Polarity of heteroduplex formation promoted by Escherichia coli recA protein.

When recA protein pairs circular single strands with linear duplex DNA, the circular strand displaces its homolog from only one end of the duplex molecule and rapidly creates heteroduplex joints that are thousands of base pairs long [DasGupta, C., Shibata, T., Cunningham, R. P. & Radding, C. M. (1980) Cell 22, 437-446]. To examine this apparently polar reaction, we prepared chimeric duplex fragments of DNA that had M13 nucleotide sequences at one end and G4 sequences at the other. Circular single strands homologous to M13 DNA paired with a chimeric fragment when M13 sequences were located at the 3' end of the complementary strand but did not pair when the M13 sequences were located at the 5' end. Likewise circular single-stranded G4 DNA paired with chimeric fragments only when G4 sequences were located at the 3' end of the complementary strand. To confirm these observations, we prepared fd DNA labeled only at the 5' or 3' end of the plus strand, and we examined the susceptibility of these labeled ends to digestion by exonucleases when joint molecules were formed. Eighty percent of the 5' label in joint molecules became sensitive to exonuclease VII. Displacement of that 5' end by recA protein was concerted because it did not occur in the absence of single-stranded DNA or in the presence of heterologous single strands. By contrast, only a small fraction of the 3' label became sensitive to exonuclease VII or exonuclease I. These observations show that recA protein forms heteroduplex joints in a concerted and polarized way.     

MECHANISM OF DNA CHAIN GROWTH, IV. DIRECTION OF SYNTHESIS OF T4 SHORT DNA CHAINS AS REVEALED BY EXONUCLEOLYTIC DEGRADATION

T4 nascent short chains labeled at their growing ends with H(3)-thymidine and uniformly with C(14)-thymidine were prepared, separated into complementary strands, and degraded by E. coli exonuclease I in the 3' to 5' direction or by B. subtilis nuclease in the 5' to 3' direction. The kinetics of release of H(3) and C(14) labels by both enzymes was consistent with the conclusion that the H(3) label is at the 3' end of the nascent short chains of both strands and that the short chains are products of discontinuous synthesis in the 5' to 3' direction along the two template strands.     

A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo.

2'-Deoxyadenosine 5'-O-(1-thiotriphosphate) (dATP[alpha S]) was introduced into the 3' ends of DNA restriction fragments with Escherichia coli DNA polymerase I to give phosphorothioate internucleotide linkages. Such "capped" 3' ends were found to be resistant to exonuclease III digestion. Moreover, the resistance to digestion is great enough that, under conditions used by us, just one strand of a double helix is digested by exonuclease III when a cap is placed at only one end; when digestion is carried to completion, this results in production of intact single strands. When digestion with exonuclease III is limited and is followed by S1 nuclease treatment, double-stranded DNA fragments asymmetrically shortened from just one side are produced. In this was thousands of nucleotides can be selectively removed from one end of a restriction fragment. In vitro introduction of phosphorothioate linkages into one end of a linearized replicative plasmid, followed by exonuclease III and S1 nuclease treatments, gives rise to truncated forms that, upon circularization by blunt-end ligation, transform E. coli and replicate in vivo.     

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.     

A Unique Form of Terminal Redundancy in Adenovirus DNA Molecules

A unique form of terminal redundancy has been observed in DNA molecules extracted from several human adenovirus serotypes. Electron microscopic studies reveal that single-stranded circular molecules are formed when native DNA is denatured and then annealed. Temperatures approaching the T(m) of native DNA are required to convert circles to linear molecules, indicating a high degree of self-complementarity between terminal base sequences of DNA strands. Single-stranded circles are not generated if a limited number of nucleotides (2-4%) are removed from the 3' ends of native DNA by digestion with Escherichia coli exonuclease III before denaturation and annealing. The lenght of the redundant segment appears to differ among major serotypic groups, and a possible association between increased length of the redundant segment and increased oncogenic capability of virus serotype is suggested. Evidence for the configuration of the duplex closure region of circular molecules is also presented.     

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.     

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.     

A protein subunit of human RNase P, Rpp14, and its interacting partner, OIP2, have 3'-->5' exoribonuclease activity

The processing of precursor tRNAs at their 5' and 3' termini is a fundamental event in the biosynthesis of tRNA. RNase P is generally responsible for endonucleolytic removal of a leader sequence of precursor tRNA to generate the mature 5' terminus. However, much less is known about the RNase P counterparts or other proteins that are active at the tRNA 3' terminus. Here we show that one of the human RNase P subunits, Rpp14, together with one of its interacting protein partners, OIP2, is a 3'-->5' exoribonuclease with a phosphorolytic activity that processes the 3' terminus of precursor tRNA. Immunoprecipitates of a crude human RNase P complex can process both ends of precursor tRNA by hydrolysis, but purified RNase P has no exonuclease activity. Rpp14 and OIP2 may be part of an exosome activity.     

Mechanism of Action of Ribonuclease H Isolated from Avian Myeloblastosis Virus and Escherichia coli

Purified preparations of RNA-dependent DNA polymerase isolated from avain myeloblastosis virus contain RNase H activity. Labeled ribohomopolymers are degraded in the presence of their complementary deoxyribopolymer, except [(3)H]poly(U).poly(dA). The degradation products formed from [(3)H]poly(A).poly(dT) were identified as oligonucleotides containing 3'-hydroxyl and 5'-phosphate termini, while AMP was not detected. The nuclease has been characterized as a processive exonuclease that requires ends of poly(A) chains for activity. Exonucleolytic attack occurs in both 5' to 3' and 3' to 5' directions.RNase H has also been purified from E. coli. This nuclease degrades all homoribopolymers tested in the presence of their complementary deoxyribopolymers to yield oligonucleotides with 5'-phosphate and 3'-hydroxyl termini. E. coli RNase H has been characterized as an endonuclease.     

Homologous recombination catalyzed by a nuclear extract from Xenopus oocytes.

Xenopus laevis oocytes efficiently recombine linear DNA injected into their nuclei (germinal vesicles). This process requires homologous sequences at or near the molecular ends. Here we report that a cell-free extract made from germinal vesicles is capable of accomplishing the complete recombination reaction in vitro. Like the in vivo process, the extract converts the overlapping ends of linear substrate molecules into covalently closed products. Establishment of this cell-free system has allowed examination of the cofactors required for recombination. The first step involves a 5'----3' exonuclease activity that requires a divalent cation but not NTPs. Completion of recombination requires a hydrolyzable NTP; maximal product formation occurs in the presence of millimolar levels of ATP or dATP. At submillimolar levels of all four dNTPs, homologous recombination is inefficient, and a side reaction produces end-joined products. This cell-free system should facilitate a step-by-step understanding of an homologous recombination pathway that operates not only in Xenopus laevis oocytes but also in cells from a wide variety of organisms.     

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.     

Eukaryotic DNA polymerase amino acid sequence required for 3'----5' exonuclease activity.

We have identified an amino-proximal sequence motif, Phe-Asp-Ile-Glu-Thr, in Saccharomyces cerevisiae DNA polymerase II that is almost identical to a sequence comprising part of the 3'----5' exonuclease active site of Escherichia coli DNA polymerase I. Similar motifs were identified by amino acid sequence alignment in related, aphidicolin-sensitive DNA polymerases possessing 3'----5' proofreading exonuclease activity. Substitution of Ala for the Asp and Glu residues in the motif reduced the exonuclease activity of partially purified DNA polymerase II at least 100-fold while preserving the polymerase activity. Yeast strains expressing the exonuclease-deficient DNA polymerase II had on average about a 22-fold increase in spontaneous mutation rate, consistent with a presumed proofreading role in vivo. In multiple amino acid sequence alignments of this and two other conserved motifs described previously, five residues of the 3'----5' exonuclease active site of E. coli DNA polymerase I appeared to be invariant in aphidicolin-sensitive DNA polymerases known to possess 3'----5' proofreading exonuclease activity. None of these residues, however, appeared to be identifiable in the catalytic subunits of human, yeast, or Drosophila alpha DNA polymerases.     

The major 5' end of HIV type 1 RNA corresponds to G456

In the course of studies on HIV-1 RNA structure, we determined that the main 5' end of viral RNA from virions and virus producer cells corresponds to G456 in the proviral DNA sequence, one or two nucleotides down-stream from the reported ends that correspond to G454 and G455. We mapped 5' ends using the highly accurate RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) method. The reactivity of the 5' ends indicates that they are mainly capped, although the presence of some uncapped (5'-triphosphorylated) RNA cannot formally be excluded. When we used a 5' mapping method susceptible to incorporating a cytosine at the 3' end of cDNA first strands, at a position templated by the 7-methylguanosine cap, 50% of clones derived from virion RNA had incorporated the additional cytosine. Reassignment of the 5' end has consequences for the design of short RNAs used to study HIV-1 RNA structural dynamics.     

A separate editing exonuclease for DNA replication: the epsilon subunit of Escherichia coli DNA polymerase III holoenzyme.

DNA polymerase III (polIII) holoenzyme of Escherichia coli has 3'----5' exonuclease ("editing") activity in addition to its polymerase activity, a property shared by other prokaryotic DNA polymerases. The polymerization activity is carried by the large alpha subunit, the product of the dnaE gene. Mutations affecting the fidelity of DNA replication in vivo and the activity of 3'----5' exonuclease assayed in vitro are found in the dnaQ gene, which specifies the epsilon subunit. To determine whether epsilon carries the 3'----5' exonuclease activity, we have used an overproduction protocol to purify epsilon separately from the other subunits of polIII holoenzyme. We find that epsilon has 3'----5' exonuclease activity indistinguishable from that of polIII core, the subassembly of polIII holoenzyme consisting of the alpha, epsilon, and theta subunits. We conclude that the editing and polymerization activities of polIII holoenzyme reside on distinct subunits, in contrast to DNA polymerase I of E. coli and DNA polymerase of phage T4. This functional separation may provide for regulation of exonucleolytic editing independently of polymerization, allowing cellular control of replication fidelity.     

Formation In Vitro of Infective Joint Molecules of λ DNA by T4 Gene-32 Protein

Half molecules of lambda DNA that had been partially digested by lambda exonuclease to expose homologous single strands were rejoined by the action of T4 gene-32 protein at 37 degrees in the presence of Mg(++). Measurements of infectivity in su(-) spheroplasts and sedimentation in sucrose demonstrated the formation of sus(+) joint molecules from two preparations of sheared lambda DNA that carried sus mutations at opposite ends of the genome. The biological activity of joint molecules made by annealing at 75 degrees was diminished by the addition of the gene-32 protein in the absence of Mg(++), and largely restored by the subsequent addition of Mg(++). The specific infectivity of joint molecules made by gene-32 protein at 37 degrees was similar to that of joint molecules made by annealing at 75 degrees . The experimental system described provides a possible model for simulating early steps in genetic recombination.     

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.     

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.     

RecA protein filaments can juxtapose DNA ends: an activity that may reflect a function in DNA repair.

To further characterize the role of RecA protein-DNA filaments in general recombination and DNA repair, we have examined interactions of these filaments with themselves following formation. When linear double-stranded DNA was incubated with RecA in the presence of Mg2+ and adenosine 5'-[gamma-thio]triphosphate, monomer-length (1n) nucleoprotein filaments were observed. Following continued incubation, filaments having 2n, 3n, ... lengths were observed, indicating that an end-to-end joining of the monomer-length filaments had occurred. When linear single-stranded DNA was covered by RecA protein under several conditions, the ends of the resulting filaments joined together rapidly, producing circular filaments. The end-to-end joining of single-stranded DNA-RecA filaments appeared to require that 3' DNA ends be juxtaposed with 5' DNA ends, because double-stranded DNA molecules having long single-stranded DNA tails with only 3' or 5' termini did not join end-to-end. However, when both 5' and 3' ends were present in the reaction, joining was observed. We suggest that this end-to-end joining activity may help explain the role of RecA protein in both the protection of damaged DNA ends and the repair of double-stranded DNA breaks.