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PubMed2003

p53-associated 3'-->5' exonuclease activity in nuclear and cytoplasmic compartments of cells.

The tumor suppressor protein p53 plays an important role in maintenance of the genomic integrity of cells. p53 possesses an intrinsic 3'-->5' exonuclease activity. p53 was found in the nucleus and in the cytoplasm of the cell. In order to evaluate the subcellular location and extent of p53-associated 3'--> 5' exonuclease activity, we established an in vitro experimental system of cell lines with different nuclear/cytoplasmic distribution of p53. Nuclear and cytoplasmic extracts obtained from LCC2 cells (expressing a high level of cytoplasmic wild-type p53), MCF-7 cells (expressing a high level of wild-type nuclear p53), MDA cells (expressing mutant p53) and H1299 cells (p53-null) were subjected to the analysis of exonuclease activity. Interestingly, 3'-->5' exonuclease was predominantly cytoplasmic; the nuclear extracts derived from all cell lines tested, exerted a low level of exonuclease activity. Cytoplasmic extracts of LCC2 cells, with a high level of wild-type p53, showed an enhanced exonuclease activity in comparison to those expressing either a low level of wild-type p53 (in MCF-7 cells) or the mutant p53 (in MDA cells). Evidence that exonuclease function detected in cytoplasmic extracts is attributed to the p53 is supported by several facts: First, this activity closely parallels with levels and status of endogenous cytoplasmic p53. Second, immunoprecipitation of p53 from cytoplasmic extracts of LCC2 cells markedly reduced the exonuclease activity. Third, the observed 3'-->5' exonuclease in cytoplasmic fraction of LCC2 cells displays identical biochemical properties characteristic of recombinant wild-type p53. The biochemical functions include: (a) substrate specificity; exonuclease hydrolyzes single-stranded DNA in preference to double-stranded DNA and RNA/DNA template-primers, (b) efficient excision of 3'-terminal mispairs from DNA/DNA and RNA/DNA substrates, (c) the preferential excision of purine-purine mispairs over purine-pyrimidine mispairs and (d) functional interaction with exonuclease-deficient DNA polymerase, for example, murine leukemia virus reverse transcriptase (representing a relatively low fidelity enzyme), thus enhancing the fidelity of DNA synthesis by excision of mismatched nucleotides from the nascent DNA strand. Taken together, the data demonstrate that wild-type p53 in cytoplasm, in its noninduced state, is functional; it displays intrinsic 3'-->5' exonuclease activity. The possible role of p53-associated 3'-->5' exonuclease activity in DNA repair in nucleus and cytoplasm is discussed.

Breast Neoplasms↗
PubMed1993

Digestion of damaged DNA by the T7 DNA polymerase-exonuclease.

We have investigated the 3'-5'-exonuclease activity of phage T7 DNA polymerase for its usefulness as an approach for the detection of lesions in DNA. Unlike the T4 DNA polymerase-exonuclease, which is commonly used to map the position and frequency of lesions in very small DNA fragments, T7 DNA polymerase-exonuclease is able to hydrolyse almost completely the large fragments from KpnI-restricted mammalian DNA. However, we found that the exonuclease was also able to hydrolyse DNA containing several kinds of lesions: cyclobutane pyrimidine dimers, thymine glycols, and mono-adducts of 4'-hydroxymethyl-4,5',8-trimethylpsoralen and 5'-methyl-isopsoralen. Modifications of the reaction conditions did not significantly alter the extent of hydrolysis. These properties distinguish the T7 DNA polymerase-exonuclease from the T4 DNA polymerase-exonuclease and make the T7 DNA polymerase-exonuclease unsuitable for detecting several types of lesions in DNA.

Animals↗
PubMed1998

Recombinant pseudorabies virus DNase exhibits a RecBCD-like catalytic function.

The pseudorabies virus (PRV) DNase gene has previously been mapped within the PRV genome. To characterize further the enzymic properties of PRV DNase, this enzyme was expressed in Escherichia coli with the use of a pET expression vector. The protein was purified to homogeneity and assayed for nuclease activity in vitro. Recombinant PRV DNase exhibited an alkaline pH preference and an absolute requirement for Mg2+ ions that could not be replaced by Ca2+ and Na+ ions. Further studies showed that PRV DNase exhibited endonuclease, 5'-exonuclease and 3'-exonuclease activities in both single-stranded and double-stranded DNA. This activity occurred randomly and no significant base preference was demonstrated. The multiple biochemical activities of PRV DNase are similar to the activities of Neurospora crassa endo-exonuclease and E. coli RecBCD, two additional enzymes that are involved in recombination. Taken together, the similarity of action between N. crassa endo-exonuclease, E. coli RecBCD, and PRV DNase suggests that PRV DNase might have a role in the process of recombination that occurs during PRV infection.

Catalysis↗
PubMed1997

Chi-activated RecBCD enzyme possesses 5'-->3' nucleolytic activity, but RecBC enzyme does not: evidence suggesting that the alteration induced by Chi is not simply ejection of the RecD subunit.

BACKGROUND: Homologous recombination in Escherichia coli is initiated by the RecBCD enzyme, and is stimulated by DNA elements known as Chi (chi) sites. The RecBCD enzyme is both a helicase and a nuclease. Recognition of chi causes both attenuation of the 3'-->5' exonuclease activity of the RecBCD enzyme, and activation of an exonuclease activity with 5'-->3' polarity, while leaving the helicase activity unaffected. A variety of evidence suggests that chi-recognition by RecBCD enzyme is accompanied by ejection of the RecD subunit. RESULTS: Through examination of RecBCD exonuclease activity under a variety of conditions, we have shown that recognition of chi by the RecBCD enzyme results in a net reduction of nuclease activity. In addition, the exact location of the first cleavage event elicited by chi-activation of the 5'-->3' nuclease is dependent upon the concentration of free magnesium ions. Finally, we have demonstrated that purified RecBC enzyme (i.e. without the RecD subunit) possesses no significant exonuclease activity under conditions where the chi-modified RecBCD enzyme is an active 5'-->3' exonuclease. CONCLUSIONS: We have shown that, despite the activation of a 5'-->3' exonuclease, recognition of chi by the RecBCD enzyme results in a net preservation of DNA. This new chi-activated nucleolytic action shows surprising variability in the exact location of its initial cleavage. We have demonstrated that purified RecBC enzyme is not an exact analogue of the chi-activated RecBCD enzyme, suggesting that the biochemical basis of chi-activation is not simply ejection of the RecD subunit.

DNA↗
PubMed1997

Chi-star, a chi-related 11-mer sequence partially active in an E. coli recC1004 strain.

BACKGROUND: chi sequence (5'GCTGGTGG) of Escherichia coli was first identified as a site that increased the plaque size of bacteriophage lambda. Subsequent studies showed that this site is responsible for both the attenuation ofRecBCD exonuclease activity and the promotion of RecA, RecBCD-mediated recombination. It is known that bacteriophage lambda containing the chi site makes very small plaques on a recC* (recC1004) mutant because chi is not recognized by the RecBC*D mutant enzyme. RESULTS: We cloned E. coli chromosomal fragments in lambda which allowed lambda to form larger plaques on this recC1004 mutant. The fragments were found to share a chi-like 11-mer sequence, 5'GCTGGTGCTCG. Substitution of these fragments with a synthetic 11-mer of this sequence and single-base-pair substitution analysis of its last four nucleotides demonstrated that this sequence is both necessary and sufficient for the observed activity. The sequence, designated X* (chi-star), protected rolling-circle DNA replication in the recC1004 mutant and in the recBCD+ strain, most likely because it attenuated the exonuclease activity of the RecBC*D and RecBCD+ enzyme. chi-star, did not significantly stimulate lambda recombination in two assays. CONCLUSION: We have discovered that a mutant RecBCD enzyme responds, in vivo, to a longer chi variant.

Bacteriophage lambda↗
PubMed1998

Abortive recombination in Escherichia coli ruv mutants blocks chromosome partitioning.

BACKGROUND: All the ruvA, ruvB and ruvC mutants of Escherichia coli are sensitive to treatments that damage DNA, and are mildly defective in homologous recombination. It has been reported that the ruv mutants form nonseptate, multinuclear filaments after low doses of UV irradiation, dependent on the sfiA gene product. In vitro, the RuvAB complex promotes the branch migration of Holliday junctions, and RuvC resolves the junctions endonucleolytically. RESULTS: After a low UV dose (5 J/m2), both delta ruvAB and delta ruvC mutant cells became filamentous, with their chromosomes aggregated in the central region. This corresponded to an increase in nonmigrating DNA on pulsed field gel electrophoresis of the XbaI digested chromosome. Upon further incubation, they produced a large number of anucleoid cells of normal size. A recA mutation, but not a recB mutation, suppressed these phenotypes of the ruv mutants. The ruv polA12(Ts) double mutants were inviable at the nonpermissive temperature and mimicked the morphological phenotypes of the UV irradiated ruv mutants. CONCLUSION: ruvA, B and C mutations block chromosome partitioning in UV irradiated cells because the abortive homologous recombination covalently links chromosomes together. There is a recBCD independent pathway for the recA dependent formation of recombination intermediates. An Ruv-mediated resolution of recombination intermediates is required for the repair of strand breaks produced in UV irradiated cells and in the polA mutant cells.

Adenosine Triphosphatases↗
PubMed2000

Orientation specificity of the Lactococcus lactis Chi site.

BACKGROUND: In Escherichia coli, the Chi sequence modulates the activity of RecBCD, a powerful double-stranded (ds) DNA exonuclease/helicase. Chi attenuates RecBCD exonuclease activity and stimulates homologous recombination in an orientation-dependent manner. ChiEc is frequent and over-represented on its genome, which is thought to be related to its role in dsDNA break repair. We previously identified a Chi-like sequence (referred to as ChiLl) and an exonuclease/helicase in the Gram-positive bacterium Lactococcus lactis. ChiLl and RexAB are functional analogues of ChiEc and RecBCD. RESULTS: We report that ChiLl attenuates RexAB exonuclease activity and stimulates homologous recombination in an orientation-dependent manner. Analysis of ChiLl distribution on the L. lactis chromosome reveals that ChiLl is frequent, highly over-represented, and oriented with respect to the direction of replication. CONCLUSION: Our results show that a single orientation of ChiLl interacts with RexAB. The active orientation is preferentially found on the replication leading strand of the L. lactis genome, consistent with a primary role of ChiLl in repair of dsDNA breaks at the replication fork. We propose that orientation-dependence of Chi activity and over-representation of Chi sequences on bacterial genomes may be conserved properties of exonuclease/helicase-Chi couples. Other properties of the Chi sequence distribution on the genomes might reflect more specific characteristics of each couple and of the host.

Chromosome Inversion↗
PubMed1996

Activation of stable DNA replication in rapidly growing Escherichia coli at the time of entry to stationary phase.

The conditions are described in which DNA replication can occur, in the absence of protein synthesis, in wild-type Escherichia coli cells. Chromosome replication, which is normally inhibited by addition of chloramphenicol, becomes resistant to this drug after nutritional shiftup, e.g. from minimal medium to Luria broth. This replication activity appears transiently when nutritionally upshifted cells enter stationary phase. The activity strictly requires recA+, but it is independent of recB+ and dnaA+. It can occur in the absence of concomitant transcription. Activation of the replication does not result from induction of the SOS response. As the characteristics of this DNA replication resemble those of the previously characterized stable DNA replication, it is termed nutritional shiftup-activatable stable DNA replication, nSDR. Possible mechanisms of the activation of nSDR in rapidly growing cells at the time of entry to stationary phase are discussed.

Bacterial Proteins↗
PubMed1996

A class of gyrase mutants of Salmonella typhimurium show quinolone-like lethality and require rec functions for viability.

We have identified a new class of DNA gyrase mutants of Salmonella typhimurium that show chronic derepression of the SOS regulon. Thus, these mutants mimic the response of wild-type cells to gyrase inhibitors of the quinolone family. SOS induction by conditional lethal mutations gyrA208 or gyrB652, like that mediated by quinolones, is completely dependent on the function of the recB gene product. Introduction of recA or recB null mutations into these strains exacerbates their temperature-sensitive phenotype and prevents growth at the otherwise permissive temperature of 37 degrees C. Selection of suppressors that concomitantly restore growth at 37 degrees C and SOS induction in a recB- background yielded mutations that relleve the RecB requirement for homologous recombination; namely, sbcB mutations as well as mutations at a new locus that was named sbcE. Such mutations also restore SOS induction in quinolone-treated gyr+ recB- strains. These findings indicate that Rec functions are needed for growth of the gyrase mutants at 37 degrees C and suggest that recombinational repair intermediates constitute the SOS-inducing signal in the mutants as well as in quinolone-treated wild-type bacteria. Unlike quinolones, however, the gyr mutations described in this study do not cause detectable accumulation of "cleavable' gyrase-DNA complexes in plasmid or chromosomal DNA. Yet gyrA208 (the only allele tested) was found to trigger RecB-mediated reckless degradation of chromosomal DNA in recA-cells at restrictive temperatures. Indirect evidence suggests that double-stranded DNA ends, entry sites for the RecBCD enzyme, are generated in the gyr mutants by the breakage of DNA-replication forks. We discuss how this could occur and how recombinational rescue of collapsed replication forks could account for cell survival (and SOS induction) in the gyr mutants as well as in quinolone-treated bacteria.

4-Quinolones↗
PubMed1997

Unsuspected prophage-like elements in Salmonella typhimurium.

We present evidence for the existence of two large (approximately 50 kb) excisable segments in the chromosome of Salmonella typhimurium. The two elements--designated Gifsy-1 and Gifsy-2--cover, respectively, the 57 units and the 24 units of the genetic map where they contribute indicative rare restriction sites. The two elements are closely interrelated and both contain a region of sequence similarity to the recE locus of the Rac prophage of Escherichia coli. Mutations within this region of Gifsy-1 yield the classical 'Sbc' phenotype: they suppress the recombination defect of recB mutants, apparently by activating a normally silent recE-like gene. At the same time, these 'sbcE' mutations activate a Xis-type function that promotes excision of one or other of the two elements. Predictably, curing of Gifsy-1 results in the loss of recB mutant suppression. Surprisingly, the suppressor phenotype is also lost in cells cured for Gifsy-2 even though the Gifsy-1-associated sbcE mutation is still present. Moreover, the excision frequency of Gifsy-1 drops dramatically in Gifsy-2-cured cells. Thus, both elements must co-operate in the activation of recombination and excision functions. Overall, the data presented here suggest that Gifsy-1 and Gifsy-2 are cryptic prophages. They are distinct from previously described Fels prophages. Unlike Fels, they are not specific to S. typhimurium strain LT2 since they are both also found in a virulent S. typhimurium isolate (ATCC 14028s).

Amino Acid Sequence↗
PubMed1997

Repair by recombination of DNA containing a palindromic sequence.

We report here that homologous recombination functions are required for the viability of Escherichia coli cells maintaining a 240 bp chromosomal inverted repeat (palindromic) sequence. Wild-type cells can successfully replicate this palindrome but recA, recB or recC mutants carrying the palindrome are unviable. The dependence on homologous recombination for cell viability is overcome in sbcC mutants. Directly repeated copies of the DNA containing the palindrome are rapidly resolved to single copies in wild-type cells but not in sbcC mutants. Our results suggest that double-strand breaks introduced at the palindromic DNA sequence by the SbcCD nuclease are repaired by homologous recombination. The repair is conservative and the palindrome is retained in the repaired chromosome. We conclude that SbcCD can attack secondary structures but that repair conserves the DNA sequence with the potential to fold.

Bacterial Proteins↗
PubMed1998

Identification of the Chi site of Haemophilus influenzae as several sequences related to the Escherichia coli Chi site.

The Escherichia coli Chi site 5'-GCTGGTGG-3' modulates the activity of the powerful dsDNA exonuclease and helicase RecBCD. Genome sequence analyses revealed that Chi is frequent on the chromosome and oriented with respect to replication on the E. coli genome. Chi is also present much more frequently than predicted statistically for a random 8-mer sequence. Although it is assumed that Chi is ubiquitous, there is virtually no proof that its features are conserved in other microorganisms. We therefore identified and analysed the Chi sequence of an organism for which the full genome sequence was available, Haemophilus influenzae. The biological test we used is based on our finding that rolling circle plasmids provide a specific substrate for RecBCD analogues in different microorganisms. Unexpectedly, several related sequences, corresponding to 5'-GNTGGTGG-3' and 5'-G(G/C)TGGAGG-3', showed Chi activity. As in E. coli, the H. influenzae Chi sites are frequent on the genome, which is in keeping with the need for frequent Chi sites for dsDNA break repair of chromosomal DNA. Although statistically over-represented, this feature is less marked than that of the E. coli Chi site. In contrast to E. coli, the H. influenzae Chi motifs are only slightly oriented with respect to the replication strand. Thus, although Chi appears to have a highly conserved biological role in attenuating exonuclease activity, its sequence characteristics and statistical representation on the genome may differ according to the particular features of the host.

Base Sequence↗
PubMed1998

A five-nucleotide sequence protects DNA from exonucleolytic degradation by AddAB, the RecBCD analogue of Bacillus subtilis.

Homologous recombination in Bacillus subtilis requires the product of the addA and addB genes, the AddAB enzyme. This enzyme, which is both a helicase and a powerful nuclease, is thought to be the counterpart of the Escherichia coli RecBCD enzyme. From this analogy, it is expected that the nuclease activity of AddAB can be downregulated by a specific DNA sequence, which would correspond to the chi site in E. coli. Using protection of linear double-stranded DNA as a criterion, we identified the five-nucleotide sequence 5'-AGCGG-3', or its complement 5'-CCGCT-3', as being sufficient for AddAB nuclease attenuation. We have shown further that this attenuation occurs only if the sequence is properly oriented with respect to the translocating AddAB enzyme. Finally, inspection of the complete B. subtilis genome revealed that this five-nucleotide sequence is over-represented and is, in a majority of cases, co-oriented with DNA replication. Based on these observations, we propose that 5'-AGCGG-3', or its complement, is the B. subtilis analogue of the E. coli chi sequence.

Bacillus subtilis↗
PubMed1998

Differential roles of homologous recombination pathways in Neisseria gonorrhoeae pilin antigenic variation, DNA transformation and DNA repair.

Neisseria gonorrhoeae (Gc) pili undergo antigenic variation when the amino acid sequence of the pilin protein is changed, aiding in immune avoidance and altering pilus expression. Pilin antigenic variation occurs by RecA-dependent unidirectional transfer of DNA sequences from a silent pilin locus to the expressed pilin gene through high-frequency recombination events that occur at limited regions of homology. We show that the Gc recQ and recO genes are essential for pilin antigenic and phase variation and DNA repair but are not involved in natural DNA transformation. This suggests that a RecF-like pathway of recombination exists in Gc. In addition, mutations in the Gc recB, recC or recD genes revealed that a Gc RecBCD pathway also exists and is involved in DNA transformation and DNA repair but not in pilin antigenic variation.

Adenosine Triphosphatases↗
PubMed1999

The mutA mistranslator tRNA-induced mutator phenotype requires recA and recB genes, but not the derepression of lexA-regulated functions.

The mapping of mutA and mutC mutator alleles to the glyV and glyW glycine tRNA genes, respectively, and the subsequent discovery that the mutA phenotype is abolished in a DeltarecA strain raise the possibility that asp --> gly misinsertion may induce a novel mutagenic pathway. The recA requirement suggests three possibilities: (i) the SOS mutagenesis pathway is activated in mutA cells; (ii) loss of recA function interferes with mutA-promoted asp --> gly misinsertion; or (iii) a hitherto unrecognized recA-dependent mutagenic pathway is activated by translational stress. By assaying the expression levels of a reporter plasmid bearing a umuC :lacZ fusion, we show that the SOS regulon is not in a derepressed state in mutA cells. Neither overexpression of the lexA gene through a multicopy plasmid nor replacement of the wild-type lexA allele with the lexA1[Ind-] allele interferes with the expression of the mutA phenotype. The mutA phenotype is unaffected in cells defective for dinB, as shown here, and is unaffected in cells defective for umuD and umuC genes, as shown previously. We show that mutA-promoted asp --> gly misinsertion occurs in recA- cells and, therefore, the requirement for recA is 'downstream' of mistranslation. Finally, we show that the mutA phenotype is abolished in cells deficient for recB, suggesting that cellular recombination functions may be required for the expression of the mutator phenotype. We propose that translational stress induces a previously unrecognized mutagenic pathway in Escherichia coli.

Alleles↗
PubMed1999

Escherichia coli cells bearing mutA, a mutant glyV tRNA gene, express a recA-dependent error-prone DNA replication activity.

A base substitution mutation (mutA) in the Escherichia coli glyV tRNA gene potentiates asp --> gly mistranslation and confers a strong mutator phenotype that is SOS independent, but requires recA, recB and recC genes. Here, we demonstrate that mutA cells express an error-prone DNA polymerase by using an in vitro experimental system based on the conversion of phage M13 single-stranded viral DNA bearing a model mutagenic lesion to the double-stranded replicative form. Amplification of the newly synthesized strand followed by multiplex DNA sequence analysis revealed that mutation fixation at 3, N4-ethenocytosine (varepsilonC) was approximately 3% when the DNA was replicated by normal cell extracts, approximately 48% when replicated by mutA cell extracts and approximately 3% when replicated by mutA recA double mutant cell extracts, in complete agreement with previous in vivo results. Mutagenesis at undamaged DNA sites was significantly elevated by mutA cell-free extracts in the M13 lacZ(alpha) forward mutagenesis system. Neither polA (DNA polymerase I) nor polB (DNA polymerase II) genes are required for the mutA phenotype, suggesting that the phenotype is mediated through a modification of DNA polymerase III or the activation of a previously unidentified DNA polymerase. These findings define the major features of a novel mutagenic pathway and imply the existence of previously unrecognized links between translation, recombination and replication.

Animals↗