MMS1 protects against replication-dependent DNA damage in Saccharomyces cerevisiae

A series of yeast mutants were isolated that are sensitive to killing by the monofunctional DNA-alkylating agent methyl methanesulfonate (MMS) but not by UV or X-radiation. We have cloned and characterized one of the corresponding genes, MMS1, and show that the mms1 Delta mutant is dramatically sensitive to killing by MMS and mildly sensitive to UV radiation. mms1 Delta mutants display an elevated level of spontaneous DNA damage and genomic instability. Furthermore, the mms1 Delta cells are sensitive to killing by conditions that induce replication-dependent double-strand breaks, such as treatment with camptothecin, and incubation of a cdc2-2 strain at the restrictive temperature. rad52 Delta is epistatic to mms1 Delta for MMS and camptothecin sensitivity, indicating that Mms1 acts in concert with Rad52. However, unlike mutants of the RAD52 group, mms1 Delta cells are not sensitive to gamma-rays, which induce double-strand breaks independently of DNA replication. Together these results suggest a role for an Mms1-dependent, Rad52-mediated, pathway in protecting cells against replication-dependent DNA damage.     

den V gene of bacteriophage T4 codes for both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities.

Recent studies have shown purified preparations of phage T4 UV DNA-incising activity (T4 UV endonuclease or endonuclease V of phage T4) contain a pyrimidine dimer-DNA glycosylase activity that catalyzes hydrolysis of the 5' glycosyl bond of dimerized pyrimidines in UV-irradiated DNA. Such enzyme preparations have also been shown to catalyze the hydrolysis of phosphodiester bonds in UV-irradiated DNA at a neutral pH, presumably reflecting the action of an apurinic/apyrimidinic endonuclease at the apyrimidinic sites created by the pyrimidine dimer-DNA glycosylase. In this study we found that preparations of T4 UV DNA-incising activity contained apurinic/apyrimidinic endonuclease activity that nicked depurinated form I simian virus 40 DNA. Apurinic/apyrimidinic endonuclease activity was also found in extracts of Escherichia coli infected with T4 denV+ phage. Extracts of cells infected with T4 denV mutants contained significantly lower levels of apurinic/apyrimidinic endonuclease activity; these levels were no greater than the levels present in extracts of uninfected cells. Furthermore, the addition of DNA containing apurinic or apyrimidinic sites to reactions containing UV-irradiated DNA and T4 enzyme resulted in competition for pyrimidine dimer-DNA glycosylase activity against the UV-irradiated DNA. On the basis of these results, we concluded that apurinic/apyrimidinic endonuclease activity is encoded by the denV gene of phage T4, the same gene that codes for pyrimidine dimer-DNA glycosylase activity.     

Construction and study of a new type of phage lambda DNA vector molecule]

A group of lambda mutants (mutants lambda 0) harbouring lesser number of EcoRI restriction sites on DNA molecules was selected. lambda3-1 recombinant (genotype lambdab221amgamma210Sr1lambda3+c-Px) was created by crosses of lambda02 phage with other lambda mutants. This phage DNA may be used as a vector molecule which makes it possible to select easily phages harbouring insertions of EcoRI DNA fragments. The maximal size of DNA fragment, the insertion of which would not decrease lambda3-1 viability, is 7.7 megadaltone. Lambda3-1 DNA has three regions heterological to lambda DNA, two of which probably include sites SRIlambda4 and SRIlambda5 and some juxtaposed genes. For example, Ptgene of lambda phage in juxtaposition with site SRIlambda4 is substituted by Px gene on the lambda3-1 DNA molecule.     

Relationships between yeast Rad27 and Apn1 in response to apurinic/apyrimidinic (AP) sites in DNA.

Yeast Rad27 is a 5'-->3' exonuclease and a flap endo-nuclease. Apn1 is the major apurinic/apyrimidinic (AP) endonuclease in yeast. The rad27 deletion mutants are highly sensitive to methylmethane sulfonate (MMS). By examining the role of Rad27 in different modes of DNA excision repair, we wish to understand why the cytotoxic effect of MMS is dramatically enhanced in the absence of Rad27. Base excision repair (BER) of uracil-containing DNA was deficient in rad27 mutant extracts in that (i) the Apn1 activity was reduced, and (ii) after DNA incision by Apn1, hydrolysis of 1-5 nucleotides 3' to the baseless sugar phosphate was deficient. Thus, some AP sites may lead to unprocessed DNA strand breaks in rad27 mutant cells. The severe MMS sensitivity of rad27 mutants is not caused by a reduction of the Apn1 activity. Surprisingly, we found that Apn1 endonuclease sensitizes rad27 mutant cells to MMS. Deleting the APN1 gene largely restored the resistance of rad27 mutants to MMS. These results suggest that unprocessed DNA strand breaks at AP sites are mainly responsible for the MMS sensitivity of rad27 mutants. In contrast, nucleotide excision repair and BER of oxidative damage were not affected in rad27 mutant extracts, indicating that Rad27 is specifically required for BER of AP sites in DNA.     

Phosphotransferase-mediated regulation of carbohydrate utilization in Escherichia coli K12: location of the gsr (tgs) and iex (crr) genes by specialized transduction

A lysogen of Escherichia coli K12 with lambda cI857 S7 xis6 nin5 b515 b519 integrated into ptsI was induced and the lysates plated on a Pel- host [on which lambda strains with less than the wild-type amount of DNA form plaques at low frequency (Cameron et al., 1977)]. All of the 40 plaques examined contained phage able to transduce at least two of the genes known from bacteriophage P1 transduction experiments to be closely linked to ptsI. Assuming that each specialized transducing phage arose by a single illegitimate recombination event, the distribution of phage types showed that the gene order is cysA gsr ptsI (ptsH, iex) cysZ lig; both gsr+ and iex+ were dominant. Analysis of restriction endonuclease digests of the transducing phage confirmed that no unexpected DNA rearrangements had taken place and allowed the construction of a map of the sites of action of the restriction endonucleases EcoRI, HindIII, BamI and Kpn for over 20 kilobases of E. coli DNA. In an Appendix, we show cysA and cysZ mutants to be deficient in sulphate assimilation.     

Behavior of a temperate bacteriophage in differentiating cells of Bacillus subtilis.

During the first 6 hr of sporulation, infection of Bacillus subtilis by by phi105 wild type or the clear-plaque mutant phi105 c30 was nonproductive, but phage DNA was trapped inside developing spores. After infection with either wild-type or mutant phage at early times of sporulation (T1-T3), phage DNA entered the developing spores in a heat-stable form, which may represent integration of the phage DNA into the host chromosome. Phage DNA in carrier spores produced by infection at later times (T4-T6) was much more heat sensitive. Spore preparations containing either phi105 wild type or phi105 c30 carrier spores gave rise to a spontaneous burst of phage during outgrowth, although the fraction of carried wild-type phage that chose lysis over lysogeny at germination has not been determined. Heat induction of the thermoinducible lysogen 3610 (phi105 cts23) was also abortive during sporulation. Furthermore, induction neither prevented eventual spore formation nor resulted in the conversion of prophage DNA to the carrier state; during outgrowth, the previously induced lysogenic spores remained stable lysogens. However, if the sporulating lysogenic cells were plated immediately after induction, they did not form colonies at high efficiency, as though transfer to fresh medium allowed sufficient phage expression to kill the host.     

Host- and Phage-Mediated Repair of Radiation Damage in Bacteriophage T4

T4+ exhibits increased ultraviolet sensitivity on derivatives of Escherichia coli K12 or B lacking deoxyribonucleic acid (DNA) polymerase I. However, the sensitivity of T4v is not affected by the absence of host DNA polymerase. T4x and T4y also show increased sensitivity on DNA polymerase-deficient strains, but to a lesser extent than observed with wild-type T4. When T4x or T4y, but not T4+, are plated on a double mutant lacking both DNA polymerase and the uvrA gene product, a partial suppression of the polymerase effect is observed. Host ligase appears to be able to suppress to some extent the T4y phenotype but has no effect on wild-type T4 or other T4 mutants. T4xv incubated in E. coli B or B(s-1) in the presence of chloramphenicol (50 mug/ml) shows increased resistance over directly plated irradiated phage. Increased survival under the same conditions was not observed with T4+ or other T4 mutants. The repair of X-ray-damaged T4 was investigated by examining survival curves of T4+, T4x, T4y, T4ts43, and T4ts30. The repair processes were further defined by observing the effects of plating irradiated phage on various hosts including strains lacking DNA polymerase I or polynucleotide ligase. Two classes of effects were observed. Firstly, the x and y gene products seem to be involved in a repair system utilizing host ligase. Secondly, in the absence of host DNA polymerase, phage sensitivity is increased in an unknown manner which is enhanced by the presence of host uvrA gene product.     

Luria effect in bacteriophages and the role of the products of recA+ and lexA+ genes in its induction

An analysis of UV-damages accumulation in the phages as revealed by delay of intracellular growth is represented using temperate lambda phage. The maximum of growth delay of phage lambda at given UV-dose was found with lambda red+, infecting Escherichia coli AB1886 uvrA strain. The growth delay was absent, when a strain RH-1 uvrA-recA- was infected with UV-irradiated phage lambda red3. A moderate growth delay was obtained with the phages lambda red+, infecting E. coli RH-1 uvrA-recA- or phage lambda red3, infecting E. coli AB1886 uvrA-. THe growth delay was also absent when wild type, recA- and uvrA mutants of E. coli were infected with phage lambda after 8-metnoxypsoralen + light (lambda > 310 nm) treatment. It is known that the crosslinks appear to be the DNA defects which give rise to the observed biological inactivation following psoralen + light treatment. However, a considerable growth delay of phage lambda, treated by 8-metnoxypsoralen + light, was only found under condition of crosslinks repair (W-reactivation and prophage-reactivation). The results obtained are best explained by the assumption that the growth delay reflects the time required for the postreplication repair (RecA, LexA, Red) of any lethal UV-lesion.     

Host-cell reactivation of alkylated T7 bacteriophage.

Purified T7 phage, treated with methyl methanesulfonate, was assayed on Escherichia coli K-12 host cells deficient in base excision repair. Phage survival, measured immediately after alkylation or following incubation to induce depurination, was lowest on a mutant defective in the polymerase activity of DNA polymerase I (p3478). Strains defective in endonuclease for apurinic sites (AB3027, BW2001) gave a significantly higher level of phage survival, as did the strain defective in the 5'--3' exonuclease activity of DNA polymerase I (RS5065). Highest survival of alkylated T7 phage was observed on the two wild-type strains (AB1157, W3110). These results show that alkylated T7 phage is subject to repair via the base excision repair pathway.     

Isolation and Characterization of Mms-Sensitive Mutants of SACCHAROMYCES CEREVISIAE

We have isolated mutants sensitive to methyl methanesulfonate (MMS) in Saccharomyces cerevisiae. Alleles of rad1, rad4, rad52, rad55 and rad57 were found amoung these mms mutants. Twenty-nine of the mms mutants which complement the existing radiation-sensitive (rad and rev) mutants belong to 22 new complementation groups. Mutants from five complementation groups are sensitive only to MMS. Mutants of 11 complementation groups are sensitive to UV or X rays in addition to MMS, mutants of six complementation groups are sensitive to all three agents. The cross-sensitivities of these mms mutants to UV and X rays are discussed in terms of their possible involvement in DNA repair. Sporulation is reduced or absent in homozygous diploids of mms mutants from nine complementation groups.     

Development of T7 phage and T7 phage containing apurinic sites in an exonuclease III, endonuclease IV double mutant of Escherichia coli.

The development of bacteriophage T7 was examined in an Escherichia coli double mutant defective for the two major apurinic, apyrimidinic endonucleases (exonuclease III and endonuclease IV, xth nfo). In cells infected with phages containing apurinic sites, the defect in repair enzymes led to a decrease of phage survival and a total absence of bacterial DNA degradation and of phage DNA synthesis. These results directly demonstrate the toxic action of apurinic sites on bacteriophage T7 at the intracellular level and its alleviation by DNA repair. In addition, untreated T7 phage unexpectedly displayed reduced plating efficiency and decreased DNA synthesis in the xth nfo double mutant.     

Role of AP endonuclease in DNA breakage and cell inactivation of Escherichia coli subjected to mild heat (52 degrees C)

The molecular mechanism of DNA injury by mild heat was investigated using matched isogenic mutants of E. coli. On heating at 52 degrees C for 1 h, the number of DNA single-strand breaks (SSBs) detected by the alkaline sucrose gradient sedimentation technique was consistently smaller in mutants NH5016 and BW2001, both deficient in the AP (apurinic/apyrimidinic) endonuclease of exonuclease III, as compared with their wild-type parent AB1157. The greater number of SSBs in the wild type was accompanied by more extensive cell death as compared with the AP-deficient mutants. Heating of endonuclease-free DNA systems, viz., T4 phage and T4 DNA, at 52 degrees C for up to 4 h did not result in any detectable SSB. Apparently, cellular injury by mild heat is self-inflicted through an AP-endonuclease-mediated process and hence depends on the cell's genetic complement of AP endonuclease. Mild heat is believed to activate the nucleolytic attack, and the resultant DNA-strand breaks, if not repaired, will eventually lead to cell death.     

Effects of x irradiation on a temperate bacteriophage of Haemophilus influenzae.

The inactivation of bacteriophage HP1c1 by X rays in a complex medium was found to be exponential, with a D0 (the X-ray exposure necessary to reduce the survival of the phage to 37%) of approximately 90 kR. Analysis of results of sucrose sedimentation of DNA from X-irradiated whole phage showed that the D0 for intactness of single strands was about 105kR, and for intactness of double strands, it was much higher. The D0 for attachment of X-irradiated phage to the host was roughly estimated as about 1,100 kR. Loss of DNA from the phage occurred and was probably due to lysis of the phage by X irradiation, but the significance of the damage is not clear. The production of single-strand breaks approaches the rate of survival loss after X irradiation. However, single-strand breaks produced by UV irradiation, in the presence of H2O2, equivalent to 215 kR of X rays, showed no lethal effect on the phage. Although UV-sensitive mutants of the host cell, Haemophilus influenzae, have been shown to reactivate UV-irradiated phage less than does the wild-type host cell, X-irradiated phage survive equally well on the mutants as on the wild type, a fact suggesting that other repair systems are involved in X-ray repair.     

Efficient breakage of DNA apurinic sites by the indoleamine related 9-amino-ellipticine

The aromatic amine, 9-NH2-ellipticine, is a synthetic DNA intercalating derivative of the antitumor agent ellipticine, which breaks circular DNA containing apurinic sites. This breakage is inhibited when the apurinic (AP) sites are reduced. The concentration of 9-NH2-ellipticine required to get a significant effect (0.1 microM) is the lowest known among chemicals which induce the same breakage reaction. Comparison with the action of structurally related amines shows that the amino-indole structure is specific for AP sites. The ability of ellipticine derivatives to induce breakage in DNA containing apurinic sites is related to the nucleophile substituent in position 9. Two ellipticine derivatives with known antitumor activity, BD 40 and 9-OH-ellipticine, were able to break purified DNA at apurinic sites.     

Effect of in vitro cleavage of apurinic/apyrimidinic sites on bleomycin-induced mutagenesis of repackaged lambda phage.

Previous studies have revealed bleomycin to be a potent base-substitution mutagen in repackaged phage lambda. In order to assess the role of apurinic/apyrimidinic (AP) sites in bleomycin-induced mutagenesis, bleomycin-damaged lambda DNA was treated with putrescine or endonuclease IV to effect cleavage of bleomycin-induced AP sites. The DNA was then packaged, the phage grown in SOS-induced E. coli, and the frequency of clear-plaque mutants in the progeny was determined. Bleomycin-induced mutagenesis was decreased approx. 2-fold by treating the DNA with putrescine, but was unaffected by endonuclease IV. The results are consistent with the production of bleomycin-induced mutation at certain AP sites having a closely opposed single-strand break, since such sites are cleaved by putrescine but not by endonuclease IV.     

Effect of in vitro cleavage of apurinic/apyrimidinic sites on bleomycin-induced mutagenesis of repackaged lambda phage

Previous studies have revealed bleomycin to be a potent base-substitution mutagen in repackaged phage lambda. In order to assess the role of apurinic/apyrimidinic (AP) sites in bleomycin-induced mutagenesis, bleomycin-damaged lambda DNA was treated with putrescine or endonuclease IV to effect cleavage of bleomycin-induced AP sites. The DNA was then packaged, the phage grown in SOS-induced E. coli, and the frequency of clear-plaque mutants in the progeny was determined. Bleomycin-induced mutagenesis was decreased approx. 2-fold by treating the DNA with putrescine, but was unaffected by endonuclease IV. The results are consistent with the production of bleomycin-induced mutation at certain AP sites having a closely opposed single-strand break, since such sites are cleaved by putrescine but not by endonuclease IV.     

A simple technique for the isolation of deletion mutants of phage lambda.

We describe a simple technique for isolating deletion mutants of phage lambda and use it to dissect a cloned fragment of foreign DNA. The technique is based on our previous finding that the normally essential product of lambda head gene D is dispensible for phage growth if the DNA content of the phage is less than 82% that of lambda wild-type (Sternberg and Weisberg, 1977). A significant fraction of the few phage that form plaques when a D amber mutant is plated on a nonsuppressing host contains deletions that reduce the phage chromosome size to less than 82% that of wild-type. It is possible to isolate deletions ranging in size from less than 1.5 kb to 14 kb (3 to 27% of wild-type lambda), and the size range can be restricted by an appropriate choice of the DNA content of the starting phage. This method, unlike the older EDTA or heat resistance methods, permits the scoring of deletions because of the absence of phenotypic variants. We investigated the effect of several host and phage mutations on deletion frequency and type and have determined that a host polA mutation increases the frequency of deletions about 30-50-fold without changing the type of deletions. A host mutD mutation or thymine deprivation increases deletion frequency about 10-fold. In contrast, a host ligts mutation has no effect on the frequency of deletions. We have also determined that the size of the smallest lambda chromosome packageable in a plaque-forming phage particle is 72-73% that of lambda wild-type.     

A single apurinic site can elicit BAL 31 nuclease-catalyzed cleavage in duplex DNA

The extracellular nuclease from Alteromonas espejiana BAL 31 is a highly sensitive endonucleolytic probe for lesions that distort the helical structure of duplex DNA. The nuclease can be isolated as two distinct molecular species, the 'fast' (F) and 'slow' (S) species, which have different kinetic properties. When nonsupercoiled, covalently closed circular phage PM2 DNA containing apurinic sites introduced by heating at acid pH was incubated with individual fractions from a chromatographic column which separated the two nuclease species, cleavage of the DNA was observed which was greatly in excess of control levels using nonmodified DNA. The initial rates of such cleavage clearly paralleled the peaks of both absorbance and nuclease activity against single-stranded and linear duplex substrates. When samples of apurinic DNA were incubated with pooled fractions from the same column representing pure F and S nucleases, respectively, the rate and extent of the cleavage observed was dependent upon the average number of apurinic sites per molecule. Cleavage was readily detectable in samples containing an average of 1.1 apurinic sites per molecule with both species of the enzyme. These results indicate that either species of the BAL 31 nuclease can recognize and cleave in response to a single apurinic site in duplex DNA. The F nuclease appears to be approx. 2.5-times as efficient in cleaving DNA containing apurinic lesions as the S enzyme in extended incubations.     

In vivo restriction. Sequence and structure of endonuclease II-dependent cleavage sites in bacteriophage T4 DNA.

Endonuclease II of bacteriophage T4 is required for in vivo restriction of cytosine-containing DNA from its host, Escherichia coli, (as well as from phage mutants lacking cytosine modification), normally the first step in the reutilization of host DNA nucleotides for synthesis of phage DNA in infected cells. The phage cytosine-DNA is fragmented incompletely to yield genetically defined fragments. This restriction is different from that of type I, II, or III restriction enzymes. We have located seven major endonuclease II-dependent restriction sites in the T4 genome, of which three were analyzed in detail; in addition, abundant sites were cleaved in less than or equal to 5% of all molecules. Sites I, II, and III shared the sequence 5'-CCGNNTTGGC-3' and were cleaved in about 25% (I and III) and 65% (II) of all molecules, predominantly staggered around the first or second of the central unspecified base pairs to yield fragments with one 5' base. The less frequently cleaved sites I and III deviated from site II in predicted helical structure when viewed from the consensus strand, and in sequence when viewed from the opposite strand. Thus, interaction with a particular helical structure as well as recognition of the bases in DNA appears important for efficient cleavage.     

Neocarzinostatin-induced DNA base release accompanied by staggered oxidative cleavage of the complementary strand

Treatment of an end-labeled DNA restriction fragment with the nonprotein chromophore of neocarzinostatin induced lesions which, after treatment with endonuclease IV or putrescine, were expressed as site-specific double-strand breaks. Analysis of the termini at cleavage sites in each strand showed that the neocarzinostatin-induced lesions consisted of an apurinic/apyrimidinic site plus a closely opposed break in the complementary strand. The break always occurred opposite the base two positions upstream from the apurinic/apyrimidinic site and had the 3'-phosphate and 5'-aldehyde termini characteristic of neocarzinostatin-induced breaks. This positioning suggests that neocarzinostatin simultaneously attacks two DNA sugars on opposite edges of the minor groove. The sequence specificity for formation of apurinic/apyrimidinic sites with closely opposed breaks reflected that of neocarzinostatin-induced mutagenesis. The potent mutagenicity of these lesions may be attributable to the presence of closely opposed damage in both DNA strands.