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PubMed1995

Dynamics of bacteriophage T4 DNA polymerase function: identification of amino acid residues that affect switching between polymerase and 3' --> 5' exonuclease activities.

Many DNA polymerases are multifunctional with the ability to replicate DNA as well as to proofread misincorporated nucleotides. Since polymerase and 3'--> 5' exonuclease activities appear to reside in spatially distinct active centers, there must be some mechanism for coordinating replication with proofreading and for transferring DNA between the two active centers. We have designed a genetic selection scheme to isolate bacteriophage T4 mutant DNA polymerases that are defective in "switching" between polymerase and exonuclease activities. Amino acid residues that affected active-site-switching were identified in four regions of the T4 DNA polymerase: two regions in the proposed exonuclease domain. Representative mutant DNA polymerases from each region were purified for biochemical studies. We propose that amino acid substitutions identified by mutational analysis affect critical contacts between T4 DNA polymerase and DNA that are required for transfer of DNA between the polymerase and exonuclease active centers.

Amino Acids↗

PubMed1996

Homologous recombination between the tuf genes of Salmonella typhimurium.

The genes coding for the translation factor EF-Tu, tufA and tufB are separated by over 700 kb on the circular chromosome of Salmonella typhimurium. The coding regions of these genes have 99% identity at the nucleotide level in spite of the presumed ancient origin of the gene duplication. Sequence comparisons between S. typhimurium and Escherichia coli suggest that within each species the two tuf genes are evolving in concert. Here we show that each of the S. typhimurium tuf genes can transfer genetic information to the other. In our genetic system the transfers are seen as non-reciprocal, i.e. as gene conversion events. However, the mechanism of recombination could be reciprocal, with sister chromosome segregation and selection leading to the isolation of a particular class of recombinant. The amount of sequence information transferred in individual recombination events varies, but can be close to the entire length of the gene. The recombination is RecABCD-dependent, and is opposed by MutSHLU mismatch repair. In the wild-type, this type of recombination occurs at a rate that is two or three orders of magnitude greater than the nucleotide substitution rate. The rate of recombination differs by six orders of magnitude between a recA and a mutS strain. Mismatch repair reduces the rate of this recombination 1000-fold. The rate of recombination also differs by one order of magnitude depending on which tuf gene is donating the sequence selected for. We discuss three classes of model that could, in principle, account for the sequence transfers: (1) tuf mRNA mediated recombination; (2) non-allelic reciprocal recombination involving sister chromosomes; (3) non-allelic gene conversion involving sister chromosomes, initiated by a double-strand break close to one tuf gene. Although the mechanism remains to be determined, the effect on the bacterial cells is tuf gene sequence homogenisation. This recombination phenomenon can account for the concerted evolution of the tuf genes.

Base Sequence↗

PubMed1997

Study of plasmid replication in Escherichia coli with a combination of 2D gel electrophoresis and electron microscopy.

We studied theta-mode DNA replication in p15A-based Escherichia coli plasmids by analyzing their replication intermediates using a combination of neutral agarose 2D gel electrophoresis and electron microscopy. Our analysis: (1) confirms the original assignment of various features of the 2D gel pattern; (2) shows that while one replication fork progresses around the plasmid DNA, the other is immobile, as if the replication were unidirectional; and (3) reveals that termination often occurs at a location away from the replication origin, suggesting that the replication of our plasmids is, in fact, bidirectional, the two forks being active at different times.

DNA Replication↗

PubMed1997

The RecBCD enzyme initiation complex for DNA unwinding: enzyme positioning and DNA opening.

The Escherichia coli RecBCD enzyme unwinds DNA from a free double-stranded DNA end to produce single-stranded DNA intermediates of homologous recombination. In the absence of ATP RecBCD binds to a free DNA end to form an initiation complex for DNA unwinding. We studied the structure of these complexes formed with blunt-ended, 5'-extended, and 3'-extended DNA. Reactivity to the single-stranded DNA-specific reagents KMnO4 and dimethyl sulfate indicated that RecBCD opened, in a Mg(2+)-dependent manner, the terminal five or six base-pairs in each substrate. Thymine residues located four to six nucleotides from the 5' end were only partially reactive to KMnO4, suggesting that part of the 5'-terminated strand was partially shielded by the enzyme. DNase I footprinting indicated that the enzyme positions itself relative to the end of the longer of the two strands, although an exception was noted. These results imply flexibility in the ability of RecBCD to open the DNA and position itself for unwinding on DNA with different types of ends. They also imply conformational differences of RecBCD enzyme bound to different types of ends; these conformational differences may be related to those occurring during the unwinding cycle.

Adenosine Triphosphate↗

PubMed1998

Structural principles for the inhibition of the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates.

A two-metal-ion catalytic mechanism has previously been proposed for several phosphoryl-transfer enzymes. In order to extend the structural basis of this mechanism, crystal structures of three single-stranded DNA substrates bound to the 3'-5' exonucleolytic active site of the large fragment of DNA polymerase I from Escherichia coli have been elucidated. The first is a 2.1 A resolution structure of a Michaelis complex between the large fragment (or Klenow fragment, KF) and a single-stranded DNA substrate, stabilized by low pH and flash-freezing. The positions and identities of the catalytic metal ions, a Zn2+ at site A and a Mg2+ at site B, have been clearly established. The structural and kinetic consequences of sulfur substitutions in the scissile phosphate have been explored. A complex with the Rp isomer of phosphorothioate DNA, refined at 2.2 A resolution, shows Zn2+ bound to both metal sites and a mispositioning of the substrate and attacking nucleophile. The complex with the Sp phosphorothioate at 2. 3 A resolution reveals that metal ions do not bind in the active site, having been displaced by a bulky sulfur atom. Steady-state kinetic experiments show that catalyzed hydrolysis of the Rp isomer was reduced only about 15-fold, while no enzyme activity could be detected with the Sp phosphorothioate, consistent with the structural observations. Furthermore, Mn2+ could not rescue the activity of the exonuclease on the Sp phosphorothioate. Taken together, these studies confirm and extend the proposed two-metal-ion exonuclease mechanism and provide a structural context to explain the effects of sulfur substitutions on this and other phosphoryl-transfer enzymes. These experiments also suggest that the possibility of metal-ion exclusion be taken into account when interpreting the results of Mn2+ rescue experiments.

Catalysis↗

PubMed1998

Functions of the ATP hydrolysis subunits (RecB and RecD) in the nuclease reactions catalyzed by the RecBCD enzyme from Escherichia coli.

The RecBCD enzyme from Escherichia coli is an ATP-dependent nuclease and helicase. Two of its subunits, the RecB and RecD proteins, are DNA-dependent ATPases. We have purified RecB and RecD proteins with mutations in their consensus ATP binding sites to study the functions of these subunits in the ATP-dependent nuclease activities of RecBCD. Reconstituted heterotrimeric enzymes were prepared by mixing wild-type RecB or RecB-K29Q mutant protein (RecB*) with purified RecC protein, and with a histidine-tagged wild-type RecD (hD) or mutant hRecD-K177Q (hD*) protein. RecBCD and all four reconstituted enzymes (wild-type, two single mutants, and the double mutant) cleave a single-stranded DNA oligomer substrate (25-mer) in the absence of ATP at rates of 0.03 to 0.06 min-1. The nuclease reaction catalyzed by RecB*ChD* is not stimulated significantly by ATP, while the reactions catalyzed by RecBCD, RecBChD, RecBChD*, and RecB*ChD are 300 to 3000 fold faster in the presence of 0.5 mM ATP. RecB*ChD* also has very low ATP hydrolysis activity (approximately 10(3)-fold less than RecBCD), as do the individual mutant RecB* and hRecD* proteins (approximately 100-fold less than RecB or hRecD). The products from the ATP-stimulated nuclease reaction with the oligomer substrate suggest a mechanism where two DNA molecules bind to the enzyme in opposite orientations and are cleaved by the nuclease active site. Cleavage towards the 3'-end of one oligomer (observed with RecBChD*) depends on the wild-type RecB subunit, while RecD-dependent cleavage (observed with RecB*ChD) occurs towards the 5'-end of the second bound oligomer.

Adenosine Triphosphate↗

PubMed1998

Phi 29 DNA polymerase requires the N-terminal domain to bind terminal protein and DNA primer substrates.

A 44 kDa C-terminal fragment of phi 29 DNA polymerase has been separately expressed and purified from Escherichia coli cells. As expected, the truncated protein lacked the 3'-5' exonuclease activity and strand-displacement capacity, previously mapped in the N-terminal domain of phi 29 DNA polymerase. On the other hand, the 44 kDa C-terminal fragment retained polymerase activity when using Mn2+ as metal activator, although the catalytic efficiency was greatly reduced with respect to that of the complete enzyme. Moreover, and in contrast to the high processivity exhibited by phi 29 DNA polymerase (> 70 kb), polymerization by its C-terminal domain was completely distributive. All these polymerization defects were related to a strong impairment of DNA binding, suggesting that additional contacts present in the N-terminal domain are important for an optimal stabilization and translocation of the DNA during polymerization. Moreover, the C-terminal domain showed a very reduced capacity to initiate terminal protein (TP)-primed DNA replication, as a consequence of a weakened interaction with the TP primer, and a lack of activation by protein p6, the initiator of phi 29 DNA replication. We conclude that the C-terminal portion of phi 29 DNA polymerase (residues 188 to 575), although having a structural entity as the domain responsible for the synthetic activities, requires the N-terminal domain to provide important contacts for the two different substrates, DNA and TP, that prime DNA synthesis. These results support the hypothesis of a modular organization of enzymatic activities in DNA-dependent DNA polymerases, but emphasize the functional coordination required for coupling DNA synthesis and proofreading, and for the more specific functions (TP-priming, high processivity and strand-displacement) inherent to phi 29 DNA polymerase.

Bacillus Phages↗

PubMed1998

SSB protein controls RecBCD enzyme nuclease activity during unwinding: a new role for looped intermediates.

The RecBCD enzyme of Escherichia coli initiates homologous recombination by unwinding and simultaneously degrading DNA from a double-stranded DNA end. Single-stranded DNA loops are intermediates of this unwinding process. Here we show that SSB protein reduces the level of DNA degradation by RecBCD enzyme during unwinding, by binding to these ssDNA intermediates. Prior to interaction with the recombination hot spot chi, RecBCD enzyme has both 3'-->5' exonuclease and a weaker 5'-->3' exonuclease activity. We show that degradation of the 5'-terminal strand at the entry site is much more extensive in the absence of SSB protein. After interaction with chi, the level of 5'-->3' exonuclease activity is increased; as expected, degradation of the 5'-strand is also elevated in the absence of SSB protein. Furthermore, we show that, in the absence of SSB protein, the RecBCD enzyme is inhibited by the ssDNA products of unwinding; SSB protein alleviates this inhibition. These results provide insight into the organization of helicase and nuclease domains within the RecBCD enzyme, and also suggest a new level at which the nuclease activity of RecBCD enzyme is controlled. Hence, they offer new insight into the role of SSB protein in the initiation phase of recombination.

DNA Helicases↗

PubMed1998

Identification of the nuclease active site in the multifunctional RecBCD enzyme by creation of a chimeric enzyme.

The recombinational hot spot chi modulates the nuclease and helicase activities of the RecBCD enzyme, leading to generation of an early DNA intermediate for homologous recombination. Here we identify the subunit location of the nuclease active site in RecBCD. The isolated RecB protein cleaves circular single-stranded M13 phage DNA, but RecB1-929, comprising only the 100 kDa N-terminal domain of RecB, does not. We reported previously that the reconstituted RecB1-929CD enzyme also is not a nuclease, suggesting that the C-terminal 30 kDa domain of RecB is a non-specific ssDNA endonuclease. However, we were unable to detect nuclease activity with the subtilisin-generated C-terminal 30 kDa fragment of RecB. Since the subtilisin-generated fragment did not bind to a ssDNA-agarose column, we designed a chimeric enzyme by attaching the C-terminal 30 kDa domain of RecB to the gene 32 protein of T4 phage, a ssDNA binding protein that does not have strand scission ability. In addition, Asp427 in the chimeric enzyme (Asp1080 in RecB), a residue that is conserved among several RecB homologs, was substituted to alanine (the D427A mutant). The wild-type chimeric enzyme cleaves the M13 DNA and the D427A mutation abolishes the endonuclease activity of the chimeric enzyme but does not affect its DNA binding ability. This finding indicates an unusual bipartite nature in the structural organization of RecB, in which the DNA-binding function is located in the N-terminal 100 kDa domain and the nuclease catalytic domain is located in the C-terminal 30 kDa domain. The purified RecBD1080ACD mutant is a processive helicase but not a nuclease, demonstrating that RecBCD has a single nuclease active site in the C-terminal 30 kDa domain of RecB.

Amino Acid Sequence↗

PubMed2000

Bacteriophage P22 Abc2 protein binds to RecC increases the 5' strand nicking activity of RecBCD and together with lambda bet, promotes Chi-independent recombination.

Bacteriophage P22 Abc2 protein binds to the RecBCD enzyme from Escherichia coli to promote phage growth and recombination. Overproduction of the RecC subunit in vivo, but not RecB or RecD, interfered with Abc2-induced UV sensitization, revealing that RecC is the target for Abc2 in vivo. UV-induced ATP crosslinking experiments revealed that Abc2 protein does not interfere with the binding of ATP to either the RecB or RecD subunits in the absence of DNA, though it partially inhibits RecBCD ATPase activity. Productive growth of phage P22 in wild-type Salmonella typhimurium correlates with the presence of Abc2, but is independent of the absolute level of ATP-dependent nuclease activity, suggesting a qualitative change in the nature of Abc2-modified RecBCD nuclease activity relative to the native enzyme. In lambda phage crosses, Abc2-modified RecBCD could substitute for lambda exonuclease in Red-promoted recombination; lambda Gam could not. In exonuclease assays designed to examine the polarity of digestion, Abc2 protein qualitatively changes the nature of RecBCD double-stranded DNA exonuclease by increasing the rate of digestion of the 5' strand. In this respect, Abc2-modified RecBCD resembles a RecBCD molecule that has encountered the recombination hotspot Chi. However, unlike Chi-modified RecBCD, Abc2-modified RecBCD still possesses 3' exonuclease activity. These results are discussed in terms of a model in which Abc2 converts the RecBCD exonuclease for use in the P22 phage recombination pathway. This mechanism of P22-mediated recombination distinguishes it from phage lambda recombination, in which the phage recombination system (Red) and its anti-RecBCD function (Gam) work independently.

Adenosine Triphosphatases↗

PubMed2000

The Bacillus subtilis AddAB helicase/nuclease is regulated by its cognate Chi sequence in vitro.

The AddAB enzyme is important to homologous DNA recombination in Bacillus subtilis, where it is thought to be the functional counterpart of the RecBCD enzyme of Escherichia coli. In vivo, AddAB responds to a specific five-nucleotide sequence (5'-AGCGG-3' or its complement) in a manner analogous to the response of the RecBCD enzyme to interaction with chi sequences. Here, we show that purified AddAB enzyme is able to load at a double-stranded DNA end and is both a DNA helicase and nuclease, whose combined action results in the degradation of both strands of the DNA duplex. During translocation, recognition of the properly oriented sequence 5'-AGCGG-3' causes attenuation of the AddAB enzyme nuclease activity that is responsible for degradation of the strand 3'-terminal at the entry site. Therefore, we conclude that 5'-AGCGG-3' is the B. subtilis Chi site and it is hereafter referred to as chi(Bs). After encountering chi(Bs), both the degradation of the 5'-terminal strand and the helicase activity persist. Thus, processing of a double-stranded DNA end by the AddAB enzyme produces a duplex DNA molecule with a protruding 3'-terminated single-stranded tail, a universal intermediate of the recombination process.

Attachment Sites, Microbiological↗

PubMed2000

Co-evolution of the tuf genes links gene conversion with the generation of chromosomal inversions.

The tufA and tufB genes in Salmonella typhimurium co-evolve by recombination and exchange of genetic material. A model is presented which predicts that co-evolution is achieved by gene conversions and chromosomal inversions. Analysis of recombinants reveals that conversion and inversion each occur with similar rates and each depends on RecBCD activity. The model predicts sequence structures for different classes of post-recombination tuf genes. Sequence analysis reveals the presence of each of these structures and classes, with a predicted bias in the absence of mismatch repair. An implication of these data is that co-evolution of gene families can be linked with the generation of chromosomal rearrangements.

Alleles↗

PubMed2000

Identification of the RecA protein-loading domain of RecBCD enzyme.

Genetic recombination in Escherichia coli is stimulated by the recombination hotspot Chi (chi), a regulatory element that modifies the activities of the RecBCD enzyme and leads to loading of the DNA strand exchange protein, RecA, onto the chi-containing DNA strand. The RecBC enzyme, which lacks the RecD subunit, loads RecA protein constitutively, in a manner that is independent of chi. Using a truncated RecBC enzyme lacking the 30 kDa C-terminal domain of the RecB subunit, we show that this domain is necessary for RecA protein-loading. We propose that this domain harbors a site that interacts with RecA protein, recruiting it to single-stranded DNA during unwinding. This ability of a translocating enzyme to deliver material (RecA protein) to a specific target site (the chi sequence) parallels that of other cellular motor proteins.

Base Pairing↗

PubMed2000

A novel, 11 nucleotide variant of chi, chi*: one of a class of sequences defining the Escherichia coli recombination hotspot chi.

In wild-type Escherichia coli, recognition of the recombination hotspot, chi (5'-GCTGGTGG-3'), by the RecBCD enzyme is central to homologous recombination. However, in the recC* class of RecBCD mutants, stimulation of recombination by the canonical chi sequence is not detectable, but the levels of homologous recombination are nearly wild-type. In vivo studies demonstrate that a member of this class of mutants, the recC1004 allele, encodes an enzyme that responds to a novel variant of chi, termed chi* (5'-GCTGGTGCTCG-3'). Here, we establish that, in vitro, the chi* sequence is recognized more efficiently by the RecBC(1004)D enzyme than is the wild-type chi. This is manifest by both a greater modification of nuclease activity and a higher stimulation of RecA protein-mediated joint molecule formation at chi* than at chi. Sequencing of the recC1004 gene revealed that it contains a frameshift mutation, which results in a replacement of nine of the wild-type amino acid residues by eight in the mutant protein, and defines a locus that is important for the specificity of chi-recognition. In addition, we show that this novel, 11 nucleotide chi* sequence also regulates the wild-type RecBCD enzyme, supporting the notion that variants of the canonical chi constitute a class of sequences that regulate the recombination function of RecBCD enzyme.

Alleles↗

PubMed2002

Mistranslation induced by streptomycin provokes a RecABC/RuvABC-dependent mutator phenotype in Escherichia coli cells.

Translational stress-induced mutagenesis (TSM) refers to the mutator phenotype observed in Escherichia coli cells expressing a mutant allele (mutA or mutC) of the glycine tRNA gene glyV (or glyW). Because of an anticodon mutation, expression of the mutA allele results in low levels of Asp-->Gly mistranslation. The mutA phenotype does not require lexA-regulated SOS mutagenesis functions, and appears to be suppressed in cells defective for RecABC-dependent homologous recombination functions. To test the hypothesis that the TSM response is mediated by non-specific mistranslation rather than specific Asp-->Gly misreading, we asked if streptomycin (Str), an aminoglycoside antibiotic known to promote mistranslation, can provoke a mutator phenotype. We report that Str induces a strong mutator phenotype in cells bearing certain alleles of rpsL, the gene encoding S12, an essential component of the ribosomal 30 S subunit. The phenotype is strikingly similar to that observed in mutA cells in its mutational specificity, as well as in its requirement for RecABC-mediated homologous recombination functions. Expression of Str-inducible mutator phenotype correlates with mistranslation efficiency in response to Str. Thus, mistranslation in general is able to induce the TSM response. The Str-inducible mutator phenotype described here defines a new functional class of rpsL alleles, and raises interesting questions on the mechanism of action of Str, and on bacterial response to antibiotic stress.

Anti-Bacterial Agents↗

PubMed1996

A positive selection vector for the analysis of structural plasmid instability in Bacillus subtilis.

A system for the positive selection of structural plasmid rearrangements in Bacillus subtilis was developed. Random deletions removing a transcription terminator structure in the assay plasmid, designated pGP100, resulted in expression of the cat-86 gene, under control of a constitutive bacteriophage promoter. The resulting chlorampenicol-resistant colonies were analyzed for plasmid contents and were shown, by restriction analysis, to contain initially both the intact parental plasmid and a deletion variant. Sequence analysis of deletion derivatives revealed a consensus target site (5'-A-T-T-A-A/T-3') at or near deletion termini, which resembles topoisomerase I target sites. Endpoints on one side of the deletions were found to be clustered in the promoter region of the tetracycline resistance gene present on pGP100, the gene product of which is an integral membrane protein. Furthermore, deletion of the genes encoding the ATP-dependent exonuclease, AddAB, severely reduced the structural stability of pGP100. The data indicate that similar mechanisms underlie deletion formation in pGP100, and a different plasmid-based system, pGP1, which we have analyzed previously.

Bacillus subtilis↗

PubMed1994

Overexpression and purification of the vaccinia virus DNA polymerase.

We have overexpressed the vaccinia virus DNA polymerase using the hybrid vaccinia virus/T7 expression system. Accumulation of the DNA polymerase to levels as high as 10% of the total protein was observed following coinfection of BSC40 cells with the appropriate vaccinia recombinants. Although the DNA polymerase produced at 37 degrees C was largely insoluble, 25% of the recombinant protein could be recovered as soluble protein when infected cultures were maintained at 32 degrees C. Starting with cytoplasmic lysates of coinfected cells, a rapid and reproducible purification protocol that yielded apparently homogeneous preparations of the DNA polymerase after four chromatographic steps was established. Typically, 0.3 mg of purified DNA polymerase was obtained from 27 mg of total protein within 10 h after harvesting infected cells. As was previously described for the DNA polymerase purified from vaccinia-infected cells (Challberg and Englund, J. Biol. Chem., 254, 7812-7819, 1979), the purified recombinant enzyme displayed both polymerase and 3'-5' exonuclease activities but lacked detectable 5'-3' exonuclease activity. Kinetic analysis of nucleotide incorporation catalyzed by the vaccinia enzyme revealed apparent Km values of 0.9, 2.9, 4.0, and 2.7 microM for dGTP, dATP, TTP, and dCTP, respectively.

Animals↗

PubMed1998

Mutations in the Exo III motif of the herpes simplex virus DNA polymerase gene can confer altered drug sensitivities.

Two herpes simplex virus mutants containing mutated residues within the conserved Exo III motif of the polymerase gene were previously shown to be defective in 3'-5' exonuclease activity and exhibited extremely high mutation frequencies. In this study, we have shown that these mutants also exhibited higher resistance to phosphonoacetic acid and sensitivity to aphidicolin and all nucleoside analogs tested, including acyclovir and gangciclovir, compared to wild-type virus. Marker transfer experiments and sequencing analyses demonstrated that these altered phenotypes were the result of mutations within the Exo III motif. The data indicate that, aside from leading to exonuclease deficiency, mutations in the Exo III motif may also affect interaction of nucleoside triphosphates with the catalytic sites of polymerase activity.

Amino Acid Sequence↗