An alternative pathway of recombination of chromosomal fragments precedes recA-dependent recombination in the radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans R1 and other members of this genus are able to repair and survive extreme DNA damage induced by ionizing radiation and many other DNA-damaging agents. The ability of R1 to repair completely > 100 double-strand breaks in its chromosome without lethality or mutagenesis is recA dependent. However, during the first 1.5 h after irradiation, recA+ and recA cells show similar increases in the average size of chromosomal fragments. In recA+ cells, DNA continues to enlarge to wild-type size within 29 h. However, in recA cells, no DNA repair is observed following the first 1.5 h postirradiation. This recA-independent effect was studied further, using two slightly different Escherichia coli plasmids forming adjacent duplication insertions in the chromosome, providing repetitive sequences suitable for circularization by non-recA-dependent pathways following irradiation. After exposure to 1.75 Mrad (17,500 Gy), circular derivatives of the integration units were detected in both recA+ and recA cells. These DNA circles were formed in the first 1.5 h postirradiation, several hours before the onset of detectable recA-dependent homologous recombination. By comparison, D. radiodurans strains containing the same E. coli plasmids as nonrepetitive direct insertions did not form circular derivatives of the integration units before or after irradiation in recA+ or recA cells. The circular derivatives of the tandemly integrated plasmids were formed before the onset of recA-dependent repair and have structures consistent with the hypothesis that DNA repair occurring immediately postirradiation is by a recA-independent single-strand annealing reaction and may be a preparatory step for further DNA repair in wild-type D. radiodurans.     

Reexamination of phenotypic defects in rec-1 and rec-2 mutants of Haemophilus influenzae Rd.

Radiolabeled donor DNA is efficiently taken up into competent H. influenzae Rd rec-2 mutant cells but does not undergo the rapid degradation observed in wild-type cells. Furthermore, donor label is not recovered in the chromosome even after 1 h. The donor DNA appears to remain in a protected state in a compartment that can be separated from the rest of the cell. We interpret this as a failure of the donor DNA to be translocated out of the transformasome. In contrast, rec-1 cells translocate labeled donor DNA normally. The donor label accumulates in the recipient chromosome, but, as expected for cells with a recombination defect, there is no preferential localization of the label in sites homologous to the donor DNA. In addition, we have observed two enzymatic activities that act on transformasome-associated DNA of rec-2 cells, an endonuclease which may play a role in the translocation of closed circular DNA and a phosphatase.     

Mutant Rec-1 Eliminates the Meiotic Pattern of Crossing over in Caenorhabditis Elegans

Meiotic crossovers are not randomly distributed along the chromosome. In Caenorhabditis elegans the central portions of the autosomes have relatively few crossovers compared to the flanking regions. We have measured the frequency of crossing over for several intervals across chromosome I in strains mutant for rec-1. The chromosome is ~50 map units in both wild-type and rec-1 homozygotes, however, the distribution of exchanges is very different in rec-1. Map distances expand across the gene cluster and contract near the right end of the chromosome, resulting in a genetic map more consistent with the physical map. Mutations in two other genes, him-6 and him-14, also disrupted the distribution of exchanges. Unlike rec-1, individuals homozygous for him-6 and him-14 had an overall reduction in the amount of crossing over accompanied by a high frequency of nondisjunction and reduced egg hatching. In rec-1; him-6 and rec-1; him-14 homozygotes the frequency of crossing over was characteristic of the Him mutant phenotype, whereas the distribution of the reduced number of exchanges was characteristic of the Rec-1 pattern. It appears that these gene products play a role in establishing the meiotic pattern of exchange events.     

Electron microscopy of single-stranded structures in the DNA of competent Haemophilus influenzae cells.

Chromosomal DNAs from exponential-phase and competent cells of Haemophilus influenzae were examined by electron microscopy to determine whether the chromosome undergoes structural changes during competence development. Single-stranded gaps and single-stranded tails formed in chromosomal DNA during competence development. The generation of gaps was dependent on the rec-2 function. Since the rec-2 mutant is defective in the translocation of donor DNA, it was inferred that the gaps were involved in the translocation step of transformation. The generation of single-stranded tails was independent of the rec-1 and rec-2 genes. Therefore, these structures were assumed to play no direct role in the interaction of donor and recipient DNAs during transformation. Gaps were preferentially associated with a readily denaturable, possibly A + T-rich fraction of the genome. This finding raised the possibility that hot spots for transformation might be associated with A + T-rich DNA.     

Time of Recombination in the DROSOPHILA MELANOGASTER Oocyte. III. Selection and Characterization of Temperature-Sensitive and -Insensitive, Recombination-Deficient Alleles in Drosophila

The procedure for the selection of a temperature-sensitive recombination mutant in Drosophila is described. Use of this procedure has led to the recovery of three alleles at a new recombination locus called rec-1, located within the region of chromosome 3 circumscribed by Deficiency (3R)sbd¹⁰⁵. One allele, rec-1²⁶, is temperature sensitive, and the other two alleles, rec-1⁶ and rec-1¹⁶, are temperature insensitive. Gene dosage studies reveal rec-1²⁶ to be a leaky mutant with greater recombination activity in two doses than in one. The other two alleles show no dose response, implying that they may be null mutants. The temperature response curves of rec-1²⁶ as a homozygote and in heteroallelic combination with rec-1¹⁶ suggest that the sharp decrease in recombination between 28° and 31° indicates temperature denaturation of an enzyme or other protein specified by the mutant and associated with the recombination process. The ability of small changes in temperature to reverse or abolish polarity in recombination along the X chromosome arm in rec-1 ²⁶/rec-1¹⁶ females brings into question the use of the "polarity" criterion to partition mutants into two functional types, i.e., precondition mutants that display polarity and exchange mutants that do not. Evidence that rec-1 may be part of a complex locus residing in a chromosome segment harboring a variety of recombination-related genes is presented.     

Recombination-deficient mutants of Bacillus subtilis.

Two mutant strains of Bacillus subtilis Marburg, NIG43 and NIG45, were isolated. They showed high sensitivities to gamma rays, ultraviolet light (UV), and chemicals. Deficiencies in genetic recombination of these two mutants were shown by the experiments on their capacity in transformation. SPO2 transfection, and PBS1 phage transduction, as well as on their radiation and drug sensitivities and their Hcr+ capacity for UV-exposed phage M2. Some of these characteristics were compared with those of the known strains possessing the recA1 or recB2 alleles. Mapping studies revealed that the mutation rec-43 of strain NIG43 lies in the region of chromosome replication origin. The order was purA dna-8132 rec-43. Another mutation, rec-45, of strain NIG45 was found to be tightly linked to recA1. The mutation rec-43 reduced mainly the frequency of PBS1 transduction. On the other hand, the mutation rec-45 reduced the frequency of recombination involved both in transformation and PBS1 transduction. The mutation rec-43 of strain NIG43 is conditional, but rec-45 of strain NIG45 is not. The UV impairment in cellular survival of strain NIG43 was gradually reverted at higher salt or sucrose concentrations, suggesting cellular possession of a mutated gene produce whose function is conditional. In contrast to several other recombination-deficient strains, SPO2 lysogens of strain NIG43 and NIG45 were not inducible, indicating involvement of rec-43+ or rec-45+ gene product in the development of SPO2 prophage to a vegetative form. The UV-induced deoxyribonucleic acid degradation in vegetative cells was higher in rec-43 and rec-45 strains.     

In vivo damage and recA-dependent repair of plasmid and chromosomal DNA in the radiation-resistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans R1 and other members of this genus share extraordinary resistance to the lethal and mutagenic effects of ionizing radiation. We have recently identified a RecA homolog in strain R1 and have shown that mutation of the corresponding gene causes marked radiosensitivity. We show here that following high-level exposure to gamma irradiation (1.75 megarads, the dose required to yield 37% of CFU for plateau-phase wild-type R1), the wild-type strain repairs > 150 double-strand breaks per chromosome, whereas a recA-defective mutant (rec30) repairs very few or none. A heterologous Escherichia coli-D. radiodurans shuttle plasmid (pMD68) was constructed and found to be retained in surviving D. radiodurans R1 and rec30 following any radiation exposure up to the highest dose tested, 3 megarads. Plasmid repair was monitored in vivo following irradiation with 1.75 megarads in both R1/pMD68 and rec30/pMD68. Immediately after irradiation, plasmids from both strains contained numerous breaks and failed to transform E. coli. While irradiation with 1.75 megarads was lethal to rec30 cultures, a small amount of supercoiled plasmid was regenerated, but it lacked the ability to transform E. coli. In contrast, wild-type cultures showed a cell division arrest of about 10 h, followed by exponential growth. Supercoiled plasmid was regenerated at normal levels, and it readily transformed E. coli. These studies show that D. radiodurans retains a heterologous plasmid following irradiation and repairs it with the same high efficiency as its chromosomal DNA, while the repair defect in rec30 prevents repair of the plasmid. Taken together, the results of this study suggest that plasmid DNA damaged in vivo in D. radiodurans is repaired by recA-dependent mechanisms similar to those employed in the repair of chromosomal DNA.     

Mechanism of acquisition of chromosomal markers by plasmids in Haemophilus influenzae.

The hybrid plasmid pNov1 readily acquired genetic information from the chromosome of wild-type, but not rec-2, cells. Most of the recombination had taken place 1 h after entrance of the plasmid into the cell, as judged by transformation of rec-2 by lysates made from wild-type cells exposed to pNov1. Measurement of physical transfer from radioactively labeled cellular DNA to plasmids recombining in wild-type cells failed, since there was little more radioactivity in plasmids from such cells than from labeled rec-2 recipients, in which no recombination took place. EcoRI digestion of pNov1 divided the DNA into a 1.7-kilobase-pair fragment containing the novobiocin resistance marker and a 13-kilobase-pair fragment containing all of the original vector and considerable portions homologous to the chromosome. Transformation by the large fragment alone resulted in a plasmid the size of the original pNov1. Our hypothesis to explain the data is that genetic transfer from chromosome to plasmid took place by a copy choice mechanism.     

Transformation of Haemophilus influenzae by plasmid RSF0885 containing a cloned segment of chromosomal deoxyribonucleic acid.

A plasmid containing a single cloned insertion of Haemophilus influenzae chromosomal deoxyribonucleic acid that carried a novobiocin resistance marker was 2.6 times larger than the parent plasmid, RSF0885, which conferred ampicillin resistance. The most frequent type of transformation by this plasmid (designated pNov1) was the transfer of novobiocin resistance to the chromosome, with the loss of the plasmid from the recipient. In accord with this observation, after radioactively labeled pNov1 entered a competent cell, it lost acid-insoluble counts, as well as biological activity. The level of ampicillin transformation, which involved establishment of the plasmid, was almost two orders of magnitude lower than the level of novobiocin transformation. Both types of transformation were depressed profoundly in rec-1 and rec-2 mutants. Ampicillin transformants of wild-type cells always contained plasmids that were the same size as pNov1, although most of these transformants were not novobiocin resistant. Plasmid pNov1 in wild-type cells but not in rec-1 or rec-2 cells often recombined with the chromosome, causing a homologous region of the chromosome to be substituted for part of the plasmid, as shown by restriction and genetic analyses. Our data suggested that plasmid-chromosome recombination took place only around the time when the plasmid entered a cell, rather than after it became established.     

rpe, a cis-acting element from the strA region of the Haemophilus influenzae chromosome that makes plasmid establishment independent of recombination.

Plasmids that share homology with the Haemophilus influenzae chromosome transform wild-type cells more efficiently than they transform recombination-defective mutants. A 5.2-kilobase-pair chromosomal fragment containing the strA gene of H. influenzae was found to promote efficient plasmid establishment in recombination-defective mutants. A cis-acting element in the insert, called rpe for rec-less plasmid establishment, promoted plasmid transformation in rec-1 and rec-2 mutants without suppressing the recombination defects of these strains. The rpe locus increased plasmid transformation in wild-type cells without interfering with the pathway of plasmid establishment that is dependent on recombination functions.     

Cloning of the rec-2 locus of Haemophilus influenzae

A collection of transposon mutants of Haemophilus influenzae was constructed by additive transformation with mutagenized chromosomal DNA. A rec-2::miniTn10 km mutation was cloned from a transformation-defective member of the mutant collection, followed by the reconstruction of the wild-type rec-2 locus by recombination to create pDM62. Southern blots showed that the commonly studied Rec-2 mutant, Rd(DB117)rec-, contained either a large deletion or a substitution that removed part of rec-2 locus. A collection of transposon mutations in pDM62 was used to characterize the rec-2 locus by complementation. A corresponding collection of mutants was also constructed. A single segment was required to complement the transformation defect in Rd(DB117)rec-. All of the transformation-defective transposon mutants failed to translocate donor DNA into then cell, in agreement with previous studies of Rd(DB117)rec-.     

Mechanism of gap-filling during postreplication repair of ultraviolet damage in Haemophilus influenzae.

Deoxyribonucleic acid (DNA), pulse labeled after ultraviolet irradiation of excision-defective mutants of Haemophilus influenzae, is of lower single strand molecular weight than that of unirradiated cells but approaches the size of DNA from unirradiated cells upon further incubation in growth medium. This gap-filling process is controlled by the rec-1 gene. Gap-filling occurs normally in a temperature-sensitive DNA synthesis mutant at the restrictive temperature showing that normal semiconservative DNA synthesis is not necessary for gap-filling. To test for recombinational events after irradiation, the DNA synthesized after irradiation was radioactively labeled for a short time in medium containing 5-bromodeoxyuridine followed by incubation for various times in non-radioactive, 5-bromodeoxyuridine-containing medium. The DNA was denatured and analyzed isopycnically. The labeled DNA was initially "heavy," but later shifted toward lighter densities. This shift occurred in the temperature-sensitive DNA synthesis mutant at the restrictive temperature and in the recombination-defective mutant rec-2, but was not seen in the rec-1 mutant. The density shift can be interpreted as evidence that rather extensive exchanges occurred between parental DNA and the DNA made after irradiation. These results suggest that such exchanges are important for gap-filling in H. influenzae.     

Radiation response mechanisms of the extremely radioresistant bacterium Deinococcus radiodurans.

Effect of microgravity on recovery of bacterial cells from radiation damage was examined in IML-2, S/MM-4 and S/MM-9 experiments using the extremely radioresistant bacterium Deinococcus radiodurans. The cells were irradiated with gamma rays before the space flight and incubated on board the Space Shuttle. The survival of the wild type cells incubated in space increased compared with the ground controls, suggesting that the recovery of this bacterium from radiation damage was enhanced under the space environment. No difference was observed between the survivals of radiosensitive mutant rec30 cells incubated in space and on the ground. The amount of DNA-repair related RecA protein induced under microgravity was similar to those of ground controls, however, induction of PprA protein, product of a unique radiation-inducible gene (designated pprA) responsible for loss of radiation resistance in repair-deficient mutant, KH311, was enhanced under microgravity compared with ground controls. Recent investigation in vitro showed that PprA preferentially bound to double-stranded DNA carrying strand breaks, inhibited Escherichia coli exonuclease III activity, and stimulated the DNA end-joining reaction catalyzed by DNA ligases. These results suggest that D. radiodurans has a radiation-induced non-homologous end-joining (NHEJ) repair mechanism in which PprA plays a critical role.     

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.     

Roles of the uvsC, uvsD, uvsE, and mtcA genes in the two pyrimidine dimer excision repair pathways of Deinococcus radiodurans.

In Deinococcus radiodurans, the genes uvsC, uvsD, uvsE, and mtcA are all involved in the single-strand incision of UV-irradiated DNA, and mutations in at least two of them were required to produce an incisionless strain. One mutation must be in mtcA and one in uvsC, uvsD, or uvsE. Strains carrying single mutations in any one of the genes can incise DNA to the same extent as the wild-type strain. Neither the presence of EDTA nor the absence of protein synthesis affected the incision step. Strains deficient in DNA incision have greatly reduced DNA degradation after UV irradiation, and upon addition of chloramphenicol to the postirradiation medium, they do not undergo excessive DNA degradation as is seen in the wild-type strain and strains singly mutant in uvsC, uvsD, or uvsE. The strain singly mutant in mtcA also lacked chloramphenicol-enhanced DNA degradation and loss of viability but behaved similarly to the wild-type strain with respect to resumption of DNA synthesis and DNA degradation in the absence of chloramphenicol. It is proposed that two constitutive, cation-independent UV endonucleases are present in D. radiodurans: UV endonuclease alpha (the product of the mtcA gene), which incises in response to pyrimidine dimers, mitomycin C cross-links, bromomethylbenzanthracene adducts, and other alkylation damage, and UV endonuclease beta (the product of the uvsC, uvsD, and uvsE genes), which incises only in response to pyrimidine dimers. Both endonucleases have associated exonuclease activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sensitivity of Deinococcus radiodurans to gamma-irradiation: a novel approach by Fourier transform infrared spectroscopy

Deinococcus radiodurans is a red-pigmented coccus known to be particularly resistant to both chemical and radiative agents. Fourier transform infrared (FT-IR) spectroscopy was used as a convenient and easy-to-run method to monitor damage induced in this bacterium by ionizing radiations. First, stationary-phase cultures were submitted to increasing doses of gamma-irradiation ((137)Cs source). Beyond a threshold of 11 kGy, striking changes occurred in spectra of irradiated samples compared with unirradiated ones, especially in the 1750-900 cm(-1) region, which is spectroscopically assigned to amide I and II components, nucleotide bases, the phosphodiester backbone, and the sugar ring. Second, bacterial cultures were postirradiation reincubated. After a reincubation time of 15 h, the oxidative stress was in part overwhelmed, and the growth of D. radiodurans again occurred, although some biocellular components remained altered. Consequently, FT-IR analysis is an accurate means to rapidly visualize biomolecular changes undergone by cells both after gamma-irradiation and during the repair mechanism.     

Localization of a determinant mediating partial macrolide resistance in Staphylococcus aureus

Four out of more than 8,200 Staphylococcus aureus strains isolated in Japan between 1961 and 1980 were constitutively resistant to a variety of macrolide antibiotics except tylosin and rokitamycin, but susceptible to lincosamide and streptogramin type B antibiotics (PM). The data obtained by agarose gel electrophoresis, CsCl-ethidium bromide density gradient analysis, diagnosis with ATP-dependent deoxyribonuclease, and a test transducing into a rec- mutant with phage 80L2 propagated on PM-resistant S. aureus all suggested that the determinant for the PM-resistance is located in chromosome.     

耐辐射奇球菌dps突变株的构建和蛋白功能初步研究

Dps(DNAprotection during starvation)蛋白是原核生物中特有的一类具有铁离子结合和抗氧化损伤功能的重要蛋白。利用体外PCR扩增技术和体内同源重组方法,获得了耐辐射奇球菌(Deinococcus radiodurans)dps全基因(DRB0092)缺失突变株。对突变株和野生型分别进行不同浓度过氧化氢(H₂O₂)处理,结果表明:与野生型菌株R1相比,dps突变株在低浓度H₂O₂(≤10mmol/L)条件下存活率急剧下降,而高浓度(≥30mmol/L)下则完全致死。Native-PAGE活性染色结果显示,稳定生长期dps突变株体内两种过氧化氢酶(KatA和KatB)的活性较野生型R1分别上调2.3倍和2.6倍。通过质粒构建和大肠杆菌诱导表达,获得可溶性Dps蛋白。体外结合和DNA保护实验结果显示:Dps具有明显的DNA结合功能,并能保护质粒DNA免受羟自由基攻击。本研究证明,Dps蛋白在耐辐射奇球菌抗氧化体系中发挥重要作用,可能对该菌极端抗性机制有重要贡献。     

Expression of <Emphasis Type="Italic">recA</Emphasis> gene of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed 1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA ⁺ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein.