T mismatches. It appears that the E. coli mismatch repair enzymes recognize and repair intrahelical mismatched bases, but not the extrahelical bases in the looped-out structures.     

8-oxoguanine incorporation into DNA repeats in vitro and mismatch recognition by MutSalpha

DNA 8-oxoguanine (8-oxoG) causes transversions and is also implicated in frameshifts. We previously identified the dNTP pool as a likely source of mutagenic DNA 8-oxoG and demonstrated that DNA mismatch repair prevented oxidation-related frameshifts in mononucleotide repeats. Here, we show that both Klenow fragment and DNA polymerase α can utilize 8-oxodGTP and incorporate the oxidized purine into model frameshift targets. Both polymerases incorporated 8-oxodGMP opposite C and A in repetitive DNA sequences and efficiently extended a terminal 8-oxoG. The human MutSα mismatch repair factor recognized DNA 8-oxoG efficiently in some contexts that resembled frameshift intermediates in the same C or A repeats. DNA 8-oxoG in other slipped/mispaired structures in the same repeats adopted configurations that prevented recognition by MutSα and by the OGG1 DNA glycosylase thereby rendering it invisible to DNA repair. These findings are consistent with a contribution of oxidative DNA damage to frameshifts. They also suggest how mismatch repair might reduce the burden of DNA 8-oxoG and prevent frameshift formation.     

2-aminopurine allows interspecies recombination by a reversible inactivation of the Escherichia coli mismatch repair system

2-Aminopurine treatment of Escherichia coli induces a reversible phenotype of DNA mismatch repair deficiency. This transient phenotype results in a 300-fold increase in the frequency of interspecies conjugational recombination with a Salmonella enterica serovar Typhimurium Hfr donor. This method can be used for the generation of biodiversity by allowing recombination between diverged genes and genomes.