A new hyperrecombinogenic mutant of Nicotiana tabacum

We have isolated a hyperrecombinogenic Nicotiana tabacum mutant. The mutation, Hyrec, is dominant and segregates in a Mendelian fashion. In the mutant, the level of mitotic recombination between homologous chromosomes is increased by more than three orders of magnitude. Recombination between extrachromosomal substrates is increased six- to ninefold, and intrachromosomal recombination is not affected. Hyrec plants were found to perform non-homologous end joining as efficiently as the wild type, ruling out the possibility that the increase in homologous recombination is due to a defect in end joining. In addition, Hyrec plants show significant resistance to gamma-irradiation, whereas UV resistance is not different from the wild type. This suggests that homologous recombination can be strongly up-regulated in plants. Moreover, Hyrec constitutes a novel type of mutation: no similar mutant was reported in plants and hyperrecombinogenic mutants from other organisms usually show sensitivity to DNA damaging agents. We discuss the insight that this mutant provides into understanding the mechanisms of recombination plus the potential application for gene targeting in plants.     

The mmsA locus of Streptococcus pneumoniae encodes a RecG-like protein involved in DNA repair and in three-strand recombination

We describe the characterization of a mutant strain of Streptococcus pneumoniae previously isolated on the basis of its sensitivity to Methyl Methane Sulphonate (MMS). The mutant strain also exhibited increased sensitivity to UV light and to X-rays, together with a reduced capacity for recombination and Hex-mediated generalized mismatch repair. We show that the original mutant contains two unlinked mutations in the mmsA and in the pms genes. The mmsA wild-type region was cloned and the nucleotide sequence of the mmsA gene was determined. mmsA encodes a polypeptide of 671 amino acids related to a large family of DNA–RNA helicases, with the highest similarity to Escheri-chia coli RecG, a protein involved in the branch migration of Holliday junctions. A plasmid carrying the intact mmsA coding region was shown to restore UV resistance to E. coli recG mutant strains. An mmsA -null mutant constructed by insertion of a chloramphenicol-resistance gene exhibited a 25-fold reduction in recombination during transformation. We suggest that MmsA recognizes and branch migrates three-strand transformation intermediates to extend donor–recipient heteroduplex regions. The mmsA -null mutant exhibited the other phenotypes of the original mutant, apart from mismatch-repair deficiency and, in addition, an alteration in colony-forming ability was noticed. In the pms mutant background, all phenotypes caused by the mmsA mutation were attenuated. Therefore, the pms mutation, although it affected mismatch repair and, to some extent, DNA repair and recombination, acted as a suppressor of the mmsA mutation.     

The pso4-1 mutation reduces spontaneous mitotic gene conversion and reciprocal recombination in Saccharomyces cerevisiae.

Summary Spontaneous mitotic recombination was examined in the haploid pso4-1 mutant of Saccharomyces cerevisiae and in the corresponding wild-type strain. Using a genetic system involving a duplication of the his4 gene it was shown that the pso4-1 mutation decreases at least fourfold the spontaneous rate of mitotic recombination. The frequency of spontaneous recombination was reduced tenfold in pso4-1 strains, as previously observed in the rad52-1 mutant. However, whereas the rad52-1 mutation specifically reduces gene conversion, the pso4-1 mutation reduces both gene conversion and reciprocal recombination. Induced mitotic recombination was also studied in pso4-1 mutant and wild-type strains after treatment with 8-methoxypsoralen plus UVA and 254 nm UV irradiation. Consistent with previous results, the pso4-1 mutation was found strongly to affect recombination induction.     

Genetic analysis of DNA repair in Aspergillus: evidence for different types of MMS-sensitive hyperrec mutants

To identify genes which affect DNA repair and possibly recombination in Aspergillus nidulans, mutants hypersensitive to methyl methanesulphonate (MMS) were induced with ultraviolet light (UV) or gamma-rays. About half of them contained associated translocations and many were hypersensitive to UV and/or defective in meiosis. Two are alleles of the known uvsB gene while most others define new genes. In addition, among available uvs mutants many were found to be MMS-sensitive. Some of the various uncharacterized ones were identified as alleles of known uvs, but 5 of them were mapped in 2 new genes, uvsH and uvsJ. To identify functional and epistatic groups, mutants from each uvs gene were tested for effects on recombination and mutation, and double mutant uvs strains were compared for UV survival to their component single mutant strains. 3 epistatic pairs were identified, (1) uvsF and H, (2) uvsB and D, and (3) uvsC and E. Conclusive interpair tests were difficult, because such double mutant combinations were frequently lethal or nearly so. The first pair, uvsF and H, shared some of the properties of excision-defective mutants, both uvs being very highly sensitive to UV for mutation as well as survival. But unlike such mutants, uvsH was also sensitive to gamma-rays and defective in meiosis. Both uvs showed normal levels of meiotic recombination, but greatly increased spontaneous mitotic crossing-over, being the most "hyperrec" types among all uvs. The second pair, uvsB and uvsC, which was similarly hyperrec showed only slight increases of UV-induced mutation (less than 2-fold). As a main effect, these uvs caused very high frequencies of unbalanced, unstable segregants from diploid conidia (30 X), but few of these were recognizable aneuploids. The third pair, uvsC and E, which are known to be rec- for gene conversion, caused reduced mitotic crossing-over in diploids and increased levels of haploid segregants. These mutants are spontaneous mutators, but showed less UV-induced mutation than wild-type controls.     

sbcB15 And DeltasbcB mutations activate two types of recf recombination pathways in Escherichia coli

Escherichia coli cells with mutations in recBC genes are defective for the main RecBCD pathway of recombination and have severe reductions in conjugational and transductional recombination, as well as in recombinational repair of double-stranded DNA breaks. This phenotype can be corrected by suppressor mutations in sbcB and sbcC(D) genes, which activate an alternative RecF pathway of recombination. It was previously suggested that sbcB15 and DeltasbcB mutations, both of which inactivate exonuclease I, are equally efficient in suppressing the recBC phenotype. In the present work we reexamined the effects of sbcB15 and DeltasbcB mutations on DNA repair after UV and gamma irradiation, on conjugational recombination, and on the viability of recBC (sbcC) cells. We found that the sbcB15 mutation is a stronger recBC suppressor than DeltasbcB, suggesting that some unspecified activity of the mutant SbcB15 protein may be favorable for recombination in the RecF pathway. We also showed that the xonA2 mutation, a member of another class of ExoI mutations, had the same effect on recombination as DeltasbcB, suggesting that it is an sbcB null mutation. In addition, we demonstrated that recombination in a recBC sbcB15 sbcC mutant is less affected by recF and recQ mutations than recombination in recBC DeltasbcB sbcC and recBC xonA2 sbcC strains is, indicating that SbcB15 alleviates the requirement for the RecFOR complex and RecQ helicase in recombination processes. Our results suggest that two types of sbcB-sensitive RecF pathways can be distinguished in E. coli, one that is activated by the sbcB15 mutation and one that is activated by sbcB null mutations. Possible roles of SbcB15 in recombination reactions in the RecF pathway are discussed.     

Escherichia coli ras locus: its involvement in radiation repair

There are several classes of Escherichia coli mutants defective in radiation repair. These include strains defective in pyrimidine dimer excision, in photoreactivation, in recombination, in repair of X-ray damage, and ultraviolet (UV)-conditional mutants which do not divide after UV. Another mutant (ras(-)) has been isolated. The ras(-) has increased UV sensitivity, but only slightly increased X-ray sensitivity (1.5-fold increase). Ability to effect genetic recombination, to reactivate irradiated bacteriophage T1, and to be photoreactivated is normal. UV-induced mutation frequency is greatly increased in the mutant. The ras(-) apparently lacks the ability to repair some UV damage in the bacterial cell but can repair UV damage to bacteriophage DNA. The ras locus is located between lac and purE on the chromosome map.     

Defective Excision Repair of Pyrimidine Dimers in the Ultraviolet-Sensitive Escherichia coli ras− Mutant

The ras(-) mutant of Escherichia coli K-12 is sensitive to ultraviolet (UV) light but only slightly sensitive to X-irradiation (1.5-fold increase). Other phenotypic properties include normal recombination ability and normal host cell reactivation ability but an abnormally high frequency of UV-induced mutation. The response of the ras(-) mutant to UV has been studied biochemically. After low doses of UV, the ras(-) mutant degraded excessive amounts of deoxyribonucleic acid, and long delays in resumption of deoxyribonucleic acid synthesis occurred. Pyrimidine dimers were excised at the normal rate. Although the mutant had the capability of initiating repair replication, the process was not completed after the high UV dose required to allow detection of repair replication. The ras(-) mutant, after low UV doses, left three to four times as many single-strand breaks not rejoined as did the wild-type strain.     

A nuclear mutation defective in mitochondrial recombination in yeast.

Homologous recombination (crossing over and gene conversion) is generally essential for heritage and DNA repair, and occasionally causes DNA aberrations, in nuclei of eukaryotes. However, little is known about the roles of homologous recombination in the inheritance and stability of mitochondrial DNA which is continuously damaged by reactive oxygen species, by-products of respiration. Here, we report the first example of a nuclear recessive mutation which suggests an essential role for homologous recombination in the stable inheritance of mitochondrial DNA. For the detection of this class of mutants, we devised a novel procedure, 'mitochondrial crossing in haploid', which has enabled us to examine many mutant clones. Using this procedure, we examined mutants of Saccharomyces cerevisiae that showed an elevated UV induction of respiration-deficient mutations. We obtained a mutant that was defective in both the omega-intron homing and Endo.SceI-induced homologous gene conversion. We found that the mutant cells are temperature sensitive in the maintenance of mitochondrial DNA. A tetrad analysis indicated that elevated UV induction of respiration-deficient mutations, recombination deficiency and temperature sensitivity are all caused by a single nuclear mutation (mhr1) on chromosome XII. The pleiotropic characteristics of the mutant suggest an essential role for the MHR1 gene in DNA repair, recombination and the maintenance of DNA in mitochondria.     

DNA-repair characterization of cdc40-1, a cell-cycle mutant of Saccharomyces cerevisiae

The cell-cycle specific mutation cdc40-1, which has been previously shown to be sensitive to MMS at the restrictive temperature, was further characterized as a DNA-repair-deficient mutation. cdc40-1 mutants shown only slight sensitivity to UV irradiation. Double mutant studies shown that rad6-l is epistatic to cdc40-1 with respect to sensitivity to UV irradiation and MMS. rad50-1 is epistatic to cdc40-1 with respect to MMS sensitivity in G1 stationary cells, but not in logarithmic cultures. An additive effect is seen between cdc40-1 and rad50-1 with respect to UV irradiation. cdc40-1 mutants are defective in UV-induced mutagenesis at the restrictive temperature. UV-induced levels of recombination are normal at both temperatures, while MMS-induced recombination is enhanced at the restrictive temperature.     

Cloning and characterisation of the Schizosaccharomyces pombe rad32 gene: a gene required for repair of double strand breaks and recombination.

A new Schizosaccharomyces pombe mutant (rad32) which is sensitive to gamma and UV irradiation is described. Pulsed field gel electrophoresis of DNA from irradiated cells indicates that the rad32 mutant, in comparison to wild type cells, has decreased ability to repair DNA double strand breaks. The mutant also undergoes decreased meiotic recombination and displays reduced stability of minichromosomes. The rad32 gene has been cloned by complementation of the UV sensitive phenotype. The gene, which is not essential for cell viability and is expressed at a moderate level in mitotically dividing cells, has significant homology to the meiotic recombination gene MRE11 of Saccharomyces cerevisiae. Epistasis analysis indicates that rad32 functions in a pathway which includes the rhp51 gene (the S.pombe homologue to S.cerevisiae RAD51) and that cells deleted for the rad32 gene in conjunction with either the rad3 deletion (a G2 checkpoint mutation) or the rad2 deletion (a chromosome stability and potential nucleotide excision repair mutation) are not viable.     

Suppressors of Recb Mutations in Salmonella Typhimurium

Using a screen that directly assesses transductional proficiency, we have isolated suppressors of recB mutations in Salmonella typhimurium. The alleles of sbcB reported here are phenotypically distinct from those isolated in Escherichia coli in that they restore recombination proficiency (Rec(+)), resistance to ultraviolet light (UV(R)), and mitomycin C resistance (MC(R)) in the absence of an accompanying sbcCD mutation. In addition the sbcB alleles reported here are co-dominant to sbcB(+). We have also isolated insertion and deletion mutants of the sbcB locus. These null mutations suppress only the UV(S) phenotype of recB mutants. We have also isolated sbcCD mutations, which map near proC. These sbcCD mutations increase the viability, recombination proficiency and MC(R) of both the transductional recombination suppressors (sbcB1 & sbcB6) and the sbcB null mutations. S. typhimurium recB sbcB1 sbcCD8 strains are 15-fold more recombination proficient than wild-type strains. The increase in transductants in these strains is accompanied by a loss of abortive transductants suggesting that these fragments are accessible to the mutant recombination apparatus. Using tandem duplications, we have constructed sbcB merodiploids and found that, in a recB mutant sbcCD(+) genetic background, the sbcB(+) allele is dominant to sbcB1 for transductional recombination but co-dominant for UV(R) and MC(R). However, in a recB sbcCD8 genetic background, the sbcB1 mutation is co-dominant to sbcB(+) for all phenotypes. Our results lead us to suggest that the SbcB and SbcCD proteins have roles in RecBCD-dependent recombination.     

Roles of RecJ,RecO, and RecR in RecET-mediated illegitimate recombination in Escherichia coli

We analyzed effects of overexpression of RecE and RecT on illegitimate recombination during prophage induction in Escherichia coli and found that frequencies of spontaneous and UV-induced illegitimate recombination are enhanced by coexpression of RecE and RecT in the wild type, but the enhanced recombination was reduced by recJ, recO, or recR mutation. The results indicated that RecET-mediated illegitimate recombination depends on the functions of RecJ, RecO, and RecR, suggesting that the RecE and RecJ exonucleases play different roles in this recombination pathway and that the RecO and RecR proteins also play important roles in the recombination. On the other hand, the frequency of the RecET-mediated illegitimate recombination was enhanced by a recQ mutation, implying that the RecQ protein plays a role in suppression of RecET-mediated illegitimate recombination. It was also found that RecET-mediated illegitimate recombination is independent of the RecA function with UV irradiation, but it is enhanced by the recA mutation without UV irradiation. Based on these results, we propose a model for the roles of RecJOR on RecET-mediated illegitimate recombination.     

Different efficiency of UmuDC and MucAB proteins in UV light induced mutagenesis in Escherichia coli

Summary Two multicopy plasmids carrying either the umuDC or the mucAB operon were used to compare the efficiency of UmuDC and MucAB proteins in UV mutagenesis of Escherichia coli K12. It was found that in recA ⁺ uvr ⁺bacteria, plasmid pIC80, mucAB ⁺mediated UV mutagenesis more efficiently than did plasmid pSE117, umuDC ⁺. A similar result was obtained in lexA51(Def) cells, excluding the possibility that this was due to a differential regulation by LexA of the umuDC and mucAB operons. We conclude that some structural characteristic of the UmuDC and MucAB proteins determines their different efficiency in UV mutagenesis. This characteristic could be also responsible for the observation that in the recA430 mutant, pIC80 but no pSE117 can mediate UV mutagenesis. In the recA142 mutant, pIC80 also promoted UV mutagenesis more efficiently than pSE117. In this mutant, the recombination proficiency, the protease activity toward LexA and the mutation frequency were increased by the presence of adenine in the medium. In recA ⁺ uvrB5 bacteria, plasmid pSE117,umuDC caused both an increase in UV sensitivity as well as a reduction in the mutation frequency. These nagative effects resulting from the overproduction of UmuDC proteins were higher in recA142 uvrB5 than in recA ⁺ uvrB5 cells. In contrast, overproduction of MucAB proteins in excision-deficient bacteria containing pIC80 led to a large increase in the mutation frequency. We suggest that the functional differences between UmuDC and MucAB proteins might be due to their different dependence on the direct role of RecA protease in UV mutagenesis.     

The role of UvrD in RecET-mediated illegitimate recombination in Escherichia coli

To study the mechanism of RecET-mediated illegitimate recombination, we examined the formation of lambdabio-transducing phage in Escherichia coli in the presence or absence of UV irradiation. We have previously reported that coexpression of RecE and RecT enhances the frequency of recA-independent illegitimate recombination. RecJOR proteins are required for this RecET-mediated illegitimate recombination, and RecQ suppresses it. Here, we showed that the frequencies of both spontaneous and UV-induced RecET-mediated illegitimate recombination events are reduced by a uvrD mutation. It should be noted that UvrD is required for illegitimate recombination only in the presence, but not in the absence, of RecET. In contrast, frequencies of RecET-mediated illegitimate recombination were not affected by ruvAB, ruvC, recG, and recN mutations. The frequency of spontaneous and UV-induced illegitimate recombination in the uvrD recR double mutant was comparable to that of the uvrD single mutant, suggesting that UvrD works at the same step as RecR in the RecET-mediated recombination pathway. Nucleotide sequence analyses of the recombination junctions showed that RecET-mediated illegitimate recombination detected in UvrD-deficient strain is short-homology-dependent. Based on these and previous results, we propose a model for the role of UvrD on RecET-mediated illegitimate recombination.     

Isolation and characterization of a UV-sensitive mutator (mutB1) mutant of Haemophilus influenzae.

The mutB1 mutant of Haemophilus influenzae is very sensitive to UV radiation but only slightly sensitive to methylmethane sulfonate or N-methyl-N'-nitro-N-nitrosoguanidine. Cultures of mutB1 cells contain high numbers of spontaneous mutants and show hypermutability after exposure to the latter mutagen. Normally high-efficiency transforming markers, as well as low-efficiency ones, transform mutB1 recipients at similarly low efficiencies. Significant host cell reactivation was observed when mutB1 cells were exposed to UV-damaged phage; however, these mutants showed a decrease in phage recombination. This mutant did not degrade its DNA following exposure to UV. It is speculated that the mutB1 mutation is similar to the Escherichia coli uvrD mutation.     

Characterization of the hyperrecombination phenotype of the pol3-t mutation of Saccharomyces cerevisiae

The DNA polymerase delta (Pol3p/Cdc2p) allele pol3-t of Saccharomyces cerevisiae has previously been shown to increase the frequency of deletions between short repeats (several base pairs), between homologous DNA sequences separated by long inverted repeats, and between distant short repeats, increasing the frequency of genomic deletions. We found that the pol3-t mutation increased intrachromosomal recombination events between direct DNA repeats up to 36-fold and interchromosomal recombination 14-fold. The hyperrecombination phenotype of pol3-t was partially dependent on the Rad52p function but much more so on Rad1p. However, in the double-mutant rad1 Delta rad52 Delta, the pol3-t mutation still increased spontaneous intrachromosomal recombination frequencies, suggesting that a Rad1p Rad52p-independent single-strand annealing pathway is involved. UV and gamma-rays were less potent inducers of recombination in the pol3-t mutant, indicating that Pol3p is partly involved in DNA-damage-induced recombination. In contrast, while UV- and gamma-ray-induced intrachromosomal recombination was almost completely abolished in the rad52 or the rad1 rad52 mutant, there was still good induction in those mutants in the pol3-t background, indicating channeling of lesions into the above-mentioned Rad1p Rad52p-independent pathway. Finally, a heterozygous pol3-t/POL3 mutant also showed an increased frequency of deletions and MMS sensitivity at the restrictive temperature, indicating that even a heterozygous polymerase delta mutation might increase the frequency of genetic instability.     

Cloning and characterization of the Azotobacter vinelandii recA gene and construction of a recA deletion mutant

Summary The recA gene of Azotobacter vinelandii was isolated from a genomic library by heterologous complementation of an Escherichia coli recA mutation for resistance to UV radiation. The A. vinelandii recA gene was localized on adjacent PstI fragments of 1.3 and 1.7 kb. The cloned A. vinelandii recA gene was functionally analogous to the E. coli recA gene. It was also able to complement the E. coli recA mutation for homologous recombination. A recA deletion mutant of A. vinelandii was constructed. This mutant was sensitive to DNA-damaging agents like UV rays, methyl methane sulfonate (MMS) and nalidixic acid and was deficient in homologous recombination.     

An Oak Ridge legacy: the specific locus test and its role in mouse mutagenesis.

Mutations in the genes encoding single-strand DNA-specific exonucleases (ssExos) of Escherichia coli were examined for effects on mutation avoidance, UV repair, and conjugational recombination. Our results indicate complex and partially redundant roles for ssExos in these processes. Although biochemical experiments have implicated RecJ exonuclease, Exonuclease I (ExoI), and Exonuclease VII (ExoVII) in the methyl-directed mismatch repair pathway, the RecJ- ExoI- ExoVII- mutant did not exhibit a mutator phenotype in several assays for base substitution mutations. If these exonucleases do participate in mismatch excision, other exonucleases in E. coli can compensate for their loss. Frameshift mutations, however, were stimulated in the RecJ- ExoI- ExoVII- mutant. For acridine-induced frameshifts, this mutator effect was due to a synergistic effect of ExoI- and ExoVII- mutations, implicating both ExoI and ExoVII in avoidance of frameshift mutations. Although no single exonuclease mutant was especially sensitive to UV irradiation, the RecJ- ExoVII- double mutant was extremely sensitive. The addition of an ExoI- mutation augmented this sensitivity, suggesting that all three exonucleases play partially redundant roles in DNA repair. The ability to inherit genetic markers by conjugation was reduced modestly in the ExoI- RecJ- mutant, implying that the function of either ExoI or RecJ exonucleases enhances RecBCD-dependent homologous recombination.     

Isolation and properties of a recombination-deficient mutant of Micrococcus radiodurans.

A mutant of micrococcus radiodurans which is deficient in recombination has been isolated after treatment of the wild type with N-methyl-N'-nitro-N-nitrosoguanidine. We have called this mutant Micrococcus radiodurans rec30. The efficiency of recombination in this mutant, as measured by transformation, is less than 0.01% that of the wild type. It is 15 times more sensitive to the lethal action of ultraviolet radiation, 120 times more sensitive to ionizing radiation, and 300 times more sensitive to mitomycin C (MMC) than the wild type. It is probably inactivated by a single MMC-induced deoxyribonucleic acid cross-link per genome. The excision of ultraviolet-induced pyrimidine dimers is normal. There is no radiation-induced degradation of deoxyribonucleic acid. All spontaneous revertants selected for resistance to low levels of MMC had wild-type resistance to radiation and MMC, and the same efficiency of recombination as the wild type, suggesting that the recombination deficiency of the strain is due to a single mutation. Deoxyribonucleic acid from this mutant can transform M. radiodurans UV17 presumed deficient in an exr type gene to wild type.