Half-site strand transfer by step-arrest mutants of yeast site-specific recombinase Flp.

The Flp recombinase of Saccharomyces cerevisae can mediate strand transfer within a half-site, between two half-sites and between a half-site and a full-site. The ability of "step-arrest" mutants of Flp to partake in half-site reactions has been examined. Arg308 variants of Flp, which show little or no strand cleavage in reactions with normal full-sites, execute significant levels of strand transfer in half-site reactions. On the other hand, His305 variants of Flp, which normally accumulate the strand cleavage product from full-sites but do not complete strand transfer, yield only minute amounts of strand transfer products from half-sites. As would be predicted, the step-arrest mutants are unable to produce "normal" or "reverse" recombinants between a half-site and a full-site. The Flp protein is able to form higher-order complexes in association with a half-site. The step-arrest mutants of Flp show specific defects in forming these complexes.     

Isolation and characterization of mAMSA-hypersensitive mutants. Cytotoxicity of Top2 covalent complexes containing DNA single strand breaks

Topoisomerase II (Top2) is the primary target for active anti-cancer agents. We developed an efficient approach for identifying hypersensitive Top2 mutants and isolated a panel of mutants in yeast Top2 conferring hypersensitivity to the intercalator N-[4-(9-acridinylamino)-3-methoxyphenyl]methanesulphonanilide (mAMSA). Some mutants conferred hypersensitivity to etoposide as well as mAMSA, whereas other mutants exhibited hypersensitivity only to mAMSA. Two mutants in Top2, changing Pro(473) to Leu and Gly(737) to Val, conferred extraordinary hypersensitivity to mAMSA and were chosen for further characterization. The mutant proteins were purified, and their biochemical activities were assessed. Both mutants encode enzymes that are hypersensitive to inhibition by mAMSA and other intercalating agents and exhibited elevated levels of mAMSA-induced Top2:DNA covalent complexes. While Gly(737) --> Val Top2p generated elevated levels of Top2-mediated double strand breaks in vitro, the Pro(473) --> Leu mutant protein showed only a modest increase in Top2-mediated double strand breaks but much higher levels of Top2-mediated single strand breaks. In addition, the Pro(473) --> Leu mutant protein also generated high levels of mAMSA-stabilized covalent complexes in the absence of ATP. We tested the role of single strand cleavage in cell killing with alleles of Top2 that could generate single strand breaks, but not double strand breaks. Expression in yeast of a Pro(473) --> Leu mutant that could only generate single strand breaks conferred hypersensitivity to mAMSA. These results indicate that generation of single strand breaks by Top2-targeting agents can be an important component of cell killing by Top2-targeting drugs.     

DNA strand specificity for UV-induced mutations in mammalian cells.

The influence of DNA repair on the molecular nature of mutations induced by UV light (254 nm) was investigated in UV-induced hprt mutants from UV-sensitive Chinese hamster cells (V-H1) and the parental line (V79). The nature of point mutations in hprt exon sequences was determined for 19 hprt mutants of V79 and for 17 hprt mutants of V-H1 cells by sequence analysis of in vitro-amplified hprt cDNA. The mutation spectrum in V79 cells consisted of single- and tandem double-base pair changes, while in V-H1 cells three frameshift mutations were also detected. All base pair changes in V-H1 mutants were due to GC----AT transitions. In contrast, in V79 all possible classes of base pair changes except the GC----CG transversion were present. In this group, 70% of the mutations were transversions. Since all mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UV-induced photoproducts at these sites. In V79 cells, 11 out of 17 base pair changes were caused by photoproducts in the nontranscribed strand of the hprt gene. However, in V-H1 cells, which are completely deficient in the removal of pyrimidine dimers from the hprt gene and which show a UV-induced mutation frequency enhanced seven times, 10 out of 11 base pair changes were caused by photoproducts in the transcribed strand of the hprt gene. We hypothesize that this extreme strand specificity in V-H1 cells is due to differences in fidelity of DNA replication of the leading and the lagging strand. Furthermore, we propose that in normal V79 cells two processes determine the strand specificity of UV-induced mutations in the hprt gene, namely preferential repair of the transcribed strand of the hprt gene and a higher fidelity of DNA replication of the nontranscribed strand compared with the transcribed strand.     

Genotypic reversion by methylene Blue: The orientation of guanine-hydroxymethylcytosine at mutated sites in rII mutants of phage T4

Summary Methylene Blue (MB) induced reversion of rII mutants to which a GC base pair was previously assigned is investigated, both in K₁₂() and in B bacteria. The rII mutants used are the same ones whose hydroxylamine (HA) induced reversion rates have previously been investigated by Levisohn (1967) in a similar series of experiments.The results show a relationship between MB-induced and HA-induced reversion patterns which can be interpreted on the assumptions: 1. Both MB and HA produce transitions, but MB acts on Guanine (in agreement with Simon and van Vunakis 1962), while HA acts on Cytocine; 2. Only one injected DNA strand (genetic strand) transmits hereditary information to the progeny while the other strand (associated strand) is disintegrated soon after performing some early functions and its nucleotides are re-used in the formation of new DNA; 3. When the injected associated strand disintegrates, Cytocine bases altered by HA can be re-used, while Guanine bases altered by MB cannot.The assumption that only one strand of the injected DNA transmits hereditary information to the progeny (Simplex hypotheses) is also used in a new interpretation of the mutagenic-induced reversion pattern of lysozyme mutants. This new interpretation avoids a difficulty in the earlier interpretation proposed by Terzaghi, Streisinger and Stahl (1962) which assigned an AT base pair to a mutant strongly HA revertible, while none of the mutants to which a GC base pair had been assigned was HA revertible.The main advantage of the Simplex interpretations of mutagenic phenomena is that self-consistent base assignments to each mutant can be made following a series of rules which have no exceptions: 1. An early function mutant with a high HA reversion rate in non-permissive host can consistently be assigned an odd C in the genetic strand (if odd designates a base which, when replaced through a transition, causes reversion to wild type); 2. A mutant with high MB reversion rate can consistently be assigned an odd G in the genetic strand; 3. A few mutants which present both of these properties are assigned both an odd C and an odd G in a genetic strand triplet; 4. A high HA reversion rate in permissive host but not in non-permissive host is consistently indicative of an odd G with no odd C in the genetic strand triplet.This work was supported by research grant GM-12581 from the Division of General Medical Sciences of the National Institutes of Health, United States Public Health Service.     

Biochemical characteristics of functional domains using feline foamy virus integrase mutants

We constructed deletion mutants and seven point mutants by polymerase chain reaction to investigate the specificity of feline foamy virus integrase functional domains. Complementation reactions were performed for three enzymatic activities such as 3’-end processing, strand transfer, and disintegration. The complementation reactions with deletion mutants showed several activities for 3’-end processing and strand transfer. The conserved central domain and the combination of the N-terminal or C-terminal domains increased disintegration activity significantly. In the complementation reactions between deletion and point mutants, the combination between D107V and deletion mutants revealed 3’-end processing activities, but the combination with others did not have any activity, including strand transfer activities. Disintegration activity increased evenly, except the combination with glutamic acid 200. These results suggest that an intact central domain mediates enzymatic activities but fails to show these activities in the absence of the N-terminal or C-terminal domains. [BMB Reports 2013; 46(1):53-58]     

Strand-specific mutation spectra in repair-proficient and repair-deficient hamster cells

The mutation spectrum induced by UV light has been determined at the hprt locus for both cultured normal (AA8) and UV-sensitive (UV-5) Chinese hamster ovary cells to investigate the effect of DNA repair on the nature of induced mutations. DNA base-pair changes of 23 hprt mutants of AA8 and of 28 hprt mutants of UV-5 were determined by sequence analysis of in vitro amplified hprt cDNA. Almost all mutants in AA8 carried single-base substitutions, transitions and transversions accounting for 38% and 62% of the base changes, respectively. In contrast, in repair-deficient cells (UV-5) tandem and nontandem double mutations represented a considerable portion of the mutations observed (30%), whereas the vast majority of base-pair substitutions were GC greater than AT transitions (87%). Moreover, 5 splice mutants and 2 frameshift mutations were found in the UV-5 collection. In almost all mutants analyzed base changes were located at dipyrimidine sites where UV photoproducts could have been formed. In AA8 the photolesions causing mutations were predominantly located in the nontranscribed strand whereas a strong bias for mutation induction towards photolesions in the transcribed strand was found in UV-5. We hypothesize that preferential removal of lesions from the transcribed strand of the hprt gene accounts for the observed DNA strand specificity of mutations in repair-proficient cells. Furthermore, differences in the degree of misincorporation opposite a lesion for lagging and leading strand DNA synthesis may dictate the pattern of UV-induced mutations in the absence of DNA repair.     

Modulating protein folding rates in vivo and in vitro by side-chain interactions between the parallel beta strands of green fluorescent protein

We have identified pairs of residues across the two parallel beta strands of green fluorescent protein that facilitate native strand register of the surface-exposed beta barrel. After constructing a suitable host environment around two guest residues, minimizing interactions of the guest residues with surrounding side-chains yet maintaining the wild-type protein structure and the chromophore environment, we introduced a library of cross-strand pairings by cassette mutagenesis. Colonies of Escherichia coli transformed with the library differ in intracellular fluorescence. Most of the fluorescent pairs have predominantly charged and polar guest site residues. The magnitude and the rate of fluorescence acquisition in vivo from transformed E. coli cells varies among the mutants despite comparable levels of protein expression. Spectroscopic measurements of purified mutants show that the native protein structure is maintained. Kinetic studies using purified protein with fully matured chromophores demonstrate that the mutants span a 10-fold range in folding rates with undetectable differences in unfolding rates. Thus, green fluorescent protein provides an ideal system for monitoring determinants of in vivo protein folding. Cross-strand pairings affect both protein stability and folding kinetics by favoring the formation of native strand register preferentially to non-native strand alignments.     

A RecG-independent nonconservative branch migration mechanism in Escherichia coli recombination

To gain insight regarding the mechanisms that extend heteroduplex joints in Escherichia coli recombination, we investigated the effect of recG and ruv genotypes on heteroduplex strand polarity in intramolecular recombination products. We also examined the cumulative effect of mutational inactivation of RecG and single-strand-specific exonucleases on recombination proficiency and the role of Chi sites in RecG-independent recombination. All four strands of the two homologs were incorporated into heteroduplex structures in wild-type cells and in ruv mutants. However, in recG mutants heteroduplexes were generated almost exclusively by pairing the invasive 3'-ending strand with its complementary strand. To explain the dependence of strand exchange reciprocity on RecG activity, we propose that alternative mechanisms may extend the heteroduplex joints after homologous pairing: a reciprocal RecG-mediated mechanism and a nonreciprocal mechanism, mediated by RecA and single-strand-specific exonucleases. The cumulative effect of recG and recJ or xonA mutations on recombination proficiency and the inhibitory effect of recJ and xonA activities on heteroduplex formation by the 5'-ending strands are consistent with this proposal.     

Cloning and characterization of DST2, the gene for DNA strand transfer protein beta from Saccharomyces cerevisiae.

The gene encoding the 180-kDa DNA strand transfer protein beta from the yeast Saccharomyces cerevisiae was identified and sequenced. This gene, DST2 (DNA strand transferase 2), was located on chromosome VII. dst2 gene disruption mutants exhibited temperature-sensitive sporulation and a 50% longer generation time during vegetative growth than did the wild type. Spontaneous mitotic recombination in the mutants was reduced severalfold for both intrachromosomal recombination and intragenic gene conversion. The mutants also had reduced levels of the intragenic recombination that is induced during meiosis. Meiotic recombinants were, however, somewhat unstable in the mutants, with a decrease in recombinants and survival upon prolonged incubation in sporulation media. spo13 or spo13 rad50 mutations did not relieve the sporulation defect of dst2 mutations. A dst1 dst2 double mutant has the same phenotype as a dst2 single mutant. All phenotypes associated with the dst2 mutations could be complemented by a plasmid containing DST2.     

Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation

Catalysis of DNA recombination by Tn3 resolvase is conditional on prior formation of a synapse, comprising 12 resolvase subunits and two recombination sites (res). Each res binds a resolvase dimer at site I, where strand exchange takes place, and additional dimers at two adjacent 'accessory' binding sites II and III. 'Hyperactive' resolvase mutants, that catalyse strand exchange at site I without accessory sites, were selected in E. coli. Some single mutants can resolve a res x site I plasmid (that is, with one res and one site I), but two or more activating mutations are necessary for efficient resolution of a site I x site I plasmid. Site I x site I resolution by hyperactive mutants can be further stimulated by mutations at the crystallographic 2-3' interface that abolish activity of wild-type resolvase. Activating mutations may allow regulatory mechanisms of the wild-type system to be bypassed, by stabilizing or destabilizing interfaces within and between subunits in the synapse. The positions and characteristics of the mutations support a mechanism for strand exchange by serine recombinases in which the DNA is on the outside of a recombinase tetramer, and the tertiary/quaternary structure of the tetramer is reconfigured.     

Cell cycle-dependent strand bias for UV-induced mutations in the transcribed strand of excision repair-proficient human fibroblasts but not in repair-deficient cells.

To study the effect of nucleotide excision repair on the spectrum of mutations induced in diploid human fibroblasts by UV light (wavelength, 254 nm), we synchronized repair-proficient cells and irradiated them when the HPRT gene was about to be replicated (early S phase) so that there would be no time for repair in that gene before replication, or in G1 phase 6 h prior to S, and determined the kinds and location of mutations in that gene. As a control, we also compared the spectra of mutations induced in synchronized populations of xeroderma pigmentosum cells (XP12BE cells, which are unable to excise UV-induced DNA damage). Among the 84 mutants sequenced, base substitutions predominated. Of the XP mutants from S or G1 and the repair-proficient mutants from S, approximately 62% were G.C----A.T. In the repair-proficient mutants from G1, 47% were. In mutants from the repair-proficient cells irradiated in S, 71% (10 of 14) of the premutagenic lesions were located in the transcribed strand; with mutants from such cells irradiated in G1, only 20% (3 of 15) were. In contrast, there was no statistically significant difference in the fraction of premutagenic lesions located in the transcribed strand of the XP12BE cells; approximately 75% (24 of 32) of the premutagenic lesions were located in that strand, i.e., 15 of 19 (79%) in the S-phase cells and 9 of 13 (69%) in the G1-phase cells. The switch in strand bias supports preferential nucleotide excision repair of UV-induced damage in the transcribed strand of the HPRT gene.     

A general method to select for M13 clones carrying base pair substitution mutants constructed in vitro.

In this paper we describe a method to select base pair substitution mutants constructed in vitro. The mutagenesis is performed by forcing mistakes during in vitro synthesis from a primer annealed to a single stranded DNA template. The selection is based on the fact that, following transformation, the progeny of the strand elongated in vitro and the template strand have different phenotypes. The method is general and applicable to any DNA segment; the type of base pair substitution and its position can be chosen at will. The combined efficiency of mutagenesis and selection allows for 85% frequency of mutants in all analyzed clones.     

The effect of dithiothreitol on radiation-induced genetic damage in Arabidopsis thaliana (L) heynh

A study was made on the effect of dithiothreitol (DTT; present during irradiation) on M₁ ovule sterility, M₂ embryonic lethals, M₂ chlorophyll mutants and M₂ viable mutants induced with fast neutrons or X-rays in Arabidopsis thaliana. DTT provides considerable protection against both fast-neutron and X-ray-induced genetic damage. However, a higher protection was observed against M₁ ovule sterility, than against embryonic lethals, chlorophylls and viable mutants. This implies a significant DTT-induced spectral shift (0.01<p<0.05), i.e. a shift in the relative frequencies of the different genetic parameters. This spectral shift is explained on the basis of a specific DTT protection against radiation-induced strand breaks, and by differences in the ratio strand breaks/base damage for the genetic parameters concerned, i.e. a higher ratio for ovule sterility than for the other parameters.The induction of the genetic damage by ionizing radiation, either with or without DTT, is described by a mathematical model, which includes both strand breaks and base damage. The model shows that the resolving power of a test for a “mutation” spectral shift depends on the relative values of the strand-break reduction factor of -SH compounds and on the ratio strand breaks/base damage of the genetic parameters. For each genetic parameter the DTT damage reduction factor (DRF) is calculated per irradiation dose, and in addition the average (over-all doses) ratio strand breaks/base damage.     

Defective repair of DNA single- and double-strand breaks in the bleomycin- and X-ray-sensitive Chinese hamster ovary cell mutant, BLM-2

Using filter elution techniques, we have measured the level of induced single- and double-strand DNA breaks and the rate of strand break rejoining following exposure of two Chinese hamster ovary (CHO) cell mutants to bleomycin or neocarzinostatin. These mutants, designated BLM-1 and BLM-2, were isolated on the basis of hypersensitivity to bleomycin and are cross-sensitive to a range of other free radical-generating agents, but exhibit enhanced resistance to neocarzinostatin. A 1-h exposure to equimolar doses of bleomycin induces a similar level of DNA strand breaks in parental CHO-K1 and mutant BLM-1 cells, but a consistently higher level is accumulated by BLM-2 cells. The rate of rejoining of bleomycin-induced single- and double-strand DNA breaks is slower in BLM-2 cells than in CHO-K1 cells. BLM-1 cells show normal strand break repair kinetics. The level of single- and double-strand breaks induced by neocarzinostatin is lower in both BLM-1 and BLM-2 cells than in CHO-K1 cells. The rate of repair of neocarzinostatin-induced strand breaks is normal in BLM-1 cells but retarded somewhat in BLM-2 cells. Thus, there is a correlation between the level of drug-induced DNA damage in BLM-2 cells and the bleomycin-sensitive, neocarzinostatin resistant phenotype of this mutant. Strand breaks induced by both of these agents are also repaired with reduced efficiency by BLM-2 cells. The neocarzinostatin resistance of BLM-1 cells appears to be a consequence of a reduced accumulation of DNA damage. However, the bleomycin-sensitive phenotype of BLM-1 cells does not apparently correlate with any alteration in DNA strand break induction or repair, as analysed by filter elution techniques, suggesting an alternative mechanism of cell killing.     

Crossover interference in Saccharomyces cerevisiae requires a TID1/RDH54- and DMC1-dependent pathway

Two RecA-like recombinases, Rad51 and Dmc1, function together during double-strand break (DSB)-mediated meiotic recombination to promote homologous strand invasion in the budding yeast Saccharomyces cerevisiae. Two partially redundant proteins, Rad54 and Tid1/Rdh54, act as recombinase accessory factors. Here, tetrad analysis shows that mutants lacking Tid1 form four-viable-spore tetrads with levels of interhomolog crossover (CO) and noncrossover recombination similar to, or slightly greater than, those in wild type. Importantly, tid1 mutants show a marked defect in crossover interference, a mechanism that distributes crossover events nonrandomly along chromosomes during meiosis. Previous work showed that dmc1Delta mutants are strongly defective in strand invasion and meiotic progression and that these defects can be partially suppressed by increasing the copy number of RAD54. Tetrad analysis is used to show that meiotic recombination in RAD54-suppressed dmc1Delta cells is similar to that in tid1; the frequency of COs and gene conversions is near normal, but crossover interference is defective. These results support the proposal that crossover interference acts at the strand invasion stage of recombination.     

Impaired incision of ultraviolet-irradiated deoxyribonucleic acid in uvrC mutants of Escherichia coli.

The production of single-strand breaks in the deoxyribonucleic acid of irradiated uvrC mutants of Escherichia coli K-12 was studied both in vivo and in vitro. In vivo, uvrC mutants displayed a slow accumulation of breaks after irradiation, and in this respect appeared different from uvrA mutants, in which very few breaks could be detected. The breakage observed in uvrC mutants differed from that observed in wild-type strains in both the slow rate of break accumulation and the very limited dose response. The behavior of the uvrC lig-7(Ts) double mutant was shown not to be consistent with the suggestion of ligase reversal as the explanation for the lower rate and limited dose response of break formation observed in ultraviolet-irradiated uvrC mutants in vivo. Rather, there appeared to be a real defect in incision. In toluene-treated cells, we studied the effect of the ligase inhibitor nicotinamide mononucleotide on strand incision. Whereas uvrC mutants displayed more strand breakage in the presence of this inhibitor, the same amount of breakage was seen in uvrA mutants, and as such the breakage could be judged as not due to the main excision repair pathway. Experiments using a cell-free system comprising the partially purified uvr+ gene products demonstrated clearly that there is a requirement for the uvrC+ gene product for strand incision. We suggest that in vivo in the absence of the uvrC+ gene product, a partial analog of this protein may allow some abnormal incision.     

Induction and repair of DNA strand breaks in Bacteroides fragilis

Alkaline sucrose gradient sedimentation was used to establish whether strand breakage and repair take place in the DNA of UV-irradiated Bacteroides fragilis during the removal of pyrimidine dimers. A B. fragilis wild-type strain and two of its repair mutants, a mitomycin C sensitive mutant (MTC25) having wild-type levels of UV survival, and a UV-sensitive, mitomycin C sensitive mutant (UVS9), were investigated. Under anaerobic conditions, far-UV irradiation induced metabolically regulated strand breakage and resynthesis in the wild-type strain, but this was markedly reduced in both the MTC25 and UVS9 mutants. Approximately half of the strand breaks generated by the various strains were rejoined during further holding in buffer. Under replicating conditions, complete repair of strand breaks in the wild type was observed. Caffeine treatment under anaerobic conditions caused direct DNA strand breakage in B. fragilis cells but did not inhibit UV-induced breakage or repair.     

Mutagenesis at a specific position in a DNA sequence

Predefined changes in a known DNA sequence were introduced by a general method. Oligodeoxyribonucleotides complementary to positions 582 to 593 of the viral DNA strand of the bacteriophage phiX174 am3 mutant (pGTATCCTACAAA), and to the wild type sequence in this region (pGTATCCTACAAA), were synthesized and used as specific mutagens. Each of these oligonucleotides was incorporated into a complete circular complementary strand when used as primer on a genetically heterologous viral strand template, by the combined action of subtilisin-treated Escherichia coli DNA polymerase I and T4 DNA ligase. Incomplete duplexes were removed or were inactivated by nuclease S1 and the products were used to transfect spheroplasts of E. coli. Both oligonucleotides induced specific mutations at high efficiency when used with heterologous template (15% mutants among progeny phage). The am phages isolated by this procedure are phenotypically gene E mutants, and contain A at position 587 of the viral strand. They thus appear identical with am3 and provide evidence that the change G leads to A at position 587 is sufficient to produce a defective E function. Since the template for the induction of am mutants carried another genetic marker (sB1), the strains carrying the induced mutations have the new genotype am3 sB1. It should be possible to introduce the am3 mutation into any known mutant strain of phi174 using this same oligonucleotide. Both possible transition mutations were induced in these experiments. In principle, the method could also induce transversions, insertions, and deletions. The method should be applicable to other circular DNAs of similar size, for example recombinant DNA plasmids.     

Functional domains of Moloney murine leukemia virus integrase defined by mutation and complementation analysis.

Retroviral integrases perform two catalytic steps, 3' processing and strand transfer, that result in the stable insertion of the retroviral DNA into the host genome. Mutant M-MuLV integrases were constructed to define the functional domains important for 3' processing, strand transfer, and disintegration by in vitro assays. N-terminal mutants had no detectable 3' processing activity, and only one mutant which lacks the HHCC domain, Ndelta105, had strand transfer activity. Strand transfer mediated by Ndelta105 showed preference for one site in the target DNA. Disintegration activity of N-terminal mutants decreased only minimally. In contrast, all C-terminal mutants truncated by more than 28 amino acids had no integration or disintegration activity. Activity on a single-strand disintegration substrate did not require a functional HHCC domain but did require most of the C-terminal region. Complementation analysis found that the HHCC region alone was able to function in trans to a promoter containing only the DD(35)E and C-terminal regions and to enhance integration site selection. Increasing the reducing conditions or adding the HHCC domain to Ndelta105 reaction mixtures restored the wild-type strand transfer activity and range of target sites. The reducing agent affected Cys-209 in the DD(35)E region. The presence of C-209 was required for complementation of Ndelta105 by the HHCC region.