Sequestration of mammalian Rad51-recombination protein into micronuclei.

The mammalian Rad51 protein is involved in homologous recombination and in DNA damage repair. Its nuclear distribution after DNA damage is highly dynamic, and distinct foci of Rad51 protein, distributed throughout the nuclear volume, are induced within a few hours after gamma irradiation; these foci then coalesce into larger clusters. Rad51-positive cells do not undergo DNA replication. Rad51 foci colocalize with both replication protein A and sites of unscheduled DNA repair synthesis and may represent a nuclear domain for recombinational DNA repair. By 24 h postirradiation, most foci are sequestered into micronuclei or assembled into Rad51-coated DNA fibers. These micronuclei and DNA fibers display genome fragmentation typical of apoptotic cell death. Other repair proteins, such as Rad52 and Gadd45, are not eliminated from the nucleus. DNA double strand breaks in repair-deficient cells or induced by the clastogen etoposide are also accompanied by the sequestering of Rad51 protein before cell death. The spindle poison colcemid causes cell cycle arrest and Rad51-foci formation without directly damaging DNA. Collectively, these observations suggest that mammalian Rad51 protein associates with damaged DNA and/or with DNA that is temporarily or irreversibly unable to replicate and these foci may subsequently be eliminated from the nucleus.

Induction of dormant HIV-1 by sodium butyrate: involvement of the TATA box in the activation of the HIV-1 promoter.

Reactivation of latent HIV-1 is believed to play a major role in the pathogenesis of AIDS. Here we show that sodium butyrate (NaB), which can cause gene induction or cell differentiation, reactivates dormant HIV-1 in vitro in chronically infected cells of T-lymphoid and monocytoid origin. The effect of NaB on HIV-1 expression in T-lymphoid cells was apparent 3 h after addition of drug and peaked at 24 h. During this time the proportion of HIV-1 antigen expressing cells increased from less than 0.5 to greater than 90%, and virus production increased by three orders of magnitude. The virus released by the NaB-induced cells was infectious. The extent and kinetics of NaB effects were similar to effects of phorbol 12-myristate 13-acetate in T cells, but not monocytes. Transient expression assays using an indicator gene under the control of the HIV-1 long terminal repeat revealed that mutations which altered the nucleotide sequence in the TATA box significantly reduced the NaB effect. These data show that NaB is a potent inducer of dormant HIV-1 and suggest that the TATA motif is required for this activity.

Human Rad52 protein promotes single-strand DNA annealing followed by branch migration.

In the yeast, Saccharomyces cerevisiae, the Rad52 gene is important for both mitotic and meiotic recombination. Homologs of the Rad52 gene have been identified in several eukaryotic organisms, ranging from yeast to man. As reported here, human Rad52 protein binds to both single- and double-stranded DNA; and acting on a pair of single-stranded and partially duplex substrates it promotes annealing of complementary strands of DNA, which is followed by branch migration.

Rec-A protein-mediated irreversible fixation of an oligodeoxyribonucleotide to specific site in DNA.

RecA protein can polymerize on an oligodeoxyribonucleotide to form a filament that finds its homologous sequence in double-stranded DNA. When such an oligonucleotide is linked to psoralen, a photoactivatable DNA intercalator, it irreversibly binds to the homologous site in double stranded DNA as a result of psoralen photoadduct formation at thymidines. The relative efficiency of specific vs. nonspecific binding of an oligonucleotide depended upon the ratio of psoralenated oligonucleotide to total DNA. Na+ ions at concentrations greater than 50 mM eliminated specific binding. Under optimal conditions. the probability of binding of an 80-mer oligonucleotide to a specific site was > 10(5) times greater than that of binding to any single nonspecific site. Under the conditions described, RecA-mediated photoadduction was equally efficient with superhelical and linear double-stranded DNA.

[Incorporation of the ampicillin resistance transposon into the Escherichia coli K-12 chromosome and into plasmids]. / Vstraivanie transpozona ustoichivosti k ampitsillinu v khromosomu Escherichia coli K-12 i plazmidy.

The mutant pEG1 of R-factor RP4 with temperature-sensitive defect in replication, carrying a transposable ampicillin resistance element Tn1 was used to define the frequency of insertion of this element into Escherichia coli K-12 chromosome and some other plasmids. Our results indicate that the frequency of colony forming by bacteria with pEG1-factor on ampicillin medium in non-permissive conditions corresponds to the frequency of Tn1 insertion into bacterial chromosome or some other plasmid (in case when the strains are carrying a second plasmid). The frequency of Tn1 insertion into the chromosome is about 4.10(-4). The defect in recA gene produce no serious change in the frequency of Tn1 insertion into the bacterial chromosome. The translocation of Tn1 element from pEG1-factor to R483, R6 and ColE1 plasmids occurs at 10 to 100-fold-higher frequency than from the plasmid to the chromosome. The insertion of Tn1 into the F'-factor KLF10 and R-factor R64-11 occurs at far lower frequency than that to plasmids R6, R483, or ColE1.

[Integration of the RP4 factor with the chromosome in Escherichia coli K12 and the formation of 2 types of Hfr-strains]. / Integratsiia faktora RP4 s khromosomoi Escherichia coli K12 i obrazovanie dvukh tipov Hfr-shtammov.

The mutant RP4ts12, derived from the R-factor RP4 and thermosensitive in replication, is incorporated into the chromosome A3dna(ts) of E. coli K12, thus suppressing dnaA mutation. The integration of this factor into the chromosome leads to the formation of Hfr strains of two types: the strains of the first type transfer plasmid markers to recipient cells earlier than to chromosomal ones; the strains of the second type transfer plasmid markers to recipient cells after chromosomal ones. During conjugation the R-factor integrated into the chromosome dissociates from chromosomal DNA introduced into the recipient cell and becomes autonomous.

The F factor of Escherichia coli carries a locus of stable plasmid inheritance stm, similar to the parB locus of plasmid RI.

We found that a 1.4 kb fragment of the F factor of Escherichia coli (coordinates 62.8-64.2) considerably increased the stable inheritance of different plasmids which carried it. The fragment has a 589 bp DNA sequence (coordinates 63.3-63.9) with extensive homology to the parB locus of plasmid RI and, probably like the parB region, ensures the presence of plasmids in bacterial populations by killing those cells which have lost the plasmid.

Derepression of single-stranded DNA-binding protein genes on plasmids derepressed for conjugation, and complementation of an E. coli ssb- mutation by these genes.

Plasmid single-stranded DNA-binding protein genes complement the E. coli ssb-1 mutation, and partially restore capacity for DNA synthesis, DNA repair (direct role as well as role in SOS induction) and general recombination. Plasmid mutants derepressed for fertility derived from R1, R64 and R222 show a higher level of complementation compared to the parental repressed plasmids. Derepressed mutants of R222 synthesize more RNA which hybridizes with the ssb gene of the F factor than does the original R222 plasmid. This indicates that plasmid ssb genes are regulated coordinately with fertility genes.

Unrelated conjugative plasmids have sequences which are homologous to the leading region of the F factor.

Conjugative plasmids from various incompatibility groups which carry DNA homologous to the ssb gene of the F factor were found to have additional homology with the F factor. This region homologous with F was located on both sides of the ssb gene and occupied a considerable part of the leading region, i.e., the 12.9-kilobase portion of F transferred first during conjugation. This region was the only region of the F factor which has a homologous counterpart on many plasmids.

Indirect stimulation of genetic recombination.

Recombination between lacZ alleles in crosses of lambdalacZ(1) (-) x lambdalacZ(2) (-) and F(-)lacZ(1) (-) x lambdalacZ(2) (-) in Escherichia coli (lambda) can be stimulated manyfold by UV irradiation of one of the lambdalacZ phages [Porter, R. D., McLaughlin, T. & Low, B. (1979) Cold Spring Harbor Symp. Quant. Biol. 43, 1043-1048]. Analogous stimulation has now been observed by coinfection of the cells by UV-irradiated lambda phage which carries no lac region. This indirect stimulation is not dependent on induction of the SOS system. The bacterial uvr system can effectively remove the damages on the lambda DNA which cause the indirect stimulation. Among a number of mutations tested, only ssb-1 was found to cause a drastic decrease in the indirect stimulation. Indirect stimulation was caused only by using phage that had a region of homology with the recombining phage. The homologous region can be separated from the recombining region by an extended nonhomologous region (>7.9 x 10(3) base pairs). This implies that damages to the DNA molecule, which stimulate recombination, can be located very far from the recombining region of the molecule.

Electric birefringence in solutions of high molecular weight ribonucleic acid.

The electric birefringence of low ionic strength solutions of high molecular weight ribonucleic acids from various sources was studied. RNA preparations with a high helical content were found to have negative electric birefringence as a result of the segment anisotropy of the helical portions of the RNA molecule. For completely unfolded molecules of RNA, the electric birefringence is positive and results from the orientation of the macromolecular coil. In intermediate cases, the observed electric birefringence is the sum of negative and positive birefringence. The negative birefringence is caused by the segment orientation of helical sections, and the positive birefringence is caused by the orientation of the macromolecular coil as a whole. Different relaxation times for the positive and negative birefringence permit the pulsed electric birefringence method to analyze these separate phenomena.

Conjugative plasmids of enteric bacteria from many different incompatibility groups have similar genes for single-stranded DNA-binding proteins.

Among 30 conjugative plasmids of enteric bacteria from 23 incompatibility (Inc) groups, we found 19 (from 12 Inc groups) which can complement defects caused by a defective single-stranded DNA-binding protein of Escherichia coli K-12. The genes which are responsible for the complementation from three of these plasmids (Inc groups I1, Y, and 9) were cloned. These genes showed extensive homology with each other and with the E. coli F factor ssb gene (formerly denoted ssf), which codes for a single-stranded DNA binding protein. The proteins coded for by the cloned genes bound tightly to single-stranded DNA. Six other ssb- -complementing plasmids were tested for homology to the F factor ssb gene, and all of these showed homology, as did one of the ssb- -noncomplementing plasmids. Plasmids from a total of 13 different Inc groups of enteric bacteria were found to be likely to have genes with some homology to the ssb gene of the F factor. For plasmids from several different Inc groups, we found no evidence for strong homology with ssb of the F factor.

[2 phosphotransferase systems that control the second stage of phosphoenolpyruvate-dependent glucose phosphorylation in E. coli]. / Dve fosfotransferaznye sistemy, kontroliruiushchie vtoroi étap fosfoenolpiruvatzavisimogo fosforilirovaniia gliukozy u E. coli

Analysis of E. coli W mutants defective in glucose transport suggests an existence of two enzyme systems which carry out the second step of phosphoenolpyruvate (PEP)-dependent glucose phosphorylation, thus controlling the glucose transport into E. coli cells. One system (PTS-glu-alpha) controls phosphorylation and transport of both glucose and alpha-methylglucoside, the other system (PTS-glu-beta) controls phosphorylation and transport of glucose only. PTS-glu-alpha system is damaged in the mutants studied, but PTS-glu-beta system is intact. On account of this fact the mutants do not uptake 14-C-alpha-methylglucoside and their extracts are practically incapable of its phosphorylation, phosphoenolpyruvate being used as phosphate donor. At the same time the mutants are capable of 14-C-glucose uptake and PEP-dependent 14-C-glucose phosphorylation. In one of the strain, having the intact PTS-glu-alpha system, the uptake of glucose and alpha-methylglucoside was stimulated by the addiction of glucose in the cultural medium. No such effect was observed in bacteria with the disturbed PTS-glu-alpha system and the intact PTS-glu-beta system. Probably, the PTS-glu-alpha system can be inducible, in contrast with the PTS-glu-beta system. The damage of the PTS-glu-alpha system results in resistance of bacteria to glucose-induced inhibition of the enzyme synthesis.

[Mechanism of action of glucose on L-asparaginase synthesis by Escherichia coli bacteria]. / Mekhanizm vliianiia gliukozy na sintez L-asparaginazy bakteriiami Escherichia coli.

The synthesis of L-asparaginase in Escherichia coli W and E. coli K-12 was almost completely supressed if glucose was added at a concentration of 0.5 per cent to a growth medium. The level of L-asparaginase synthesis decreased by ca. 75 per cent as a result of cyamutations when the bacteria could not produce cyclo-3',5'-AMP (cAMP). Apparently, a decrease in the intracellular content of cAMP caused by glucose could not be the only factor inhibiting L-asparaginase synthesis. Lactate was found to stimulate L-asparaginase synthesis. Glucose caused the catabolite repression and catabolite inhibition of the components of a system involved in lactate transport. The inhibition of L-asparaginase synthesis by glucose seems to be due, at least partly, to the fact that it prevents the assimilation of lactate by the cells, as well as the utilization of some other compounds which stimulate synthesis of this enzyme.

Inhibition of RNA polymerase II transcription by oligonucleotide-RecA protein filaments targeted to promoter sequences.

In the presence of RecA protein, which plays a major role in genetic recombination in Escherichia coli, an oligodeoxyribonucleotide can find its homologous counterpart in double-stranded DNA and form triple-stranded structures. A triple-stranded structure formed by an oligonucleotide with a sequence overlapping essential regulatory elements of a viral promoter, such as TATA or GC boxes, inhibited in vitro transcription driven by RNA polymerase II. An oligonucleotide with eight nucleotides homologous to its target suppressed RNA polymerase II activity in HeLa cell extracts. This procedure offers a potential alternative to the usual mutational analysis of transcriptional promoters.

Joints formed by RecA protein from oligonucleotides and duplex DNA block initiation and elongation of transcription.

In the presence of the non-hydrolyzable analog of ATP, ATP gamma S, RecA protein can polymerize on an oligodeoxy-ribonucleotide to form a stable oligonucleoprotein filament that can find its homologous sequence in double-stranded DNA. The homologous joint formed by the oligonucleotide and duplex DNA is stable only if RecA protein is not removed. Such a nucleoprotein joint, covering a part or all of the promoter region of T3 or T7 phage RNA polymerase, blocked transcription directed by those polymerases. The same kind of joint, located downstream of the RNA polymerase promoter, also inhibited elongation of transcription and caused accumulation of truncated transcripts. These observations suggest that RecA protein can be used to shut off transcription from any promoter of known sequence.

[2 types of auxotrophic mutants occurring by insertion of the ampicillin transposon into the chromosome of E. coli K-12]. / Dva tipa auksotrofnykh mutantov, voznikaiushchikh pri vstraivanii ampitsillinovogo transpozona v khromosomu E. coli K-12.

Insertion of transpozone TnI determining ampicillin resistance into the E. coli K-12 chromosome resulted in formation of auxothrophic mutants of 2 types. The mutants of the first type carried thermosensitive mutation resulting in auxotrophy with respect to isoleucine at a temperature of 43 degrees C. Such mutants occurred with high frequency (up to 14 per cent with respect to the number of the survived cells with the chromosomes carrying inserted TnI) and had capacity for reversion to the phenotype of the wild type. The mutants of the second type occurred with a frequency 20--180 times lower than that of the mutants of the first type and did not reverse to the phenotype of the parent bacteria. It was found that the chromosome of E. coli K-12 possessed at least 7 sites available for transpozone TnI insertion.

Nuclear foci of mammalian recombination proteins are located at single-stranded DNA regions formed after DNA damage.

A sensitive and rapid in situ method was developed to visualize sites of single-stranded (ss) DNA in cultured cells and in experimental test animals. Anti-bromodeoxyuridine antibody recognizes the halogenated base analog incorporated into chromosomal DNA only when substituted DNA is in the single strand form. After treatment of cells with DNA-damaging agents or gamma irradiation, ssDNA molecules form nuclear foci in a dose-dependent manner within 60 min. The mammalian recombination protein Rad51 and the replication protein A then accumulate at sites of ssDNA and form foci, suggesting that these are sites of recombinational DNA repair.

Differences in the basal activity of the long terminal repeat determine different replicative capacities of two closely related human immunodeficiency virus type 1 isolates.

Two human immunodeficiency virus type 1 (HIV-1) variants derived from a single parental isolate were found to differ substantially in their ability to replicate in CD4-positive cells. Using transient chloramphenicol acetyltransferase expression assays, we show that the long terminal repeat (LTR) of the better-replicating virus has significantly higher capacity than that of the companion virus to direct gene expression in T cells. Sequence data and site-specific mutagenesis experiments demonstrate that the higher LTR activity of the better-replicating HIV-1 is due to a combined effect of two mutations: (i) a point mutation in position -94 (relative to the transcriptional start site), which is located between the two subunits of the HIV-1 enhancer, and (ii) a duplication of 24 base pairs in positions -128 to -151, which was not previously known to be involved in any regulatory function. The presence of these mutations increases the basal level of the LTR-driven gene expression and does not influence the degree of induction caused by the viral tat gene product or by cell activation. Reciprocal exchange of LTRs between the respective viral DNAs results in a change of a recombinant virus replication pattern consistent with the activity of the particular LTR. These experiments suggest that the HIV-1 LTR is one of the sites which determines the functional heterogeneity of HIV-1.