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At least 361 records · Page 20Linked to original sources
PubMed1982

Orientation of cohesive end site cos determines the active orientation of chi sequence in stimulating recA . recBC-mediated recombination in phage lambda lytic infections.

Sequence chi, 5'G-C-T-G-G-T-G-G, locally enhances homologous recombination by recA and recBC proteins of Escherichia coli. Previous work showed that, in phage lambda, chi is more active in one orientation (leftward) than in the other (rightward). Inverting cos, the sequence for the mature DNA ends of lambda, reverses this orientation dependence: the rightward chi becomes fully active, and the leftward chi becomes relatively inactive. We surmise that chi action in phage lambda is coupled with DNA packaging or injection.

Bacterial Proteins↗
PubMed1984

A new class of Escherichia coli recBC mutants: implications for the role of RecBC enzyme in homologous recombination.

Mutants of Escherichia coli sensitive to phage T4 gene 2 mutants were obtained following ethyl methanesulfonate mutagenesis. By mapping and complementation analysis, the mutations in each of the six mutants are in recB and recC. By both in vivo and in vitro analyses, the nuclease activity of RecBC enzyme is undetectable in these mutants. However, by several other criteria, such as proficiency in recombination, relative resistance to UV radiation, and viability of the cells in the culture, these mutants are almost identical to their recBC+ parent. The properties of these mutants indicate that the ATP-dependent double-stranded DNA exonuclease activity of RecBC enzyme is not required for recombination. Chi recombinational hotspots, which stimulate recombination by the RecBC pathway, have no detectable activity in the mutants. This result suggests that the nuclease activity of RecBC enzyme is required for Chi activity and is consistent with the hypothesis that Chi stimulates recombination by directing RecBC enzyme to cut DNA at or near Chi.

Crosses, Genetic↗
PubMed1985

Mini-F plasmid-induced SOS signal in Escherichia coli is RecBC dependent.

Dispensable replicons such as F plasmid [95 kilobases (kb)] or its mini-derivatives such as mini-F (9.3 kb) or lambda mini-F efficiently induced cellular SOS genes such as sfiA (sulA) when they were damaged by UV irradiation and then introduced into a recipient bacterium. To generate an SOS signal, UV light-damaged mini-F or mini-F conditional mutants deficient in replication required that the bacterial RecBC enzyme retained some activity different from the nuclease activity that was dispensable. In contrast, UV light-damaged F plasmid produced an SOS signal independently of the activity of the RecBC enzyme and of the expression of the mini-F, -H, and -G proteins. Our findings are consistent with a picture in which the SOS signal is constituted by stretches of single-stranded DNA on a replicon. Moreover, our present data combined with other data previously published lead to the hypothesis that the SOS signal induced by mini-F plasmid is located in trans on the host chromosome, whereas the one generated by UV light-damaged F plasmid is in cis on the transferred DNA.

Chromosomes, Bacterial↗
PubMed1986

A phage P1 function that stimulates homologous recombination of the Escherichia coli chromosome.

Recombination between two different defective lacZ genes in the Escherichia coli chromosome (lac- X lac- recombination) was stimulated 2- to 8-fold by prophage P1, depending on the nature of the phage c1 repressor. The P1 BamHI restriction fragment B8 in a lambda-P1:B8 hybrid phage, stimulated lac- X lac- recombination 90-fold in the absence of P1 repressor. A gene necessary for recombination enhancement, designated ref, was localized to one end of B8. Ref expression from lambda-P1:B8 was repressed in trans by a P1 c+ prophage. Two P1 regulatory mutations, bof and lxc, derepressed prophage expression of ref and depressed a prophage function that complemented an E. coli mutant (ssb) deficient in the single-stranded DNA binding protein. Ref stimulation was dependent on preexisting E. coli recombination functions (RecA-RecBC and RecA-RecF). However, other (phage and plasmid) recombination processes involving these functions were not stimulated. ref::Tn5 phages plated and formed lysogens normally. Thus ref appears to be an integral, but not essential, phage gene that stimulates recombination of the host chromosome specifically.

Bacterial Proteins↗
PubMed1986

recD: the gene for an essential third subunit of exonuclease V.

Exonuclease V (EC 3.1.11.5) of Escherichia coli, an enzyme with multiple activities promoting genetic recombination, has previously been shown to contain two polypeptides, the products of the recB and recC genes. We report here that the enzyme contains in addition a third polypeptide (alpha) with a molecular mass of about 58 kDa. The alpha polypeptide is not synthesized by a class of mutants (previously designated recB) lacking the nuclease activity of exonuclease V but retaining recombination proficiency. The gene, recD, coding for the alpha polypeptide is located near recB in the order thyA-recC-ptr-recB-recD-argA on the E. coli chromosome. The recB and recD genes appear to be governed by a common promoter to the left of recB; a weaker promoter appears to govern recD alone. In the light of these results we discuss the relation between the structure and function of the three polypeptides of exonuclease V, hereby alternatively designated RecBCD enzyme.

Bacterial Proteins↗
PubMed1987

A cryptic proofreading 3'----5' exonuclease associated with the polymerase subunit of the DNA polymerase-primase from Drosophila melanogaster.

The DNA polymerase-primase from Drosophila lacks 3'----5' exonuclease activity. However, a potent exonuclease can be detected after separating the 182-kDa polymerase subunit from the other three subunits of the enzyme (73, 60, and 50 kDa) by glycerol gradient sedimentation in the presence of 50% ethylene glycol. The exonuclease activity cosediments with the polymerase subunit, suggesting that the two activities reside in the same polypeptide. The 3'----5' exonuclease excises mismatched bases at the 3' termini of primed synthetic and natural DNA templates. Excision of a mispaired base at the 3' terminus occurs at a 10-fold greater rate than excision of the correctly paired base. When replication fidelity is measured by the bacteriophage phi X174 am3 reversion assay, the isolated polymerase subunit is at least 100-fold more accurate than either the intact polymerase-primase or a complex of the 182- and 73-kDa subunits. These results suggest that the 3'----5' exonuclease functions as a proofreading enzyme during Drosophila DNA replication in vitro and very likely in vivo.

Animals↗
PubMed1987

Large palindromes in the lambda phage genome are preserved in a rec+ host by inhibiting lambda DNA replication.

A large palindrome carried by phage lambda has been shown to prevent growth of the phage on a rec+ strain of Escherichia coli. The phage do form plaques on recBC sbcB strains, but the palindrome is not stable--deletions that either destroy the palindrome or diminish its size overgrow the original engineered palindrome-containing phage. We have prepared stocks of lambda carrying a palindrome that is 2 X 4200 base pairs long. These phage stocks are produced by induction of a lysogen in which the two halves of the palindrome are stored at opposite ends of the prophage and are of sufficient titer (10(9) phage per ml) to enable one-step growth experiments with replication-blocked phage. We find that the large palindrome as well as a lesser palindrome of 2 X 265 base pairs are recovered intact among particles carrying unreplicated chromosomes following such an infection of a rec+ host. We propose that DNA replication drives the extrusion of palindromic sequences in vivo, forming secondary structures that are substrates for the recBC and sbcB gene products.

Bacteriophage lambda↗
PubMed1989

Studies on the DNA elongation inhibitor and its proliferating cell nuclear antigen-dependent control in simian virus 40 DNA replication in vitro.

A 120-kDa protein that blocks DNA termini has been purified from extracts of HeLa cells. This protein inhibits the action of a number of enzymes that catalyze reactions involving the 5' and 3' ends of DNA (DNA ligase, 3' and 5' exonucleases, and DNA polymerase alpha). The 120-kDa protein blocks the synthesis of long DNA chains that are normally formed during simian virus 40 DNA replication, causing the accumulation of small DNA fragments. The effects of this protein can be reversed by the addition of proliferating cell nuclear antigen and other protein fractions (activators).

Antigens, Neoplasm↗
PubMed1989

Mismatch-specific 3'----5' exonuclease associated with the mitochondrial DNA polymerase from Drosophila embryos.

The mitochondrial DNA polymerase from Drosophila embryos lacks dNTP turnover activity. However, a potent 3'----5' exonuclease activity can be detected by a specific assay in which the exonuclease excises mispaired nucleotides at the 3' termini of primed synthetic and natural DNA templates. The excision of a mispaired nucleotide occurs at a significantly greater rate than excision of a correctly paired nucleotide and, under conditions of DNA synthesis, hydrolysis of a mispaired terminal nucleotide occurs prior to primer extension. The 3'----5' exonuclease copurifies quantitatively with DNA polymerase gamma and cosediments with the nearly homogeneous enzyme under native conditions. These results suggest that the 3'----5' exonuclease provides a proofreading function to enhance the fidelity of DNA synthesis during Drosophila mitochondrial DNA replication.

Animals↗
PubMed1989

Strand specificity of DNA unwinding by RecBCD enzyme.

RecBCD enzyme (exonuclease V) of Escherichia coli unwinds DNA, frequently forming asymmetric structures with two single-stranded tails of unequal length abutting a single-stranded loop at the junction with double-stranded DNA. Their lengths are consistent with the longer tail being one strand of the duplex and the loop plus the shorter tail being the other strand. The strand polarity of the unwinding was determined by labeling the 3' or 5' ends of duplex DNA with biotinylated nucleotides, reacting the DNA with RecBCD enzyme, and distinguishing the labeled ends, in the electron microscope, by their binding to streptavidin-gold complex. The shorter tail was formed from the DNA strand with its 3' terminus at the duplex end where RecBCD enzyme entered. We conclude that RecBCD enzyme unwinds DNA by forming a loop on the strand with a 3' end at the entry point. This result is concordant with a previously proposed model of recombination, which we discuss.

Base Sequence↗
PubMed1989

Proofreading by the epsilon subunit of Escherichia coli DNA polymerase III increases the fidelity of calf thymus DNA polymerase alpha.

Addition of the 3'----5' proofreading exonuclease, epsilon subunit of Escherichia coli DNA polymerase III, to DNA polymerase alpha from calf thymus has been studied. Alone, calf thymus DNA polymerase alpha terminates in vitro DNA synthesis upon insertion of noncomplementary nucleotides. Upon addition of the epsilon subunit, DNA polymerase alpha elongates the newly synthesized DNA as a result of hydrolysis of the 3'-terminal mispair. The fidelity of DNA polymerase alpha in vitro is increased 7-fold by addition of the exonuclease. The functional interaction between DNA polymerase alpha and the epsilon subunit is independent of any detectable physical association. This suggests that a mechanism for proofreading could exist in mammalian cells involving sequential catalysis by DNA polymerase alpha excision of errors by a separate 3'----5' exonuclease, and further elongation onto correctly base-paired 3' termini by DNA polymerase alpha.

Animals↗
PubMed1990

The highly conserved amino acid sequence motif Tyr-Gly-Asp-Thr-Asp-Ser in alpha-like DNA polymerases is required by phage phi 29 DNA polymerase for protein-primed initiation and polymerization.

The alpha-like DNA polymerases from bacteriophage phi 29 and other viruses, prokaryotes and eukaryotes contain an amino acid consensus sequence that has been proposed to form part of the dNTP binding site. We have used site-directed mutants to study five of the six highly conserved consecutive amino acids corresponding to the most conserved C-terminal segment (Tyr-Gly-Asp-Thr-Asp-Ser). Our results indicate that in phi 29 DNA polymerase this consensus sequence, although irrelevant for the 3'----5' exonuclease activity, is essential for initiation and elongation. Based on these results and on its homology with known or putative metal-binding amino acid sequences, we propose that in phi 29 DNA polymerase the Tyr-Gly-Asp-Thr-Asp-Ser consensus motif is part of the dNTP binding site, involved in the synthetic activities of the polymerase (i.e., initiation and polymerization), and that it is involved particularly in the metal binding associated with the dNTP site.

Amino Acid Sequence↗
PubMed1991

Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase.

The 5'----3' exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase may be employed in a polymerase chain reaction product detection system to generate a specific detectable signal concomitantly with amplification. An oligonucleotide probe, nonextendable at the 3' end, labeled at the 5' end, and designed to hybridize within the target sequence, is introduced into the polymerase chain reaction assay. Annealing of probe to one of the polymerase chain reaction product strands during the course of amplification generates a substrate suitable for exonuclease activity. During amplification, the 5'----3' exonuclease activity of T. aquaticus DNA polymerase degrades the probe into smaller fragments that can be differentiated from undegraded probe. The assay is sensitive and specific and is a significant improvement over more cumbersome detection methods.

Base Sequence↗
PubMed1991

DNA polymerization in the absence of exonucleolytic proofreading: in vivo and in vitro studies.

Classical genetic selection was combined with site-directed mutagenesis to study bacteriophage T4 DNA polymerase 3'----5' exonuclease activity. A mutant DNA polymerase with very little (less than or equal to 1%) 3'----5' exonuclease activity was generated. In vivo, the 3'----5' exonuclease-deficient DNA polymerase produced the highest level of spontaneous mutation observed in T4, 500- to 1800-fold above that of wild type. The large reduction in 3'----5' exonuclease activity appears to be due to two amino acid substitutions: Glu-191 to Ala and Asp-324 to Gly. Protein sequence similarities have been observed between sequences in the Escherichia coli DNA polymerase I 3'----5' exonuclease domain and conserved sequences in eukaryotic, viral, and phage DNA polymerases. It has been proposed that the conserved sequences contain metal ion binding ligands that are required for 3'----5' exonuclease activity; however, we find that some proposed T4 DNA polymerase metal binding residues are not essential for 3'----5' exonuclease activity. Thus, our T4 DNA polymerase studies do not support the hypothesis by Bernad et al. [Bernad, A., Blanco, L., Lazaro, J.M., Martin, G. & Salas, M. (1989) Cell 59, 219-228] that many DNA polymerases, including T4 DNA polymerase, share an extensively conserved 3'----5' exonuclease motif. Therefore, extrapolation from E. coli DNA polymerase I sequence and structure to other DNA polymerases for which there is no structural information may not be valid.

Amino Acid Sequence↗
PubMed1991

RecBCD-dependent joint molecule formation promoted by the Escherichia coli RecA and SSB proteins.

We describe the formation of homologously paired joint molecules in an in vitro reaction that is dependent on the concerted actions of purified RecA and RecBCD proteins and is stimulated by single-stranded DNA-binding protein (SSB). RecBCD enzyme initiates the process by unwinding the linear double-stranded DNA to produce single-stranded DNA, which is trapped by SSB and RecA. RecA uses this single-stranded DNA to catalyze the invasion of a supercoiled double-stranded DNA molecule, forming a homologously paired joint molecule. At low RecBCD enzyme concentrations, the rate-limiting step is the unwinding of duplex DNA by RecBCD, whereas at higher RecBCD concentrations, the rate-limiting step is RecA-catalyzed strand invasion. The behavior of mutant RecA proteins in this in vitro reaction parallels their in vivo phenotypes, suggesting that this reaction may define biochemical steps that occur during homologous recombination by the RecBCD pathway in vivo.

Adenosine Triphosphate↗
PubMed1992

RecBCD enzyme is altered upon cutting DNA at a chi recombination hotspot.

During its unidirectional unwinding of DNA, RecBCD enzyme cuts one DNA strand near a properly oriented Chi site, a hotspot of homologous genetic recombination in Escherichia coli. We report here that individual DNA molecules containing two properly oriented Chi sites were cut with about 40% efficiency at one or the other Chi site but not detectably at both Chi sites. Furthermore, initial incubation of RecBCD with Chi-containing DNA reduced its ability both to unwind DNA and to cut at Chi sites on subsequently added DNA molecules much more than did initial incubation with Chi-free DNA; the nuclease activity was less severely affected. These results imply that RecBCD loses its Chi-cutting activity upon cutting at a single Chi site and provide a mechanism for ensuring single genetic exchanges near the ends of DNA molecules.

Bacteriophage lambda↗
PubMed1992

Chi sequence protects against RecBCD degradation of DNA in vivo.

RecBCD is a multifunctional enzyme involved in DNA degradation and homologous recombination. It also produces an endonucleolytic cleavage near properly oriented chi sites (5'-GCTGGTGG-3'). Plasmids are not known to be affected by either RecBCD enzyme or the presence of a chi site. We report here that plasmids that replicate by a rolling circle mechanism accumulate large amounts of high molecular weight linear multimers (HMW), either if they contain a chi site or if RecBCD is absent. An in vivo inducible system for rolling circle replication was constructed to study RecBCD and its interactions with chi. Results show that (i) HMW accumulation is chi orientation dependent, and (ii) a succession of chi sites prevents degradation of HMW by RecBCD enzyme. These results demonstrate chi activity in plasmids. The rolling circle mechanism produces a sigma structure during plasmid replication; we propose that the double-stranded DNA tail of this sigma form allows RecBCD entry; the tail is degraded unless it is protected by a chi site. By analogy, a principal role of chi in the survival of lambda red-gam- mutants in wild-type strains may be to protect rolling circle concatemers (in late replication) from degradation by RecBCD.

Base Sequence↗
PubMed1993

Construction and characterization of a bacteriophage T4 DNA polymerase deficient in 3'-->5' exonuclease activity.

Bacteriophage T4 DNA polymerase has a proofreading 3'-->5' exonuclease that plays an important role in maintaining the accuracy of DNA replication. We have constructed a T4 DNA polymerase deficient in this exonuclease by converting Asp-219 to Ala. The exonuclease activity of the mutant T4 DNA polymerase has been reduced by a factor of at least 10(7), but it retains a polymerase activity whose kinetic parameters, kcat, Kd DNA, and Kd dATP, are very close to those of the wild-type enzyme. Bacteriophage T4 with the mutant polymerase gene has a markedly increased mutation frequency. Asp-219 in T4 DNA polymerase is within a sequence similar to those surrounding Asp residues previously shown to be essential for the exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I (Asp-424), bacteriophage phi 29 DNA polymerase (Asp-66), and Saccharomyces cerevisiae DNA polymerase delta (Asp-405). Thus, these studies support the proposal that there are similar sequences in the active sites for the proofreading exonucleases of these and related DNA polymerases.

Amino Acid Sequence↗