The premature ageing syndrome protein, WRN, is a 3'-->5' exonuclease.
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DNA↗
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Viability of lambda phages carrying a perfect palindrome in the absence of recombination nucleases.
In Escherichia coli in vitro constructions of perfect palindromes larger than 30 base pairs (bp) long have in general been unstable. A perfect palindrome has the unique possibility of forming a cruciform structure, and it is this feature which probably results in its instability. Negative supercoiling favours the formation of the cruciform conformation, which in turn causes the molecule to relax. This relaxation may render replicons containing large perfect palindromes inviable. An alternative hypothesis for inviability has been that the cruciform interferes with replication by favouring strand switching by polymerase I. Here we show that the simultaneous absence of two recombination nucleases, the recBC product, exonuclease V, and the sbcB product, exonuclease I, confers viability on a derivative of phage lambda carrying a perfect palindrome of inverted repeat length 1,600 bases. This observation suggests a third hypothesis--that nucleolytic cleavage of the cruciform is responsible for the inviability of the phage. Such an activity has been shown in vitro for T4 exonuclease VII.
Bacteriophage lambda↗
A conserved NTP-motif in putative helicases.
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Amino Acid Sequence↗
Translocation step size and mechanism of the RecBC DNA helicase.
DNA helicases are ubiquitous enzymes that unwind double-stranded DNA. They are a diverse group of proteins that move in a linear fashion along a one-dimensional polymer lattice--DNA--by using a mechanism that couples nucleoside triphosphate hydrolysis to both translocation and double-stranded DNA unwinding to produce separate strands of DNA. The RecBC enzyme is a processive DNA helicase that functions in homologous recombination in Escherichia coli; it unwinds up to 6,250 base pairs per binding event and hydrolyses slightly more than one ATP molecule per base pair unwound. Here we show, by using a series of gapped oligonucleotide substrates, that this enzyme translocates along only one strand of duplex DNA in the 3'-->5' direction. The translocating enzyme will traverse, or 'step' across, single-stranded DNA gaps in defined steps that are 23 (+/-2) nucleotides in length. This step is much larger than the amount of double-stranded DNA that can be unwound using the free energy derived from hydrolysis of one molecule of ATP, implying that translocation and DNA unwinding are separate events. We propose that the RecBC enzyme both translocates and unwinds by a quantized, two-step, inchworm-like mechanism that may have parallels for translocation by other linear motor proteins.
DNA Helicases↗
Chi-sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules.
Major pathways of recombinational DNA repair in Escherichia coli require the RecBCD protein--a heterotrimeric, ATP-driven, DNA translocating motor enzyme. RecBCD combines a highly processive and exceptionally fast helicase (DNA-unwinding) activity with a strand-specific nuclease (DNA-cleaving) activity (refs 1, 2 and references therein). Recognition of the DNA sequence 'chi' (5'-GCTGGTGG-3') switches the polarity of DNA cleavage and stimulates recombination at nearby sequences in vivo. Here we attach microscopic polystyrene beads to biotin-tagged RecD protein subunits and use tethered-particle light microscopy to observe translocation of single RecBCD molecules (with a precision of up to approximately 30 nm at 2 Hz) and to examine the mechanism by which chi modifies enzyme activity. Observed translocation is unidirectional, with each molecule moving at a constant velocity corresponding to the population-average DNA unwinding rate. These observations place strong constraints on possible movement mechanisms. Bead release at chi is negligible, showing that the activity modification at chi does not require ejection of the RecD subunit from the enzyme as previously proposed; modification may occur through an unusual, pure conformational switch mechanism.
Adenosine Triphosphatases↗
Processive translocation and DNA unwinding by individual RecBCD enzyme molecules.
RecBCD enzyme is a processive DNA helicase and nuclease that participates in the repair of chromosomal DNA through homologous recombination. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme. Here we show that unwinding is both continuous and processive, occurring at a maximum rate of 972 +/- 172 base pairs per second (0.30 microm s(-1)), with as many as 42,300 base pairs of dsDNA unwound by a single RecBCD enzyme molecule. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.
Adenosine Triphosphate↗
RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes.
Extensive work on the maturation of lagging strands during the replication of simian virus 40 DNA suggests that the initiator RNA primers of Okazaki fragments are removed by the combined action of two nucleases, RNase HI and Fen1, before the Okazaki fragments join. Despite the well established in vitro roles of these two enzymes, genetic analyses in yeast revealed that null mutants of RNase HI and/or Fen1 are not lethal, suggesting that an additional enzymatic activity may be required for the removal of RNA. One such enzyme is the Saccharomyces cerevisiae Dna2 helicase/endonuclease, which is essential for cell viability and is well suited to removing RNA primers of Okazaki fragments. In addition, Dna2 interacts genetically and physically with several proteins involved in the elongation or maturation of Okazaki fragments. Here we show that the endonucleases Dna2 and Fen1 act sequentially to facilitate the complete removal of the primer RNA. The sequential action of these enzymes is governed by a single-stranded DNA-binding protein, replication protein-A (RPA). Our results demonstrate that the processing of Okazaki fragments in eukaryotes differs significantly from, and is more complicated than, that occurring in prokaryotes. We propose a novel biochemical mechanism for the maturation of eukaryotic Okazaki fragments.
Adenosine Triphosphatases↗
Recombineering: a powerful new tool for mouse functional genomics.
Highly efficient phage-based Escherichia coli homologous recombination systems have recently been developed that enable genomic DNA in bacterial artificial chromosomes to be modified and subcloned, without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombinogenic engineering or recombineering, is efficient and greatly decreases the time it takes to create transgenic mouse models by traditional means. Recombineering also facilitates many kinds of genomic experiment that have otherwise been difficult to carry out, and should enhance functional genomic studies by providing better mouse models and a more refined genetic analysis of the mouse genome.
Animals↗
Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes.
Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.
Archaeoglobus fulgidus↗
RecBCD enzyme is a bipolar DNA helicase.
Escherichia coli RecBCD is a heterotrimeric helicase/nuclease that catalyses a complex reaction in which double-strand breaks in DNA are processed for repair by homologous recombination. For some time it has been clear that the RecB subunit possesses a 3' --> 5' DNA helicase activity, which was thought to drive DNA translocation and unwinding in the RecBCD holoenzyme. Here we show that purified RecD protein is also a DNA helicase, but one that possesses a 5' --> 3' polarity. We also show that the RecB and RecD helicases are both active in intact RecBCD, because the enzyme remains capable of processive DNA unwinding when either of these subunits is inactivated by mutation. These findings point to a bipolar translocation model for RecBCD in which the two DNA helicases are complementary, travelling with opposite polarities, but in the same direction, on each strand of the antiparallel DNA duplex. This bipolar motor organization helps to explain various biochemical properties of RecBCD, notably its exceptionally high speed and processivity, and offers a mechanistic insight into aspects of RecBCD function.
Adenosine Triphosphatases↗
RecBCD enzyme is a DNA helicase with fast and slow motors of opposite polarity.
Helicases are molecular motors that move along and unwind double-stranded nucleic acids. RecBCD enzyme is a complex helicase and nuclease, essential for the major pathway of homologous recombination and DNA repair in Escherichia coli. It has sets of helicase motifs in both RecB and RecD, two of its three subunits. This rapid, highly processive enzyme unwinds DNA in an unusual manner: the 5'-ended strand forms a long single-stranded tail, whereas the 3'-ended strand forms an ever-growing single-stranded loop and short single-stranded tail. Here we show by electron microscopy of individual molecules that RecD is a fast helicase acting on the 5'-ended strand and RecB is a slow helicase acting on the 3'-ended strand on which the single-stranded loop accumulates. Mutational inactivation of the helicase domain in RecB or in RecD, or removal of the RecD subunit, altered the rates of unwinding or the types of structure produced, or both. This dual-helicase mechanism explains how the looped recombination intermediates are generated and may serve as a general model for highly processive travelling machines with two active motors, such as other helicases and kinesins.
DNA↗
Towards genetic genome projects: genomic library screening and gene-targeting vector construction in a single step.
We have developed technologies that simplify genomic library construction and screening, substantially reducing both the time and the cost associated with traditional library screening methods and facilitating the generation of gene-targeting constructs. By taking advantage of homologous recombination in Escherichia coli, we were able to use as little as 80 bp of total sequence homology to screen for a specific gene from a genomic library in plasmid or phage form. This method, called recombination cloning (REC), takes only a few days instead of the several weeks required for traditional plaque-lift methods. In addition, because every clone in the mouse genomic library we have constructed has a negative selection marker adjacent to the genomic insert, REC screening can generate gene-targeting vectors in one step, from library screening to finished construct. Conditional targeting constructs can be generated easily with minimal additional manipulation.
Animals↗
The 3' 5' exonucleases.
Over the past few years, several new 3' 5' exonucleases have been identified. In vitro studies of these enzymes have uncovered much about their potential functions in vivo, and certain organisms with a defect in 3' 5' exonucleases have an increased susceptibility to cancer, especially under conditions of stress. Here, we look at not only the newly discovered enzymes, but also at the roles of other 3' 5' exonucleases in the quality control of DNA synthesis, where they act as proofreading exonucleases for DNA polymerases during DNA replication, repair and recombination.
Amino Acid Sequence↗
Dual engines moving on antiparallel tracks.
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DNA, Bacterial↗
Homologous regions of Fen1 and p21Cip1 compete for binding to the same site on PCNA: a potential mechanism to co-ordinate DNA replication and repair.
Following genomic damage, the cessation of DNA replication is co-ordinated with onset of DNA repair; this co-ordination is essential to avoid mutation and genomic instability. To investigate these phenomena, we have analysed proteins that interact with PCNA, which is required for both DNA replication and repair. One such protein is p21Cip1, which inhibits DNA replication through its interaction with PCNA, while allowing repair to continue. We have identified an interaction between PCNA and the structure specific nuclease, Fen1, which is involved in DNA replication. Deletion analysis suggests that p21Cip1 and Fen1 bind to the same region of PCNA. Within Fen1 and its homologues a small region (10 amino acids) is sufficient for PCNA binding, which contains an 8 amino acid conserved PCNA-binding motif. This motif shares critical residues with the PCNA-binding region of p21Cip1. A PCNA binding peptide from p21Cip1 competes with Fen1 peptides for binding to PCNA, disrupts the Fen1-PCNA complex in replicating cell extracts, and concomitantly inhibits DNA synthesis. Competition between homologous regions of Fen1 and p21Cip1 for binding to the same site on PCNA may provide a mechanism to co-ordinate the functions of PCNA in DNA replication and repair.
Amino Acid Sequence↗
Excision of mismatched nucleotides from DNA: a potential mechanism for enhancing DNA replication fidelity by the wild-type p53 protein.
The tumor suppressor p53 plays a critical role in the regulation of the cell cycle and the maintenance of genetic stability. The 3'-->5' exonuclease activity of p53 has recently been recognized as a novel biochemical function of this molecule, but the biological significance of this activity remains elusive. Using an in vitro DNA replication assay with purified human DNA polymerases, p53 protein, and defined DNA primer/templates, we demonstrated that the wild-type (wt) p53 protein, but not the mutant p53 protein, specifically enhanced the DNA replication fidelity of polymerase (pol) alpha, an enzyme that lacks 3'-->5' exonuclease activity. The misincorporation of non-complementary deoxynucleotides into DNA by pol alpha was substantially decreased by p53. In contrast, wt p53 showed no significant effect on replication fidelity or the rates of DNA synthesis by human pol epsilon or the bacterial enzyme pol I. Inhibition of 3'-->5' exonuclease activity by guanosine monophosphate (GMP) abolished the ability of p53 to enhance the replication fidelity of pol alpha. Quantitative analyses revealed that the 3'-->5' exonuclease activity of p53 effectively removed mismatched nucleotides from DNA in preference over matched nucleotides. Furthermore, study in intact cells revealed that the wt p53 protein was co-localized with DNA synthesis activity in S phase cells. These results suggest a possibility that the 3'-->5' exonuclease of the wt p53 protein might provide the proof-reading function for DNA pol alpha. The preferential excision of mismatched nucleotides from the replicating DNA strand appears to be a potential biochemical mechanism by which p53 enhances DNA replication fidelity and thereby helps to maintain genomic integrity.
Cell Nucleus↗
Substrate specificity of the p53-associated 3'-5' exonuclease.
p53 exhibits 3'-5' exonuclease activity and the significance of this biochemical function is currently not defined. In order to gain information about the potential role(s) of this exonuclease activity, recombinant and wild-type human p53 was examined for excision of nucleotides from defined synthetic DNA substrates. p53 removes nucleotides threefold faster from single-strand DNA than from DNA duplexes, exhibits a 1.5-fold preference for 3'-terminals of DNA that contain a single nucleotide mispair (mismatch) as compared to correctly paired DNA and efficiently excises nucleotides from 3'-ends of blunt and cohesive (staggered) DNA double-strand breaks. The p53 exonuclease is predominantly non-processive on DNA which is 17 nucleotides long (or shorter) and processive on the longer 30-mers. The processivity of nucleotide excision is decreased in the presence of 50 mM potassium phosphate and eliminated when full-length p53 is replaced with the core domain, comprised of amino acids 82-292. Photoaffinity labeling indicates that (1) p53 monomers, rather than dimers, bind to single-strand forms of these oligomers; (2) complexes between p53 and 30-mers are more stable than those formed with 17-mers. The stability of these complexes determines processivity during nucleotide removal and modulates the 3'-5' exonuclease activity of p53. The relevance of substrate specificity of the p53 exonuclease to DNA repair is discussed.
Animals↗
p53 enhances the fidelity of DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase.
The tumor suppressor protein p53 plays a critical role in the maintenance of genetic integrity. p53 possesses 3'-->5' exonuclease activity, however, the significance of this function in DNA replication process remains elusive. It was suggested that 3'-->5' exonuclease activity of p53 may provide a proofreading function for DNA polymerases. In order to better understand the significance of this activity, the purified wild-type recombinant p53 was further evaluated for substrate specificity and for contribution to the accuracy of DNA synthesis. p53-associated 3'-->5' exonuclease displays 3' terminal nucleotide excision from RNA/DNA template-primer using ribosomal RNA as a template. The data demonstrate that p53 is highly efficient in removing a terminal mispair. Analysis of mispair excision opposite the template adenine residue shows that p53 catalyzes 3' terminal mismatch excision with a specificity of A : G>A : A>A : C. Hence, the observed specificity of mismatch excision indicates that p53 exonucleolytic proofreading preferentially repairs transversion mutations. The influence of the p53 on the accuracy of DNA synthesis was determined with exonuclease-deficient human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT), a key enzyme in the life cycle of the virus, that contributes significantly to the low accuracy of proviral DNA synthesis. Using an in vitro biochemical assay with recombinant purified HIV-1 RT, p53 and defined RNA/DNA or DNA/DNA template-primers, two basic features related to fidelity of DNA synthesis were studied: the misinsertion and mispair extension. The misincorporation of non-complementary deoxynucleotides into nascent DNA and subsequent mispair extension by HIV-1 RT were substantially decreased in the presence of p53 with both RNA/DNA and DNA/DNA template-primers. In addition, the productive interaction between polymerization (by HIV-1 RT) and exonuclease (by p53) activities was observed; p53 preferentially hydrolyzes mispaired 3'-termini, permitting subsequent extension of the correctly paired 3'-terminus by HIV-1 RT. Taken together the data demonstrate that preferential excision of mismatched nucleotides by 3'-->5' exonuclease activity of wild-type p53 enhances the fidelity of DNA synthesis by HIV-1 RT in vitro, thus providing a biochemical mechanism to reduce mutations caused by incorporation of mismatched nucleotides. The fact that p53 is reactive with both RNA/DNA and DNA/DNA template-primers raises an interesting possibility of the existence of functional cooperation between p53 and HIV-1 RT in cytoplasm during the reverse transcription process, which may be important for maintaining HIV genomic integrity.
Base Pair Mismatch↗