The nature of the 5'-terminus is a major determinant for DNA processing by Schizosaccharomyces pombe Rad2p, a FEN-1 family nuclease

The nuclease activity of FEN-1 is essential for both DNA replication and repair. Intermediate DNA products formed during these processes possess a variety of structures and termini. We have previously demonstrated that the 5′→3′ exonuclease activity of the Schizosaccharomyces pombe FEN-1 protein Rad2p requires a 5′-phosphoryl moiety to efficiently degrade a nick-containing substrate in a reconstituted alternative excision repair system. Here we report the effect of different 5′-terminal moieties of a variety of DNA substrates on Rad2p activity. We also show that Rad2p possesses a 5′→3′ single-stranded exonuclease activity, similar to Saccharomyces cerevisiae Rad27p and phage T5 5′→3′ exonuclease (also a FEN-1 homolog). FEN-1 nucleases have been associated with the base excision repair pathway, specifically processing cleaved abasic sites. Because several enzymes cleave abasic sites through different mechanisms resulting in different 5′-termini, we investigated the ability of Rad2p to process several different types of cleaved abasic sites. With varying efficiency, Rad2p degrades the products of an abasic site cleaved by Escherichia coli endonuclease III and endonuclease IV (prototype AP endonucleases) and S.pombe Uve1p. These results provide important insights into the roles of Rad2p in DNA repair processes in S.pombe.     

Methanococcus jannaschii Flap Endonuclease: Expression, Purification, and Substrate Requirements

The flap endonuclease (FEN) of the hyperthermophilic archaeon Methanococcus jannaschii was expressed in Escherichia coli and purified to homogeneity. FEN retained activity after preincubation at 95°C for 15 min. A pseudo-Y-shaped substrate was formed by hybridization of two partially complementary oligonucleotides. FEN cleaved the strand with the free 5′ end adjacent to the single-strand–duplex junction. Deletion of the free 3′ end prevented cleavage. Hybridization of a complementary oligonucleotide to the free 3′ end moved the cleavage site by 1 to 2 nucleotides. Hybridization of excess complementary oligonucleotide to the free 5′ end failed to block cleavage, although this substrate was refractory to cleavage by the 5′-3′ exonuclease activity of Taq DNA polymerase. For verification, the free 5′ end was replaced by an internally labeled hairpin structure. This structure was a substrate for FEN but became a substrate for Taq DNA polymerase only after exonucleolytic cleavage had destabilized the hairpin. A circular duplex substrate with a 5′ single-stranded branch was formed by primer extension of a partially complementary oligonucleotide on virion X174. This denaturation-resistant substrate was used to examine the effects of temperature and solution properties, such as pH, salt, and divalent ion concentration on the turnover number of the enzyme.     

DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction

Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5′-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5′-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5′-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5′-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Tyr⁴⁰, Asp¹⁸¹, and Arg¹⁰⁰ and a reacting duplex 5′-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5′-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage.     

Dna2 is a structure-specific nuclease,with affinity for 5′-flap intermediates

Dna2 is a nuclease/helicase with proposed roles in DNA replication, double-strand break repair and telomere maintenance. For each role Dna2 is proposed to process DNA substrates with a 5′-flap. To date, however, Dna2 has not revealed a preference for binding or cleavage of flaps over single-stranded DNA. Using DNA binding competition assays we found that Dna2 has substrate structure specificity. The nuclease displayed a strong preference for binding substrates with a 5′-flap or some variations of flap structure. Further analysis revealed that Dna2 recognized and bound both the single-stranded flap and portions of the duplex region immediately downstream of the flap. A model is proposed in which Dna2 first binds to a flap base, and then the flap threads through the protein with periodic cleavage, to a terminal flap length of ∼5 nt. This resembles the mechanism of flap endonuclease 1, consistent with cooperation of these two proteins in flap processing.     

Evidence That the DNA Endonuclease ARTEMIS also Has Intrinsic 5′-Exonuclease Activity

ARTEMIS is a member of the metallo-β-lactamase protein family. ARTEMIS has endonuclease activity at DNA hairpins and at 5′- and 3′-DNA overhangs of duplex DNA, and this endonucleolytic activity is dependent upon DNA-PKcs. There has been uncertainty about whether ARTEMIS also has 5′-exonuclease activity on single-stranded DNA and 5′-overhangs, because this 5′-exonuclease is not dependent upon DNA-PKcs. Here, we show that the 5′-exonuclease and the endonuclease activities co-purify. Second, we show that a point mutant of ARTEMIS at a putative active site residue (H115A) markedly reduces both the endonuclease activity and the 5′-exonuclease activity. Third, divalent cation effects on the 5′-exonuclease and the endonuclease parallel one another. Fourth, both the endonuclease activity and 5′-exonuclease activity of ARTEMIS can be blocked in parallel by small molecule inhibitors, which do not block unrelated nucleases. We conclude that the 5′-exonuclease is intrinsic to ARTEMIS, making it relevant to the role of ARTEMIS in nonhomologous DNA end joining.     

Crystal structure of endonuclease G in complex with DNA reveals how it nonspecifically degrades DNA as a homodimer

Endonuclease G (EndoG) is an evolutionarily conserved mitochondrial protein in eukaryotes that digests nucleus chromosomal DNA during apoptosis and paternal mitochondrial DNA during embryogenesis. Under oxidative stress, homodimeric EndoG becomes oxidized and converts to monomers with diminished nuclease activity. However, it remains unclear why EndoG has to function as a homodimer in DNA degradation. Here, we report the crystal structure of the Caenorhabditis elegans EndoG homologue, CPS-6, in complex with single-stranded DNA at a resolution of 2.3 Å. Two separate DNA strands are bound at the ββα-metal motifs in the homodimer with their nucleobases pointing away from the enzyme, explaining why CPS-6 degrades DNA without sequence specificity. Two obligatory monomeric CPS-6 mutants (P207E and K131D/F132N) were constructed, and they degrade DNA with diminished activity due to poorer DNA-binding affinity as compared to wild-type CPS-6. Moreover, the P207E mutant exhibits predominantly 3′-to-5′ exonuclease activity, indicating a possible endonuclease to exonuclease activity change. Thus, the dimer conformation of CPS-6 is essential for maintaining its optimal DNA-binding and endonuclease activity. Compared to other non-specific endonucleases, which are usually monomeric enzymes, EndoG is a unique dimeric endonuclease, whose activity hence can be modulated by oxidation to induce conformational changes.     

Mechanistic decoupling of exonuclease III multifunctionality into AP endonuclease and exonuclease activities at the single-residue level

Bacterial exonuclease III (ExoIII) is a multifunctional enzyme that uses a single active site to perform two conspicuous activities: (i) apurinic/apyrimidinic (AP)-endonuclease and (ii) 3′→5′ exonuclease activities. The AP endonuclease activity results in AP site incision, while the exonuclease activity results in the continuous excision of 3′ terminal nucleobases to generate a partial duplex for recruiting the downstream DNA polymerase during the base excision repair process (BER). The key determinants of functional selection between the two activities are poorly understood. Here, we use a series of mutational analyses and single-molecule imaging to unravel the pivotal rules governing these endo- and exonuclease activities at the single amino acid level. An aromatic residue, either W212 or F213, recognizes AP sites to allow for the AP endonuclease activity, and the F213 residue also participates in the stabilization of the melted state of the 3′ terminal nucleobases, leading to the catalytically competent state that activates the 3′→5′ exonuclease activity. During exonucleolytic cleavage, the DNA substrate must be maintained as a B-form helix through a series of phosphate-stabilizing residues (R90, Y109, K121 and N153). Our work decouples the AP endonuclease and exonuclease activities of ExoIII and provides insights into how this multifunctional enzyme controls each function at the amino acid level.     

Molecular interactions of Escherichia coli ExoIX and identification of its associated 3'-5' exonuclease activity

The flap endonucleases (FENs) participate in a wide range of processes involving the structure-specific cleavage of branched nucleic acids. They are also able to hydrolyse DNA and RNA substrates from the 5′-end, liberating mono-, di- and polynucleotides terminating with a 5′ phosphate. Exonuclease IX is a paralogue of the small fragment of Escherichia coli DNA polymerase I, a FEN with which it shares 66% similarity. Here we show that both glutathione-S-transferase-tagged and native recombinant ExoIX are able to interact with the E. coli single-stranded DNA binding protein, SSB. Immobilized ExoIX was able to recover SSB from E. coli lysates both in the presence and absence of DNA. In vitro cross-linking studies carried out in the absence of DNA showed that the SSB tetramer appears to bind up to two molecules of ExoIX. Furthermore, we found that a 3′–5′ exodeoxyribonuclease activity previously associated with ExoIX can be separated from it by extensive liquid chromatography. The associated 3′–5′ exodeoxyribonuclease activity was excised from a 2D gel and identified as exonuclease III using matrix-assisted laser-desorption ionization mass spectrometry.     

RecJ exonuclease: substrates, products and interaction with SSB

The RecJ exonuclease from Escherichia coli degrades single-stranded DNA (ssDNA) in the 5′–3′ direction and participates in homologous recombination and mismatch repair. The experiments described here address RecJ's substrate requirements and reaction products. RecJ complexes on a variety of 5′ single-strand tailed substrates were analyzed by electrophoretic mobility shift in the absence of Mg²⁺ ion required for substrate degradation. RecJ required single-stranded tails of 7 nt or greater for robust binding; addition of Mg²⁺ confirmed that substrates with 5′ tails of 6 nt or less were poor substrates for RecJ exonuclease. RecJ is a processive exonuclease, degrading ~1000 nt after a single binding event to single-strand DNA, and releases mononucleotide products. RecJ is capable of degrading a single-stranded tail up to a double-stranded junction, although products in such reactions were heterogeneous and RecJ showed a limited ability to penetrate the duplex region. RecJ exonuclease was equally potent on 5′ phosphorylated and unphosphorylated ends. Finally, DNA binding and nuclease activity of RecJ was specifically enhanced by the pre-addition of ssDNA-binding protein and we propose that this specific interaction may aid recruitment of RecJ.     

PCNA and XPF cooperate to distort DNA substrates

XPF is a structure-specific endonuclease that preferentially cleaves 3′ DNA flaps during a variety of repair processes. The crystal structure of a crenarchaeal XPF protein bound to a DNA duplex yielded insights into how XPF might recognise branched DNA structures, and recent kinetic data have demonstrated that the sliding clamp PCNA acts as an essential cofactor, possibly by allowing XPF to distort the DNA structure into a proper conformation for efficient cleavage to occur. Here, we investigate the solution structure of the 3′-flap substrate bound to XPF in the presence and absence of PCNA using intramolecular Förster resonance energy transfer (FRET). We demonstrate that recognition of the flap substrate by XPF involves major conformational changes of the DNA, including a 90° kink of the DNA duplex and organization of the single-stranded flap. In the presence of PCNA, there is a further substantial reorganization of the flap substrate bound to XPF, providing a structural basis for the observation that PCNA has an essential catalytic role in this system. The wider implications of these observations for the plethora of PCNA-dependent enzymes are discussed.     

Studies of the 5' exonuclease and endonuclease activities of CPSF-73 in histone pre-mRNA processing

Processing of histone pre-mRNA requires a single 3′ endonucleolytic cleavage guided by the U7 snRNP that binds downstream of the cleavage site. Following cleavage, the downstream cleavage product (DCP) is rapidly degraded in vitro by a nuclease that also depends on the U7 snRNP. Our previous studies demonstrated that the endonucleolytic cleavage is catalyzed by the cleavage/polyadenylation factor CPSF-73. Here, by using RNA substrates with different nucleotide modifications, we characterize the activity that degrades the DCP. We show that the degradation is blocked by a 2′-O-methyl nucleotide and occurs in the 5′-to-3′ direction. The U7-dependent 5′ exonuclease activity is processive and continues degrading the DCP substrate even after complete removal of the U7-binding site. Thus, U7 snRNP is required only to initiate the degradation. UV cross-linking studies demonstrate that the DCP and its 5′-truncated version specifically interact with CPSF-73, strongly suggesting that in vitro, the same protein is responsible for the endonucleolytic cleavage of histone pre-mRNA and the subsequent degradation of the DCP. By using various RNA substrates, we define important space requirements upstream and downstream of the cleavage site that dictate whether CPSF-73 functions as an endonuclease or a 5′ exonuclease. RNA interference experiments with HeLa cells indicate that degradation of the DCP does not depend on the Xrn2 5′ exonuclease, suggesting that CPSF-73 degrades the DCP both in vitro and in vivo.     

Dissecting endonuclease and exonuclease activities in endonuclease V from Thermotoga maritima

Endonuclease V is an enzyme that initiates a conserved DNA repair pathway by making an endonucleolytic incision at the 3′-side 1 nt from a deaminated base lesion. DNA cleavage analysis using mutants defective in DNA binding and Mn²⁺ as a metal cofactor reveals a novel 3′-exonuclease activity in endonuclease V [Feng,H., Dong,L., Klutz,A.M., Aghaebrahim,N. and Cao,W. (2005) Defining amino acid residues involved in DNA-protein interactions and revelation of 3′-exonuclease activity in endonuclease V. Biochemistry, 44, 11486–11495.]. This study defines the enzymatic nature of the endonuclease and exonuclease activity in endonuclease V from Thermotoga maritima. In addition to its well-known inosine-dependent endonuclease, Tma endonuclease V also exhibits inosine-dependent 3′-exonuclease activity. The dependence on an inosine site and the exonuclease nature of the 3′-exonuclease activity was demonstrated using 5′-labeled and internally-labeled inosine-containing DNA and a H214D mutant that is defective in non-specific nuclease activity. Detailed kinetic analysis using 3′-labeled DNA indicates that Tma endonuclease V also possesses non-specific 5′-exonuclease activity. The multiplicity of the endonuclease and exonuclease activity is discussed with respect to deaminated base repair.     

NurA,a novel 5'-3' nuclease gene linked to rad50 and mre11 homologs of thermophilic Archaea

We isolated and characterized a new nuclease (NurA) exhibiting both single-stranded endonuclease activity and 5′–3′ exonuclease activity on single-stranded and double-stranded DNA from the hyperthermophilic archaeon Sulfolobus acidocaldarius. Nuclease homologs are detected in all thermophilic archaea and, in most species, the nurA gene is organized in an operon-like structure with rad50 and mre11 archaeal homologs. This nuclease might thus act in concert with Rad50 and Mre11 proteins in archaeal recombination/repair. To our knowledge, this is the first report of a 5′–3′ nuclease potentially associated with Rad50 and Mre11-like proteins that may lead to the processing of double-stranded breaks in 3′ single-stranded tails.     

Endonuclease IV Is the Major Apurinic/Apyrimidinic Endonuclease in Mycobacterium tuberculosis and Is Important for Protection against Oxidative Damage

During the establishment of an infection, bacterial pathogens encounter oxidative stress resulting in the production of DNA lesions. Majority of these lesions are repaired by base excision repair (BER) pathway. Amongst these, abasic sites are the most frequent lesions in DNA. Class II apurinic/apyrimidinic (AP) endonucleases play a major role in BER of damaged DNA comprising of abasic sites. Mycobacterium tuberculosis, a deadly pathogen, resides in the human macrophages and is continually subjected to oxidative assaults. We have characterized for the first time two AP endonucleases namely Endonuclease IV (End) and Exonuclease III (XthA) that perform distinct functions in M.tuberculosis. We demonstrate that M.tuberculosis End is a typical AP endonuclease while XthA is predominantly a 3′→5′ exonuclease. The AP endonuclease activity of End and XthA was stimulated by Mg²⁺ and Ca²⁺ and displayed a preferential recognition for abasic site paired opposite to a cytosine residue in DNA. Moreover, End exhibited metal ion independent 3′→5′ exonuclease activity while in the case of XthA this activity was metal ion dependent. We demonstrate that End is not only a more efficient AP endonuclease than XthA but it also represents the major AP endonuclease activity in M.tuberculosis and plays a crucial role in defense against oxidative stress.     

Characterization of the endonuclease and ATP-dependent flap endo/exonuclease of Dna2

Two processes, DNA replication and DNA damage repair, are key to maintaining genomic fidelity. The Dna2 enzyme lies at the heart of both of these processes, acting in conjunction with flap endonuclease 1 and replication protein A in DNA lagging strand replication and with BLM/Sgs1 and MRN/X in double strand break repair. In vitro, Dna2 helicase and flap endo/exonuclease activities require an unblocked 5' single-stranded DNA end to unwind or cleave DNA. In this study we characterize a Dna2 nuclease activity that does not require, and in fact can create, 5' single-stranded DNA ends. Both endonuclease and flap endo/exonuclease are abolished by the Dna2-K677R mutation, implicating the same active site in catalysis. In addition, we define a novel ATP-dependent flap endo/exonuclease activity, which is observed only in the presence of Mn(2+). The endonuclease is blocked by ATP and is thus experimentally distinguishable from the flap endo/exonuclease function. Thus, Dna2 activities resemble those of RecB and AddAB nucleases even more closely than previously appreciated. This work has important implications for understanding the mechanism of action of Dna2 in multiprotein complexes, where dissection of enzymatic activities and cofactor requirements of individual components contributing to orderly and precise execution of multistep replication/repair processes depends on detailed characterization of each individual activity.     

A structure-specific endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence.

A protein with structure-specific endonuclease activity has been purified to near homogeneity from cauliflower ( Brassica oleracea var. botrytis) inflorescence through five successive column chromatographies. The protein is a single polypeptide with a molecular mass of 40 kDa. Using three different branched DNA structures (flap, pseudo-Y and stem-loop) we found that the enzyme, a cauliflower structure-specific endonuclease, cleaved the single-stranded tail in the 5'-flap and 5'-pseudo-Y structures, whereas it could not incise the 3'-flap and 3'-pseudo-Y structures. The incision points occur around the single strand-duplex junction in these DNA substrates and the enzyme leaves 5'-PO4 and 3'-OH termini on DNA. The protein also endonucleolytically cleaves on the 3'-side of the single-stranded region at the junction of unpaired and duplex DNA in the stem-loop structure. The structure-specific endonuclease activity is stimulated by Mg2+ and by Mn2+, but not by Ca2+. Like mammalian FEN-1, the protein has weak 5'-->3' double-stranded DNA-specific exonuclease activity. These results indicate that the cauliflower protein is a plant structure-specific endonuclease like mammalian FEN-1 or may be the plant alternative.     

Molecular and Functional Characterization of RecD,a Novel Member of the SF1 Family of Helicases,from Mycobacterium tuberculosis

The annotated whole-genome sequence of Mycobacterium tuberculosis revealed the presence of a putative recD gene; however, the biochemical characteristics of its encoded protein product (MtRecD) remain largely unknown. Here, we show that MtRecD exists in solution as a stable homodimer. Protein-DNA binding assays revealed that MtRecD binds efficiently to single-stranded DNA and linear duplexes containing 5′ overhangs relative to the 3′ overhangs but not to blunt-ended duplex. Furthermore, MtRecD bound more robustly to a variety of Y-shaped DNA structures having ≥18-nucleotide overhangs but not to a similar substrate containing 5-nucleotide overhangs. MtRecD formed more salt-tolerant complexes with Y-shaped structures compared with linear duplex having 3′ overhangs. The intrinsic ATPase activity of MtRecD was stimulated by single-stranded DNA. Site-specific mutagenesis of Lys-179 in motif I abolished the ATPase activity of MtRecD. Interestingly, although MtRecD-catalyzed unwinding showed a markedly higher preference for duplex substrates with 5′ overhangs, it could also catalyze significant unwinding of substrates containing 3′ overhangs. These results support the notion that MtRecD is a bipolar helicase with strong 5′ → 3′ and weak 3′ → 5′ unwinding activities. The extent of unwinding of Y-shaped DNA structures was ∼3-fold lower compared with duplexes with 5′ overhangs. Notably, direct interaction between MtRecD and its cognate RecA led to inhibition of DNA strand exchange promoted by RecA. Altogether, these studies provide the first detailed characterization of MtRecD and present important insights into the type of DNA structure the enzyme is likely to act upon during the processes of DNA repair or homologous recombination.     

Bacillus subtilis RNase J1 endonuclease and 5′ exonuclease activities in the turnover of ΔermC mRNA

RNase J1, a ribonuclease with 5′ exonuclease and endonuclease activities, is an important factor in Bacillus subtilis mRNA decay. A model for RNase J1 endonuclease activity in mRNA turnover has RNase J1 binding to the 5′ end and tracking to a target site downstream, where it makes a decay-initiating cleavage. The upstream fragment from this cleavage is degraded by 3′ exonucleases; the downstream fragment is degraded by RNase J1 5′ exonuclease activity. Previously, ΔermC mRNA was used to show 5′-end dependence of mRNA turnover. Here we used ΔermC mRNA to probe RNase J1-dependent degradation, and the results were consistent with aspects of the model. ΔermC mRNA showed increased stability in a mutant strain that contained a reduced level of RNase J1. In agreement with the tracking concept, insertion of a strong stem–loop structure at +65 resulted in increased stability. Weakening this stem–loop structure resulted in reversion to wild-type stability. RNA fragments containing the 3′ end were detected in a strain with reduced RNase J1 expression, but were undetectable in the wild type. The 5′ ends of these fragments mapped to the upstream side of predicted stem–loop structures, consistent with an impediment to RNase J1 5′ exonuclease processivity. A ΔermC mRNA deletion analysis suggested that decay-initiating endonuclease cleavage could occur at several sites near the 3′ end. However, even in the absence of these sites, stability was further increased in a strain with reduced RNase J1, suggesting alternate pathways for decay that could include exonucleolytic decay from the 5′ end.     

Coordinate 5' and 3' endonucleolytic trimming of terminally blocked blunt DNA double-strand break ends by Artemis nuclease and DNA-dependent protein kinase

Previous work showed that, in the presence of DNA-dependent protein kinase (DNA-PK), Artemis slowly trims 3′-phosphoglycolate-terminated blunt ends. To examine the trimming reaction in more detail, long internally labeled DNA substrates were treated with Artemis. In the absence of DNA-PK, Artemis catalyzed extensive 5′→3′ exonucleolytic resection of double-stranded DNA. This resection required a 5′-phosphate, but did not require ATP, and was accompanied by endonucleolytic cleavage of the resulting 3′ overhang. In the presence of DNA-PK, Artemis-mediated trimming was more limited, was ATP-dependent and did not require a 5′-phosphate. For a blunt end with either a 3′-phosphoglycolate or 3′-hydroxyl terminus, endonucleolytic trimming of 2–4 nucleotides from the 3′-terminal strand was accompanied by trimming of 6 nt from the 5′-terminal strand. The results suggest that autophosphorylated DNA-PK suppresses the exonuclease activity of Artemis toward blunt-ended DNA, and promotes slow and limited endonucleolytic trimming of the 5′-terminal strand, resulting in short 3′ overhangs that are trimmed endonucleolytically. Thus, Artemis and DNA-PK can convert terminally blocked DNA ends of diverse geometry and chemical structure to a form suitable for polymerase-mediated patching and ligation, with minimal loss of terminal sequence. Such processing could account for the very small deletions often found at DNA double-strand break repair sites.