Single-stranded DNA Exonuclease Research Articles

Role for DNA double strand end-resection activity of RecBCD in control of aberrant chromosomal replication initiation in Escherichia coli.

Replication of the circular bacterial chromosome is initiated from a locus oriC with the aid of an essential protein DnaA. One approach to identify factors acting to prevent aberrant oriC-independent replication initiation in Escherichia coli has been that to obtain mutants which survive loss of DnaA. Here, we show that a ΔrecD mutation, associated with attenuation of RecBCD’s DNA double strand end-resection activity, provokes abnormal replication and rescues ΔdnaA lethality in two situations: (i) in absence of 5′-3′ single-strand DNA exonuclease RecJ, or (ii) when multiple two-ended DNA double strand breaks (DSBs) are generated either by I-SceI endonucleolytic cleavages or by radiomimetic agents phleomycin or bleomycin. One-ended DSBs in the ΔrecD mutant did not rescue ΔdnaA lethality. With two-ended DSBs in the ΔrecD strain, ΔdnaA viability was retained even after linearization of the chromosome. Data from genome-wide DNA copy number determinations in ΔdnaA-rescued cells lead us to propose a model that nuclease-mediated DNA resection activity of RecBCD is critical for prevention of a σ-mode of rolling-circle over-replication when convergent replication forks merge and fuse, as may be expected to occur during normal replication at the chromosomal terminus region or during repair of two-ended DSBs following ‘ends-in’ replication.

Expansion of CAG repeats in Escherichia coli is controlled by single-strand DNA exonucleases of both polarities.

The expansion of CAG·CTG repeat tracts is responsible for several neurodegenerative diseases, including Huntington disease and myotonic dystrophy. Understanding the molecular mechanism of CAG·CTG repeat tract expansion is therefore important if we are to develop medical interventions limiting expansion rates. Escherichia coli provides a simple and tractable model system to understand the fundamental properties of these DNA sequences, with the potential to suggest pathways that might be conserved in humans or to highlight differences in behavior that could signal the existence of human-specific factors affecting repeat array processing. We have addressed the genetics of CAG·CTG repeat expansion in E. coli and shown that these repeat arrays expand via an orientation-independent mechanism that contrasts with the orientation dependence of CAG·CTG repeat tract contraction. The helicase Rep contributes to the orientation dependence of repeat tract contraction and limits repeat tract expansion in both orientations. However, RuvAB-dependent fork reversal, which occurs in a rep mutant, is not responsible for the observed increase in expansions. The frequency of repeat tract expansion is controlled by both the 5′–3′ exonuclease RecJ and the 3′–5′ exonuclease ExoI, observations that suggest the importance of both 3′and 5′ single-strand ends in the pathway of CAG·CTG repeat tract expansion. We discuss the relevance of our results to two competing models of repeat tract expansion.

Avoiding chromosome pathology when replication forks collide

Chromosome duplication normally initiates via the assembly of replication fork complexes at defined origins1,2. DNA synthesis by any one fork is thought to cease when it meets another travelling in the opposite direction, at which stage the replication machinery may simply dissociate before the nascent strands are finally ligated. But what actually happens is not clear. Here we present evidence consistent with the idea that every fork collision has the potential to threaten genomic integrity. In Escherichia coli this threat is kept at bay by RecG DNA translocase3 and by single-strand DNA exonucleases. Without RecG, replication initiates where forks meet via a replisome assembly mechanism normally associated with fork repair, replication restart and recombination4,5, establishing new forks with the potential to sustain cell growth and division without an active origin. This potential is realised when roadblocks to fork progression are reduced or eliminated. It relies on the chromosome being circular, reinforcing the idea that replication initiation is triggered repeatedly by fork collision. The results reported raise the question of whether replication fork collisions have pathogenic potential for organisms that exploit multiple origins to replicate each chromosome.

The CRISPR Associated Protein Cas4 Is a 5′ to 3′ DNA Exonuclease with an Iron-Sulfur Cluster

The Cas4 protein is one of the core CRISPR-associated (Cas) proteins implicated in the prokaryotic CRISPR system for antiviral defence. Cas4 is thought to play a role in the capture of new viral DNA sequences for incorporation into the host genome. No biochemical activity has been reported for Cas4, but it is predicted to include a RecB nuclease domain. We show here that Cas4 family proteins from the archaeon Sulfolobus solfataricus utilise four conserved cysteine residues to bind an iron-sulfur cluster in an arrangement reminiscent of the AddB nuclease of Bacillus subtilis. The Cas4 family protein Sso0001 is a 5′ to 3′ single stranded DNA exonuclease in vitro that is stalled by extrahelical DNA adducts. A role for Cas4 in DNA duplex strand resectioning to generate recombinogenic 3′ single stranded DNA overhangs is proposed. Comparison of the AddB structure with that of a related bacterial nuclease from Eubacterium rectales reveals that the iron-sulfur cluster can be replaced by a zinc ion without disrupting the protein structure, with implications for the evolution of iron-sulfur binding proteins.

RecG Protein and Single-Strand DNA Exonucleases Avoid Cell Lethality Associated With PriA Helicase Activity inEscherichia coli

Replication of the Escherichia coli chromosome usually initiates at a single origin (oriC) under control of DnaA. Two forks are established and move away in opposite directions. Replication is completed when these meet in a broadly defined terminus area half way around the circular chromosome. RecG appears to consolidate this arrangement by unwinding D-loops and R-loops that PriA might otherwise exploit to initiate replication at other sites. It has been suggested that without RecG such replication generates 3' flaps as the additional forks collide and displace nascent leading strands, providing yet more potential targets for PriA. Here we show that, to stay alive, cells must have either RecG or a 3' single-stranded DNA (ssDNA) exonuclease, which can be exonuclease I, exonuclease VII, or SbcCD. Cells lacking all three nucleases are inviable without RecG. They also need RecA recombinase and a Holliday junction resolvase to survive rapid growth, but SOS induction, although elevated, is not required. Additional requirements for Rep and UvrD are identified and linked with defects in DNA mismatch repair and with the ability to cope with conflicts between replication and transcription, respectively. Eliminating PriA helicase activity removes the requirement for RecG. The data are consistent with RecG and ssDNA exonucleases acting to limit PriA-mediated re-replication of the chromosome and the consequent generation of linear DNA branches that provoke recombination and delay chromosome segregation.

Bacillus subtilis BY‐kinase PtkA controls enzyme activity and localization of its protein substrates

Bacillus subtilis BY-kinase PtkA was previously shown to phosphorylate, and thereby regulate the activity of two classes of protein substrates: UDP-glucose dehydrogenases and single-stranded DNA-binding proteins. Our recent phosphoproteome study identified nine new tyrosine-phosphorylated proteins in B. subtilis. We found that the majority of these proteins could be phosphorylated by PtkA in vitro. Among these new substrates, single-stranded DNA exonuclease YorK, and aspartate semialdehyde dehydrogenase Asd were activated by PtkA-dependent phosphorylation. Because enzyme activity was not affected in other cases, we used fluorescent protein tags to study the impact of PtkA on localization of these proteins in vivo. For several substrates colocalization with PtkA was observed, and more importantly, the localization pattern of the proteins enolase, YjoA, YnfE, YvyG, Ugd and SsbA was dramatically altered in DeltaptkA background. Our results confirm that PtkA can control enzyme activity of its substrates in some cases, but also reveal a new mode of action for PtkA, namely ensuring correct cellular localization of its targets.

The Crystal Structure of the Cytosolic Exopolyphosphatase from Saccharomyces cerevisiae Reveals the Basis for Substrate Specificity

Inorganic long-chain polyphosphate is a ubiquitous linear polymer in biology, consisting of many phosphate moieties linked by phosphoanhydride bonds. It is synthesized by polyphosphate kinase, and metabolised by a number of enzymes, including exo- and endopolyphosphatases. The Saccharomyces cerevisiae gene PPX1 encodes for a 45 kDa, metal-dependent, cytosolic exopolyphosphatase that processively cleaves the terminal phosphate group from the polyphosphate chain, until inorganic pyrophosphate is all that remains. PPX1 belongs to the DHH family of phosphoesterases, which includes: family-2 inorganic pyrophosphatases, found in Gram-positive bacteria; prune, a cyclic AMPase; and RecJ, a single-stranded DNA exonuclease. We describe the high-resolution X-ray structures of yeast PPX1, solved using the multiple isomorphous replacement with anomalous scattering (MIRAS) technique, and its complexes with phosphate (1.6 Å), sulphate (1.8 Å) and ATP (1.9 Å). Yeast PPX1 folds into two domains, and the structures reveal a strong similarity to the family-2 inorganic pyrophosphatases, particularly in the active-site region. A large, extended channel formed at the interface of the N and C-terminal domains is lined with positively charged amino acids and represents a conduit for polyphosphate and the site of phosphate hydrolysis. Structural comparisons with the inorganic pyrophosphatases and analysis of the ligand-bound complexes lead us to propose a hydrolysis mechanism. Finally, we discuss a structural basis for substrate selectivity and processivity.

Replication arrest-stimulated recombination: Dependence on the RecA paralog, RadA/Sms and translesion polymerase, DinB

Difficulties in replication can lead to breakage of the fork. Recombinational reactions restore the integrity of the fork through strand-invasion of the broken chromosome with its sister. If this occurs in the context of repeated DNA sequences, genetic rearrangements can result. We have proposed that this process accounts for stimulation of chromosomal rearrangements by mutations in Escherichia coli's replicative DNA helicase, DnaB. At its permissive temperature for growth, a dnaB107 mutant is a 1000-fold more likely to experience a deletion of a 787 bp tandem repeated segment inserted in the E. coli chromosome than is a wild-type strain. We have previously shown that enhanced deletion in a dnaB107 strain is reduced in recA, recB and recG102 (formerly known as radC102) derivatives. Here I show that this enhanced recombination is dependent on other factors: the RuvA Holliday junction helicase, the RecJ single-strand DNA exonuclease, the RadA/Sms RecA-paralog protein of unknown function and, surprisingly, the DinB translesion polymerase. The requirement for these factors in DnaB-stimulated rearrangements is much greater than that observed for recombinational events such as P1 transduction. This may be because strand invasion into the repeats limits the extent of heteroduplex DNA that can be formed in the initial stage of recombination. I propose that RadA, RecG and RuvAB are critically required to stabilize the strand-invasion intermediate and that DinB polymerase extends the invading 3′ strand to aid in re-initiation. The role of DinB in bacteria may be analogous to translesion DNA polymerase η in eukaryotes, recently shown to aid recombination.

Cis and Trans-acting Effects on a Mutational Hotspot Involving a Replication Template Switch

A natural mutational hotspot in the thyA gene of Escherichia coli accounts for over half of the mutations that inactivate this gene, which can be selected by resistance to the antibiotic trimethoprim. This T to A transversion, at base 131 of the coding sequence, occurs within a 17 bp quasi-palindromic sequence. To clarify the mechanism of mutagenesis, we examine here cis and trans-acting factors affecting thyA131 mutational hotspot activity at its natural location on the E.coli chromosome. Confirming a template-switch mechanism for mutagenesis, an alteration that strengthens base-pairing between the inverted repeat DNA sequences surrounding the hotspot stimulated mutagenesis and, conversely, mutations that weakened pairing reduced hotspot activity. In addition, consistent with the idea that the hotspot mutation is templated from DNA synthesis from mispaired strands of the inverted repeats, co-mutation of multiple sites within the quasipalindrome was observed as predicted from the DNA sequence of the corresponding repeat. Surprisingly, inversion of the thyA operon on the chromosome did not abolish thyA131 hotspot mutagenesis, indicating that mutagenesis at this site occurs during both leading and lagging-strand synthesis. Loss of the SOS-induced DNA polymerases PolII, PolIV, and PolV, caused a marked increase in the hotspot mutation rate, indicating a heretofore unknown and redundant antimutagenic effect of these repair polymerases. Hotspot mutagenesis did not require the PriA replication restart factor and hence must not require fork reassembly after the template-switch reaction. Deficiency in the two major 3' single-strand DNA exonucleases, ExoI and ExoVII, stimulated hotspot mutagenesis 30-fold and extended the mutagenic tract, indicating that these exonucleases normally abort a large fraction of premutagenic events. The high frequency of quasipalindrome-associated mutations suggests that template-switching occurs readily during chromosomal replication.

Stabilization of perfect and imperfect tandem repeats by single-strand DNA exonucleases.

Rearrangements between tandemly repeated DNA sequences are a common source of genetic instability. Such rearrangements underlie several human genetic diseases. In many organisms, the mismatch-repair (MMR) system functions to stabilize repeats when the repeat unit is short or when sequence imperfections are present between the repeats. We show here that the action of single-stranded DNA (ssDNA) exonucleases plays an additional, important role in stabilizing tandem repeats, independent of their role in MMR. For perfect repeats of approximately 100 bp in Escherichia coli that are not susceptible to MMR, exonuclease (Exo)-I, ExoX, and RecJ exonuclease redundantly inhibit deletion. Our data suggest that >90% of potential deletion events are avoided by the combined action of these three exonucleases. Imperfect tandem repeats, less prone to rearrangements, are stabilized by both the MMR-pathway and ssDNA-specific exonucleases. For 100-bp repeats containing four mispairs, ExoI alone aborts most deletion events, even in the presence of a functional MMR system. By genetic analysis, we show that the inhibitory effect of ssDNA exonucleases on deletion formation is independent of the MutS and UvrD proteins. Exonuclease degradation of DNA displaced during the deletion process may abort slipped misalignment. Exonuclease action is therefore a significant force in genetic stabilization of many forms of repetitive DNA.

5' to 3' Single Strand DNA Exonuclease Activity in a Preparation of Human Ku Protein

5' to 3' Single Strand DNA Exonuclease Activity in a Preparation of Human Ku Protein

5' to 3' single strand DNA exonuclease activity in a preparation of human Ku protein.

We describe a novel 5' to 3' single-strand exonuclease activity exhibited by a Ku preparation purified from a human cell line. The enzyme removes 5' single-strand extensions from duplex DNA molecules. The exonuclease and helicase activities respond reciprocally to changes in ATP concentrations: Nuclease activity is inhibited at the ATP concentrations that are optimal for the helicase. The exonuclease activity does not require divalent cations. The potential implications of the exonuclease activity findings for repair of double-strand breaks and recombination processes are discussed.

Exonuclease X of Escherichia coli: A NOVEL 3′-5′ DNase AND DnaQ SUPERFAMILY MEMBER INVOLVED IN DNA REPAIR

DNA exonucleases are critical for DNA replication, repair, and recombination. In the bacterium Escherichia coli there are 14 DNA exonucleases including exonucleases I-IX (including the two DNA polymerase I exonucleases), RecJ exonuclease, SbcCD exonuclease, RNase T, and the exonuclease domains of DNA polymerase II and III. Here we report the discovery and characterization of a new E. coli exonuclease, exonuclease X. Exonuclease X is a member of a superfamily of proteins that have homology to the 3'-5' exonuclease proofreading subunit (DnaQ) of E. coli DNA polymerase III. We have engineered and purified a (His)(6)-exonuclease X fusion protein and characterized its activity. Exonuclease X is a potent distributive exonuclease, capable of degrading both single-stranded and duplex DNA with 3'-5' polarity. Its high affinity for single-strand DNA and its rapid catalytic rate are similar to the processive exonucleases RecJ and exonuclease I. Deletion of the exoX gene exacerbated the UV sensitivity of a strain lacking RecJ, exonuclease I, and exonuclease VII. When overexpressed, exonuclease X is capable of substituting for exonuclease I in UV repair. As we have proposed for the other single-strand DNA exonucleases, exonuclease X may facilitate recombinational repair by pre-synaptic and/or post-synaptic DNA degradation.

Identification of RNase T as a high-copy suppressor of the UV sensitivity associated with single-strand DNA exonuclease deficiency in Escherichia coli.

There are three known single-strand DNA-specific exonucleases in Escherichia coli: RecJ, exonuclease I (ExoI), and exonuclease VII (ExoVII). E. coli that are deficient in all three exonucleases are abnormally sensitive to UV irradiation, most likely because of their inability to repair lesions that block replication. We have performed an iterative screen to uncover genes capable of ameliorating the UV repair defect of xonA (ExoI-) xseA (ExoVII-) recJ triple mutants. In this screen, exonuclease-deficient cells were transformed with a high-copy E. coli genomic library and then irradiated; plasmids harvested from surviving cells were used to seed subsequent rounds of transformation and selection. After several rounds of selection, multiple plasmids containing the rnt gene, which encodes RNase T, were found. An rnt plasmid increased the UV resistance of a xonA xseA recJ mutant and uvrA and uvrC mutants; however, it did not alter the survival of xseA recJ or recA mutants. RNase T also has amino acid sequence similarity to other 3' DNA exonucleases, including ExoI. These results suggest that RNase T may possess a 3' DNase activity capable of substituting for ExoI in the recombinational repair of UV-induced lesions.

Requirement of S. pombe exonuclease II, a homologue of S. cerevisiae Sep1, for normal mitotic growth and viability.

Exonuclease II (ExoII) from Schizosaccharomyces pombe is a 5'-->3' single-stranded DNA exonuclease. We have cloned its gene, exo2, whose nucleotide sequence revealed that ExoII is a homologue of the multifunctional Saccharomyces cerevisiae Sep1 protein (also called Kem1, Xrn1, Rar5, Dst2). S. pombe exo2 null mutants were cold-sensitive for growth, had increased cell size at the restrictive temperature, were hypersensitive to the mitotic inhibitor thiabendazol and to caffeine, and died rapidly in stationary phase. Many of these phenotypes are similar to those of sep1 (kem1 or xrn1) mutants of S. cerevisiae. In contrast, the exo2 mutation had only a moderate effect on progression through meiosis and no significant effect on meiotic recombination. We discuss possible functions of the multifunctional ExoII protein.

Enhancement of RecA Strand-transfer Activity by the RecJ Exonuclease of Escherichia coli

We have examined coupled reactions with the RecA protein of Escherichia coli, which can mediate DNA strand exchange in vitro between homologous DNA molecules, and the RecJ exonuclease, a 5' to 3' single-stranded DNA exonuclease. In RecA-mediated strand-transfer reactions between circular single-stranded and duplex linear DNA, we have found that RecJ stimulates the rate of heteroduplex product formation. Because RecJ must be present concurrent with strand transfer and RecJ does not detectably stimulate the synapsis stage of the reaction, we believe that RecJ stimulates specifically the branch migration phase of the RecA strand-transfer reaction. RecJ also dramatically enhances the efficiency with which RecA is able to transverse regions of non-homology in the substrates. We propose a model where RecJ degrades the displaced strand produced by strand exchange which competes for pairing with the transferred strand, thus driving forward the unidirectional branch migration mediated by RecA protein. This suggests a new role for exonucleases in genetic recombination, facilitating the strand-transfer reaction itself.

Biochemical characterization of P22 phage-modified Escherichia coli RecBCD enzyme.

The biochemical properties of phage P22 Abc-modified RecBCD enzyme from Escherichia coli have been examined. RecBCD purified from a cell that expresses Abc (anti-RecBCD) contains all three RecBCD subunits and the 11.6-kDa Abc protein in equal stoichiometric amounts. Abc depresses the rate of RecBCD double-stranded DNA exonuclease, helicase, and ATPase activities about 3-4-fold, yet it has no effect on the rate of the single-stranded DNA exonuclease activity. Abc induces a slight increase in the ATP-independent single-stranded DNA endonuclease activity and does not induce dimerization of the RecBCD trimer. Abc-modified RecBCD helicase activity possesses reduced but significant processivity (10 kilobase pairs) relative to the native enzyme (30 kilobase pairs). In the absence of ATP, Abc-modified RecBCD shows a 2-4-fold higher affinity for double-stranded DNA ends. The RecBCD-binding Gam protein from bacteriophage lambda inhibits binding of both native and Abc-modified RecBCD to double-stranded DNA ends. Finally, unlike the native enzyme, the nonspecific nuclease activity of Abc-modified RecBCD is not suppressed by Chi sites in vitro. These findings are discussed in terms of the recombination-deficient phenotype of cells expressing Abc in vivo and the relationship between Abc-modified RecBCD and two mutant RecBCD's previously characterized: the RecBCD-K117Q and RecB2109CD mutant enzymes.

A single-stranded DNA exonuclease from Schizosaccharomyces pombe.

We have purified to near homogeneity a DNA exonuclease from meiotic cells of Schizosaccharomyces pombe. The enzyme, designated exonuclease II (ExoII), had an apparent molecular weight of 134,000 and was abundant in the cell. It specifically degraded single-stranded DNA in the 5'----3' direction with an apparent Km for 5' DNA ends of 3.6 x 10(-11) M and produced 5' deoxynucleoside monophosphates. Its mode of degradation is similar to that of the RecJ protein from Escherichia coli; ExoII may, therefore, be involved in genetic recombination and DNA damage repair.

Modulation of the action of the recBC enzyme of Escherichia coli K-12 by Ca2+.

The various activities of the recBC enzyme of Escherichia coli are differentially sensitive to reaction conditions. Although spermidine up to 50 mM and putrescine up to 40 mM in the presence of Mg/sup 2 +/ produce no significant alteration in the activities of the enzyme, low concentrations of Ca/sup 2 +/ have a dramatic effect. In the presence of 1 mM Ca/sup 2 +/, 1 mM Mg/sup 2 +/, and 5 mM ATP, the DNA-dependent ATPase is unaffected, but the double strand DNA exonuclease and the single strand DNA exonuclease and endonuclease activities are completely inhibited. Duplex phage T7 DNA which has been exposed to enzyme under these conditions remains wholly duplex and full length, although about 5 nicks are made randomly in each duplex. When DNA binding protein from E. coli is included in the reaction with enzyme and DNA, the products are, at early stages of the reaction, duplex DNA molecules with one or two single-stranded tails at a terminus; after prolonged incubation, solely single-stranded fragments of 5000 to 35,000 nucleotides are found. (Full length strands are roughly 40,000 nucleotides long.) These products can be rationalized by a model in which the enzyme binds initially to the terminusmore » of a duplex DNA molecule and then tracks along the DNA, unwinding the strands of the helix as it moves. This denaturation would be transient and localized around the enzyme molecule so that the DNA would renature after the enzyme has passed through a particular region. However, the renaturation can be prevented by the presence of DNA binding protein during the reaction. During the passage of an enzyme molecule through a DNA molecule, a limited number of nicks are randomly introduced in a manner essentially unaffected by binding protein. This mode of action is distinct from that observed for the degradation of duplex DNA with Mg/sup 2 +/ but not Ca/sup 2 +/ present.« less