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PubMed1994

Enzymatic completion of mammalian lagging-strand DNA replication.

Using purified proteins from calf and a synthetic substrate, we have reconstituted the enzymatic reactions required for mammalian Okazaki fragment processing in vitro. The required reactions are removal of initiator RNA, synthesis from an upstream fragment to generate a nick, and then ligation. With our substrate, RNase H type I (RNase HI) makes a single cut in the initiator RNA, one nucleotide 5' of the RNA-DNA junction. The double strand specific 5' to 3' exonuclease removes the remaining monoribonucleotide. After dissociation of cleaved RNA, synthesis by DNA polymerase generates a nick, which is then sealed by DNA ligase I. The unique specificities of the two nucleases for primers with initiator RNA strongly suggest that they perform the same reactions in vivo.

Animals↗
PubMed1994

Reversible inactivation of the Escherichia coli RecBCD enzyme by the recombination hotspot chi in vitro: evidence for functional inactivation or loss of the RecD subunit.

Genetic recombination in Escherichia coli is stimulated by a RecBCD enzyme-mediated event at DNA sequences known as Chi (chi) sites (5'-GCTGGTGG-3'). Previously, it was shown that chi acts to regulate the nuclease activity of RecBCD; here, we demonstrate that, under appropriate conditions, interaction with chi sites can also result in an inactivation of helicase activity of RecBCD. The unwinding of double-stranded DNA-containing chi sites, under conditions of limiting Mg2+ ion, results in the reversible inactivation of RecBCD; addition of excess Mg2+ to the reaction reactivates all activities of RecBCD. Inactivation is the consequence of a chi-dependent modification of RecBCD that appears to result from an inability of the chi-modified RecBCD to reinitiate unwinding of intact DNA molecules. This characteristic behavior of RecBCD and chi is displayed by the reconstituted RecBC (i.e., without the RecD subunit), except that it is not dependent on chi interaction. This biochemical similarity between the chi-modified RecBCD and RecBC enzymes implies that recognition of chi results in a dissociation or functional inactivation of RecD subunit and lends support to the hypothesis that interaction with chi results in ejection of the RecD subunit.

DNA Helicases↗
PubMed1995

The recombination hot spot chi activates RecBCD recombination by converting Escherichia coli to a recD mutant phenocopy.

The products of the recB and recC genes are necessary for conjugal recombination and for repair of chromosomal double-chain breaks in Escherichia coli. The recD gene product combines with the RecB and RecC proteins to comprise RecBCD enzyme but is required for neither recombination nor repair. On the contrary, RecBCD enzyme is an exonuclease that inhibits recombination by destroying linear DNA. The RecD ejection model proposes that RecBCD enzyme enters a DNA duplex at a double-chain end and travels destructively until it encounters the recombination hot spot sequence chi. Chi then alters the RecBCD enzyme by weakening the affinity of the RecD subunit for the RecBC heterodimer. With the loss of the RecD subunit, the resulting protein, RecBC(D-), becomes deficient for exonuclease activity and proficient as a recombinagenic helicase. To test the model, genetic crosses between lambda phage were conducted in cells containing chi on a nonhomologous plasmid. Upon delivering a double-chain break to the plasmid, lambda recombined as if the cells had become recD mutants. The ability of chi to alter lambda recombination in trans was reversed by overproducing the RecD subunit. These results indicate that chi can influence a recombination act without directly participating in it.

Bacteriophage lambda↗
PubMed1995

Interaction with the recombination hot spot chi in vivo converts the RecBCD enzyme of Escherichia coli into a chi-independent recombinase by inactivation of the RecD subunit.

The RecBCD enzyme of Escherichia coli promotes recombination preferentially at chi nucleotide sequences and has in vivo helicase and strong duplex DNA exonuclease (exoV) activities. The enzyme without the RecD subunit, as in a recD null mutant, promotes recombination efficiently but independently of chi and has no nucleolytic activity. Employing phage lambda red gam crosses, phage T4 2- survival measurements, and exoV assays, it is shown that E. coli cells in which RecBCD has extensive opportunity to interact with linear chi-containing DNA (produced by rolling circle replication of a plasmid with chi or by bleomycin-induced fragmentation of the cellular chromosome) acquire the phenotype of a recD mutant and maintain this for approximately 2 h. It is concluded that RecBCD is converted into RecBC during interaction with chi by irreversible inactivation of RecD. After conversion, the enzyme is released and initiates recombination on other DNA molecules in a chi-independent fashion. Overexpression of recD+ (from a plasmid) prevented the phenotypic change and providing RecD after the change restored chi-stimulated recombination. The observed recA+ dependence of the downregulation of exoV could explain the previously noted "reckless" DNA degradation of recA mutants. It is proposed that chi sites are regulatory elements for the RecBCD to RecBC switch and thereby function as cis- and trans-acting stimulators of RecBC-dependent recombination.

Bleomycin↗
PubMed1995

Proofreading-defective DNA polymerase II increases adaptive mutation in Escherichia coli.

The role of Escherichia coli DNA polymerase (Pol) II in producing or avoiding mutations was investigated by replacing the chromosomal Pol II gene (polB+) by a gene encoding an exonuclease-deficient mutant Pol II (polBex1). The polBex1 allele increased adaptive mutations on an episome in nondividing cells under lactose selection. The presence of a Pol III antimutator allele (dnaE915) reduced adaptive mutations in both polB+ cells and cells deleted for polB (polB delta 1) to below the wild-type level, suggesting that both Pol II and Pol III are synthesizing episomal DNA in nondividing cells but that in wild-type cells Pol III generates the adaptive mutations. The adaptive mutations were mainly -1 frame-shifts occurring in short homopolymeric runs and were similar in wild-type, polB delta 1, and polBex1 strains. Mutations produced by both Pol III and Pol II ex1 were corrected by the mutHLS mismatch repair system.

Acclimatization↗
PubMed1995

DNA nick processing by exonuclease and polymerase activities of bacteriophage T4 DNA polymerase accounts for acridine-induced mutation specificities in T4.

Acridine-induced frameshift mutagenesis in bacteriophage T4 has been shown to be dependent on T4 topoisomerase. In the absence of a functional T4 topoisomerase, in vivo acridine-induced mutagenesis is reduced to background levels. Further, the in vivo sites of acridine-induced deletions and duplications correlate precisely with in vitro sites of acridine-induced T4 topoisomerase cleavage. These correlations suggest that acridine-induced discontinuities introduced by topoisomerase could be processed into frameshift mutations. The induced mutations at these sites have a specific arrangement about the cleavage site. Deletions occur adjacent to the 3' end and duplications occur adjacent to the 5' end of the cleaved bond. It was proposed that at the nick, deletions could be produced by the 3'-->5' removal of bases by DNA polymerase-associated exonuclease and duplications could be produced by the 5'-->3' templated addition of bases. We have tested in vivo for T4 DNA polymerase involvement in nick processing, using T4 phage having DNA polymerases with altered ratios of exonuclease to polymerase activities. We predicted that the ratios of the deletion to duplication mutations induced by acridines in these polymerase mutant strains would reflect the altered exonuclease/polymerase ratios of the mutant T4 DNA polymerases. The results support this prediction, confirming that the two activities of the T4 DNA polymerase contribute to mutagenesis. The experiments show that the influence of T4 DNA polymerase in acridine-induced mutation specificities is due to its processing of acridine-induced 3'-hydroxyl ends to generate deletions and duplications by a mechanism that does not involve DNA slippage.

Aminacrine↗
PubMed1995

A 7-base-pair sequence protects DNA from exonucleolytic degradation in Lactococcus lactis.

Linear DNA molecules are subject to degradation by various exonucleases in vivo unless their ends are protected. It has been demonstrated that a specific 8-bp sequence, 5'-GCTGGTGG-3', named Chi, can protect linear double-stranded DNA from the major Escherichia coli exonuclease RecBCD. Chi protects linear replication products of rolling-circle plasmids from RecBCD degradation in vivo, in agreement with observations in vitro. A unique 7-bp sequence, 5'-GCGCGTG-3', is shown to protect similar replication products from degradation in Lactococcus lactis strains but not in more distantly related Gram-positive bacteria. The properties of this sequence in L. lactis correspond to those of a Chi site. Linear plasmid replication products have been detected in numerous prokaryotes, suggesting the widespread existence of short species-specific sequences that preserve linear DNA from extensive degradation by host cell exonucleases.

Base Composition↗
PubMed1996

Cloning of thermostable DNA polymerases from hyperthermophilic marine Archaea with emphasis on Thermococcus sp. 9 degrees N-7 and mutations affecting 3'-5' exonuclease activity.

Five extremely thermophilic Archaea from hydrothermal vents were isolated, and their DNA polymerases were cloned and expressed in Escherichia coli. Protein splicing elements (inteins) are present in many archaeal DNA polymerases, but only the DNA polymerase from strain GB-C contained an intein. Of the five cloned DNA polymerases, the Thermococcus sp. 9 degrees N-7 DNA polymerase was chosen for biochemical characterization. Thermococcus sp. 9 degrees N-7 DNA polymerase exhibited temperature-sensitive strand displacement activity and apparent Km values for DNA and dNTP similar to those of Thermococcus litoralis DNA polymerase. Six substitutions in the 3'-5' exonuclease motif I were constructed in an attempt to reduce the 3'-5' exonuclease activity of Thermococcus sp. 9 degrees N-7 DNA polymerase. Five mutants resulted in no detectable 3'-5' exonuclease activity, while one mutant (Glul43Asp) had <1% of wild-type activity.

Amino Acid Sequence↗
PubMed1996

p21Cip1/Waf1 disrupts the recruitment of human Fen1 by proliferating-cell nuclear antigen into the DNA replication complex.

Fen1 or maturation factor 1 is a 5'-3' exonuclease essential for the degradation of the RNA primer-DNA junctions at the 5' ends of immature Okazaki fragments prior to their ligation into a continuous DNA strand. The gene is also necessary for repair of damaged DNA in yeast. We report that human proliferating-cell nuclear antigen (PCNA) associates with human Fen1 with a Kd of 60 nM and an apparent stoichiometry of three Fen1 molecules per PCNA trimer. The Fen1-PCNA association is seen in cell extracts without overexpression of either partner and is mediated by a basic region at the C terminus of Fen1. Therefore, the polymerase delta-PCNA-Fen1 complex has all the activities associated with prokaryotic DNA polymerases involved in replication: 5'-3' polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. Although p21, a regulatory protein induced by p53 in response to DNA damage, interacts with PCNA with a comparable Kd (10 nM) and a stoichiometry of three molecules of p21 per PCNA trimer, a p21-PCNA-Fen1 complex is not formed. This mutually exclusive interaction suggests that the conformation of a PCNA trimer switches such that it can either bind p21 or Fen1. Furthermore, overexpression of p21 can disrupt Fen1-PCNA interaction in vivo. Therefore, besides interfering with the processivity of polymerase delta-PCNA, p21 also uncouples Fen1 from the PCNA scaffold.

Binding Sites↗
PubMed1996

Mutants in the Exo I motif of Escherichia coli dnaQ: defective proofreading and inviability due to error catastrophe.

The Escherichia coli dnaQ gene encodes the proofreading 3' exonuclease (epsilon subunit) of DNA polymerase III holoenzyme and is a critical determinant of chromosomal replication fidelity. We constructed by site-specific mutagenesis a mutant, dnaQ926, by changing two conserved amino acid residues (Asp-12-->Ala and Glu-14-->Ala) in the Exo I motif, which, by analogy to other proofreading exonucleases, is essential for the catalytic activity. When residing on a plasmid, dnaQ926 confers a strong, dominant mutator phenotype, suggesting that the protein, although deficient in exonuclease activity, still binds to the polymerase subunit (alpha subunit or dnaE gene product). When dnaQ926 was transferred to the chromosome, replacing the wild-type gene, the cells became inviable. However, viable dnaQ926 strains could be obtained if they contained one of the dnaE alleles previously characterized in our laboratory as antimutator alleles or if it carried a multicopy plasmid containing the E. coli mutL+ gene. These results suggest that loss of proofreading exonuclease activity in dnaQ926 is lethal due to excessive error rates (error catastrophe). Error catastrophe results from both the loss of proofreading and the subsequent saturation of DNA mismatch repair. The probability of lethality by excessive mutation is supported by calculations estimating the number of inactivating mutations in essential genes per chromosome replication.

Amino Acid Sequence↗
PubMed1997

The recombination hotspot Chi is recognized by the translocating RecBCD enzyme as the single strand of DNA containing the sequence 5'-GCTGGTGG-3'.

The RecBCD enzyme of Escherichia coli functions in the seemingly disparate roles of homologous recombination and the degradation of DNA. Which of these two roles it assumes is regulated by the 8-base recombination hotspot, Chi. Using double-stranded DNA substrates that are heteroduplex at the Chi locus we have established the determinants for Chi recognition. Our results show that an actively translocating RecBCD enzyme requires only the sequence information in the 5'-GCTGGTGG-3'-containing strand to recognize and to be regulated by Chi. Furthermore, the RecBCD enzyme can translocate through DNA heteroduplex bubbles as large as 22 bases, and still recognize a Chi sequence embedded in this region. This implies that recognition of Chi occurs following the unwinding of the DNA.

DNA, Recombinant↗
PubMed1997

Barriers to recombination between closely related bacteria: MutS and RecBCD inhibit recombination between Salmonella typhimurium and Salmonella typhi.

Previous studies have shown that inactivation of the MutS or MutL mismatch repair enzymes increases the efficiency of homeologous recombination between Escherichia coli and Salmonella typhimurium and between S. typhimurium and Salmonella typhi. However, even in mutants defective for mismatch repair the recombination frequencies are 10(2)- to 10(3)-fold less than observed during homologous recombination between a donor and recipient of the same species. In addition, the length of DNA exchanged during transduction between S. typhimurium and S. typhi is less than in transductions between strains of S. typhimurium. In homeologous transductions, mutations in the recD gene increased the frequency of transduction and the length of DNA exchanged. Furthermore, in mutS recD double mutants the frequency of homeologous recombination was nearly as high as that seen during homologous recombination. The phenotypes of the mutants indicate that the gene products of mutS and recD act independently. Because S. typhimurium and S. typhi are approximately 98-99% identical at the DNA sequence level, the inhibition of recombination is probably not due to a failure of RecA to initiate strand exchange. Instead, these results suggest that mismatches act at a subsequent step, possibly by slowing the rate of branch migration. Slowing the rate of branch migration may stimulate helicase proteins to unwind rather than extend the heteroduplex and leave uncomplexed donor DNA susceptible to further degradation by RecBCD exonuclease.

Adenosine Triphosphatases↗
PubMed1998

Identification of the lactococcal exonuclease/recombinase and its modulation by the putative Chi sequence.

Studies of RecBCD-Chi interactions in Escherichia coli have served as a model to understand recombination events in bacteria. However, the existence of similar interactions has not been demonstrated in bacteria unrelated to E. coli. We developed an in vivo model to examine components of dsDNA break repair in various microorganisms. Here, we identify the major exonuclease in Lactococcus lactis, a Gram-positive organism evolutionarily distant from E. coli, and provide evidence for exonuclease-Chi interactions. Insertional mutants of L. lactis, screened as exonuclease-deficient, affected a single locus and resulted in UV sensitivity and recombination deficiency. The cloned lactococcal genes (called rexAB) restored UV resistance, recombination proficiency, and the capacity to degrade linear DNA, to an E. coli recBCD mutant. In this context, DNA degradation is specifically blocked by the putative lactococcal Chi site (5'-GCGCGTG-3'), but not by the E. coli Chi (5'-GCTGGTGG-3') site. RexAB-mediated recombination was shown to be stimulated approximately 27-fold by lactococcal Chi. Our results reveal that RexAB fulfills the biological roles of RecBCD and indicate that its activity is modulated by a short DNA sequence. We speculate that exonuclease/recombinase enzymes whose activities are modulated by short DNA sequences are widespread among bacteria.

Base Sequence↗
PubMed1998

Efficient translesion replication in the absence of Escherichia coli Umu proteins and 3'-5' exonuclease proofreading function.

Translesion replication (TR) past a cyclobutane pyrimidine dimer in Escherichia coli normally requires the UmuD'2C complex, RecA protein, and DNA polymerase III holoenzyme (pol III). However, we find that efficient TR can occur in the absence of the Umu proteins if the 3'-5' exonuclease proofreading activity of the pol III epsilon-subunit also is disabled. TR was measured in isogenic uvrA6 DeltaumuDC strains carrying the dominant negative dnaQ allele, mutD5, or DeltadnaQ spq-2 mutations by transfecting them with single-stranded M13-based vectors containing a specifically located cis-syn T-T dimer. As expected, little TR was observed in the DeltaumuDC dnaQ+ strain. Surprisingly, 26% TR occurred in UV-irradiated DeltaumuDC mutD5 cells, one-half the frequency found in a uvrA6 umuDC+mutD5 strain. lexA3 (Ind-) derivatives of the strains showed that this TR was contingent on two inducible functions, one LexA-dependent, responsible for approximately 70% of the TR, and another LexA-independent, responsible for the remaining approximately 30%. Curiously, the DeltaumuDC DeltadnaQ spq-2 strain exhibited only the LexA-independent level of TR. The cause of this result appears to be the spq-2 allele, a dnaE mutation required for viability in DeltadnaQ strains, since introduction of spq-2 into the DeltaumuDC mutD5 strain also reduces the frequency of TR to the LexA-independent level. The molecular mechanism responsible for the LexA-independent TR is unknown but may be related to the UVM phenomenon [Palejwala, V. A., Wang, G. E., Murphy, H. S. & Humayun, M. Z. (1995) J. Bacteriol. 177, 6041-6048]. LexA-dependent TR does not result from the induction of pol II, since TR in the DeltaumuDC mutD5 strain is unchanged by introduction of a DeltapolB mutation.

Alleles↗
PubMed1998

The 30-kDa C-terminal domain of the RecB protein is critical for the nuclease activity, but not the helicase activity, of the RecBCD enzyme from Escherichia coli.

The RecBCD enzyme from Escherichia coli is an ATP-dependent helicase and an ATP-stimulated nuclease. The 3' --> 5' exonuclease activity on double-stranded DNA is suppressed when the enzyme encounters a recombinational hot spot, called chi (chi). We have prepared a RecB deletion mutant (RecB1-929) by using results of limited proteolysis experiments that indicated that the RecB subunit consists of two main domains. The RecB1-929 protein, comprising the 100-kDa N-terminal domain of RecB, is an ATP-dependent helicase and a single-stranded DNA-dependent ATPase. Reconstitution of RecB1-929 with RecC and RecD leads to processive unwinding of a linearized plasmid. However, the reconstituted RecB1-929CD enzyme has lost the single-strand endo- and exonuclease and the double-strand exonuclease activities of the RecBCD enzyme. These results show that the 30-kDa C-terminal domain of RecB has an important role in the nuclease activity of RecBCD. On the basis of these findings, we propose the RecB C-terminal domain swing model to explain RecBCD's transformation from a 3' --> 5' exonuclease to a helicase when it meets a chi site.

DNA Helicases↗
PubMed1999

Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3' exonuclease suggests separate mechanisms of endo-and exonucleolytic cleavage.

Efficient cellular DNA replication requires the activity of a 5'-3' exonuclease. These enzymes are able to hydrolyze DNA.DNA and RNA.DNA substrates exonucleolytically, and they are structure-specific endonucleases. The 5'-3' exonucleases are conserved in organisms as diverse as bacteriophage and mammals. Crystal structures of three representative enzymes identify two divalent-metal-binding sites typically separated by 8-10 A. Site-directed mutagenesis was used to investigate the roles of three lysine residues (K83, K196, and K215) situated near two metal-binding sites in bacteriophage T5 5'-3' exonuclease. Neither K196 nor K215 was essential for either the exo- or the endonuclease activity, but mutation of these residues increased the dissociation constant for the substrate from 5 nM to 200 nM (K196A) and 50 nM (K215A). Biochemical analysis demonstrated that K83 is absolutely required for exonucleolytic activity on single-stranded DNA but is not required for endonucleolytic cleavage of flap structures. Structural analysis of this mutant by x-ray crystallography showed no significant perturbations around the metal-binding sites in the active site. The wild-type protein has different pH optima for endonuclease and exonuclease activities. Taken together, these results suggest that different mechanisms for endo- and exonucleolytic hydrolysis are used by this multifunctional enzyme.

Amino Acid Sequence↗
PubMed1999

Comparison of the 5' nuclease activities of taq DNA polymerase and its isolated nuclease domain.

Many eubacterial DNA polymerases are bifunctional molecules having both polymerization (P) and 5' nuclease (N) activities, which are contained in separable domains. We previously showed that the DNA polymerase I of Thermus aquaticus (TaqNP) endonucleolytically cleaves DNA substrates, releasing unpaired 5' arms of bifurcated duplexes. Here, we compare the substrate specificities of TaqNP and the isolated 5' nuclease domain of this enzyme, TaqN. Both enzymes are significantly activated by primer oligonucleotides that are hybridized to the 3' arm of the bifurcation; optimal stimulation requires overlap of the 3' terminal nucleotide of the primer with the terminal base pair of the duplex, but the terminal nucleotide need not hybridize to the complementary strand in the substrate. In the presence of Mn2+ ions, TaqN can cleave both RNA and circular DNA at structural bifurcations. Certain anti-TaqNP mAbs block cleavage by one or both enzymes, whereas others can stimulate cleavage of nonoptimal substrates.

Base Sequence↗
PubMed1995

Role of the Escherichia coli recombination hotspot, chi, in RecABCD-dependent homologous pairing.

Genetic recombination occurring in wild type Escherichia coli is stimulated at DNA sequences known as chi sites, 5'-GCTGGTGG-3'. In vitro, homologous pairing between duplex DNA substrates dependent upon the RecA, RecBCD, and SSB proteins is stimulated by the presence of a chi sequence in the donor linear double-stranded DNA. We show that this stimulation is due to two factors: 1) the enhanced production of chi-specific single-stranded DNA fragments and 2) their preferential use in the RecA protein-promoted pairing step. Furthermore, under conditions of limiting Mg2+ concentration, joint molecule formation does not occur, even though DNA unwinding and chi-specific single-stranded DNA fragment production are observed. Also, under these conditions, chi-specific fragments derived from both the upstream and downstream regions of the DNA strand containing chi and from cleavage of the non-chi-containing DNA strand are detected. Finally, the behavior of mutant RecBCD enzymes (RecBC*D and RecBCD not equal to) in this in vitro reaction is shown to parallel their in vivo phenotypes with respect to chi stimulation of recombination. Thus we suggest that, in addition to its ability to regulate the degradative activities of RecBCD enzyme, chi itself may be a preferred site for initiation of homologous pairing in this concerted process.

DNA Helicases↗