A novel structure-specific endonuclease activity associated with polypyrimidine-tract binding (PTB) related protein from rat testis

Nucleases are involved in the processing of various intermediates generated during crucial DNA metabolic processes such as replication, repair, and recombination and also during maturation of RNA precursors. An endonuclease, degrading specifically single-stranded circular DNA, was identified earlier in rat testis nuclear extract while purifying a strand-transfer activity. We are now reporting the purification of this endonuclease, which is a monomeric 42 kDa protein, from rat testis to near-homogeneity. In addition to degrading single-stranded circular DNA, it nicks supercoiled plasmid DNA to generate relaxed DNA and does not act on linear single-stranded or double-stranded DNA. It also makes specific incisions at the single-strand/duplex junction of pseudo-Y, 3'- and 5'-overhangs and 3'- and 5'-flap structures. Other structures such as mismatch, insertion loop, and Holliday junction are not substrates for the testis endonuclease. In contrast to FEN1, the testis endonuclease makes asymmetric incisions on both strands of the branched structures, and free single-stranded ends are not necessary for the structure-specific incisions. Neither 5'-3' nor 3'-5' exonuclease activity is associated with the testis endonuclease. The amino acid sequences of tryptic peptides of the 42 kDa endonuclease show near-identity to polypyrimidine-tract binding protein (PTB) that is involved in the regulation of splicing of eukaryotic mRNA. The significance of the results on the association of structure-specific endonucleae activities with PTB-related protein is discussed.     

3'-phosphodiesterase activity of human apurinic/apyrimidinic endonuclease at DNA double-strand break ends.

In order to assess the possible role of human apurinic/apyrimidinic endonuclease (Ape) in double-strand break repair, the substrate specificity of this enzyme was investigated using short DNA duplexes and partial duplexes, each having a single 3'-phosphoglycolate terminus. Phosphoglycolate removal by Ape was detected as a shift in mobility of 5'-end-labeled DNA strands on polyacrylamide sequencing gels, and was quantified by phosphorimaging. Recombinant Ape efficiently removed phosphoglycolates from the 3'-terminus of an internal 1 base gap in a 38mer duplex, but acted more slowly on 3'-phosphoglycolates at a 19 base-recessed 3'-terminus, at an internal nick with no missing bases, and at a double-strand break end with either blunt or 2 base-recessed 3'-termini. There was no detectable activity of Ape toward 3'-phosphoglycolates on 1 or 2 base protruding single-stranded 3'-overhangs. The results suggest that both a single-base internal gap, and duplex DNA on each side of the gap are important binding/recognition determinants for Ape. While Ape may play a role in repair of terminally blocked double-strand breaks, there must also be additional factors involved in removal of at least some damaged 3'-termini, particularly those on 3'-overhangs.     

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.     

Purification and characterization of a DNA-pairing and strand transfer activity from mitotic Saccharomyces cerevisiae

An enzyme catalyzing homologous pairing of DNA chains has been extensively purified from mitotic yeast. The most highly purified fractions are enriched for a polypeptide with a molecular mass of approximately 120 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein-dependent pairing of single-stranded DNAs requires a divalent cation (Mg2+ or Ca2+) but proceeds rapidly in the absence of any nucleoside triphosphates. The kinetics of reassociation are extremely rapid, with more than 60% of the single-stranded DNA becoming resistant to S1 nuclease within 1 min at a ratio of 1 protein monomer/50 nucleotides. The results of enzyme titration and DNA challenge experiments suggest that this protein does not act catalytically during renaturation but is required stoichiometrically. The protein promotes formation of joint molecules between linear M13 replicative form DNA (form III) containing short single-stranded tails and homologous single-stranded M13 viral DNA. Removal of approximately 50 nucleotides from the ends of the linear duplex using either exonuclease III (5' ends) or T7 gene 6 exonuclease (3' ends) activates the duplex for extensive strand exchange. Electron microscopic analysis of product molecules suggests that the homologous circular DNA initially associates with the single-stranded tails of the duplexes, and the heteroduplex region is extended with displacement of the noncomplementary strand. The ability of this protein to pair and to promote strand transfer using either exonuclease III or T7 gene 6 exonuclease-treated duplex substrates suggests that this activity promotes heteroduplex extension in a nonpolar fashion. The biochemical properties of the transferase are consistent with a role for this protein in heteroduplex joint formation during mitotic recombination in Saccharomyces cerevisiae.     

DNA末端构型调控RecJ核酸外切酶活性

目的 核酸酶介导的DNA双链末端切割对同源重组修复至关重要。然而,DNA末端构型对RecJ 5’-3’核酸外切酶活性的调控尚不清楚。本研究旨在探究DNA3’端和5’端构型对RecJ核酸外切酶活性的影响及其机制。方法 为探究DNA3’端构型对RecJ核酸外切酶活性的影响,使用含有Mg²⁺的体系,对具有不同3’突出末端长度（9 nt与18 nt）和3’突出末端修饰（磷酸化和硫代磷酸酯修饰）的单链DNA分别进行RecJ核酸酶活性检测。为揭示DNA 3’端构型对RecJ外切酶活性的调控机制,在Mg²⁺缺失的体系中,使RecJ与底物结合后进行凝胶迁移实验（EMSA）。为探索其他调控因子与DNA3’端构型对RecJ的协同作用,分别检测5’端磷酸化修饰和单链DNA结合蛋白（SSB）对DNA3’突出末端修饰的影响。结果 DNA3’端构型包括突出末端的长度和修饰（磷酸化和硫代磷酸酯修饰）均会抑制RecJ外切酶活性。DNA 3’端磷酸化和硫代磷酸酯修饰通过重塑RecJ-DNA的结合模式抑制RecJ外切酶活性。DNA 5’端磷酸化修饰可增强RecJ对具有不同3’端...     

Bacteriophage T4 RNase H removes both RNA primers and adjacent DNA from the 5' end of lagging strand fragments

Bacteriophage T4 RNase H belongs to a family of prokaryotic and eukaryotic nucleases that remove RNA primers from lagging strand fragments during DNA replication. Each enzyme has a flap endonuclease activity, cutting at or near the junction between single- and double-stranded DNA, and a 5'- to 3'-exonuclease, degrading both RNA.DNA and DNA.DNA duplexes. On model substrates for lagging strand synthesis, T4 RNase H functions as an exonuclease removing short oligonucleotides, rather than as an endonuclease removing longer flaps created by the advancing polymerase. The combined length of the DNA oligonucleotides released from each fragment ranges from 3 to 30 nucleotides, which corresponds to one round of processive degradation by T4 RNase H with 32 single-stranded DNA-binding protein present. Approximately 30 nucleotides are removed from each fragment during coupled leading and lagging strand synthesis with the complete T4 replication system. We conclude that the presence of 32 protein on the single-stranded DNA between lagging strand fragments guarantees that the nuclease will degrade processively, removing adjacent DNA as well as the RNA primers, and that the difference in the relative rates of synthesis and hydrolysis ensures that there is usually only a single round of degradation during each lagging strand cycle.     

Flap endonucleases pass 5'-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 5'-ends

Flap endonucleases (FENs), essential for DNA replication and repair, recognize and remove RNA or DNA 5'-flaps. Related to FEN specificity for substrates with free 5'-ends, but controversial, is the role of the helical arch observed in varying conformations in substrate-free FEN structures. Conflicting models suggest either 5'-flaps thread through the arch, which when structured can only accommodate single-stranded (ss) DNA, or the arch acts as a clamp. Here we show that free 5'-termini are selected using a disorder-thread-order mechanism. Adding short duplexes to 5'-flaps or 3'-streptavidin does not markedly impair the FEN reaction. In contrast, reactions of 5'-streptavidin substrates are drastically slowed. However, when added to premixed FEN and 5'-biotinylated substrate, streptavidin is not inhibitory and complexes persist after challenge with unlabelled competitor substrate, regardless of flap length or the presence of a short duplex. Cross-linked flap duplexes that cannot thread through the structured arch react at modestly reduced rate, ruling out mechanisms involving resolution of secondary structure. Combined results explain how FEN avoids cutting template DNA between Okazaki fragments and link local FEN folding to catalysis and specificity: the arch is disordered when flaps are threaded to confer specificity for free 5'-ends, with subsequent ordering of the arch to catalyze hydrolysis.     

Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7

Flap endonucleases remove flap structures generated during DNA replication. Gene 6 protein of bacteriophage T7 is a 5′–3′-exonuclease specific for dsDNA. Here we show that gene 6 protein also possesses a structure-specific endonuclease activity similar to known flap endonucleases. The flap endonuclease activity is less active relative to its exonuclease activity. The major cleavage by the endonuclease activity occurs at a position one nucleotide into the duplex region adjacent to a dsDNA-ssDNA junction. The efficiency of cleavage of the flap decreases with increasing length of the 5′-overhang. A 3′-single-stranded tail arising from the same end of the duplex as the 5′-tail inhibits gene 6 protein flap endonuclease activity. The released flap is not degraded further, but the exonuclease activity then proceeds to hydrolyze the 5′-terminal strand of the duplex. T7 gene 2.5 single-stranded DNA-binding protein stimulates the exonuclease and also the endonuclease activity. This stimulation is attributed to a specific interaction between the two proteins because Escherichia coli single-stranded DNA binding protein does not produce this stimulatory effect. The ability of gene 6 protein to remove 5′-terminal overhangs as well as to remove nucleotides from the 5′-termini enables it to effectively process the 5′-termini of Okazaki fragments before they are ligated.     

Specific stimulation of the T7 gene 6 exonuclease by the phage T7 coded deoxyribonucleic acid binding protein

Bacteriophage T7 codes for a single-stranded DNA binding protein. This protein is the product of gene 2.5 and has been found previously to stimulate specifically the activity of the phage-coded DNA polymerase. We report here that the T7 DNA binding protein also stimulates the activity of the phage-coded exonuclease. The gene 6 exonuclease is a double-stranded DNA specific 5'-exonuclease that has been implicated in destruction of bacterial DNA, removal of RNA primers during DNA replication, genetic recombination, and DNA maturation. The enzyme is markedly inhibited by physiological concentrations of NaCl. This inhibition, which is due to a marked reduction in the Vmax of the enzyme, can be largely overcome by the phage-coded DNA binding protein. This stimulation is specific since the Escherichia coli DNA binding protein is without effect. The stimulation by the binding protein is apparently not due to its coating of the 3' single-stranded tails generated during the digestion. Kinetic studies show that the stimulation is due to a combined effect on both the Km and Vmax of the exonuclease. These studies are consistent with a loose binding of the binding protein to either the DNA or the exonuclease.     

The RAD2 domain of human exonuclease 1 exhibits 5' to 3' exonuclease and flap structure-specific endonuclease activities

The RAD2 family of nucleases includes human XPG (Class I), FEN1 (Class II), and HEX1/hEXO1 (Class III) products gene. These proteins exhibit a blend of substrate specific exo- and endonuclease activities and contribute to repair, recombination, and/or replication. To date, the substrate preferences of the EXO1-like Class III proteins have not been thoroughly defined. We report here that the RAD2 domain of human exonuclease 1 (HEX1-N2) exhibits both a robust 5' to 3' exonuclease activity on single- and double-stranded DNA substrates as well as a flap structure-specific endonuclease activity but does not show specific endonuclease activity at 10-base pair bubble-like structures, G:T mismatches, or uracil residues. Both the 5' to 3' exonuclease and flap endonuclease activities require a divalent metal cofactor, with Mg(2+) being the preferred metal ion. HEX1-N2 is approximately 3-fold less active in Mn(2+)-containing buffers and exhibits <5% activity in the presence of Co(2+), Zn(2+), or Ca(2+). The optimal pH range for the nuclease activities of HEX1-N2 is 7.2-8.2. The specific activity of its 5' to 3' exonuclease function is 2.5-7-fold higher on blunt end and 5'-recessed double-stranded DNA substrates compared with duplex 5'-overhang or single-stranded DNAs. The flap endonuclease activity of HEX1-N2 is similar to that of human flap endonuclease-1, both in terms of turnover efficiency (k(cat)) and site of incision, and is as efficient (k(cat)/K(m)) as its exonuclease function. The nuclease activities of HEX1-N2 described here indicate functions for the EXO1-like proteins in replication, repair, and/or recombination that may overlap with human flap endonuclease-1.     

Structures of human exonuclease 1 DNA complexes suggest a unified mechanism for nuclease family

Human exonuclease 1 (hExo1) plays important roles in DNA repair and recombination processes that maintain genomic integrity. It is a member of the 5' structure-specific nuclease family of exonucleases and endonucleases that includes FEN-1, XPG, and GEN1. We present structures of hExo1 in complex with a DNA substrate, followed by mutagenesis studies, and propose a common mechanism by which this nuclease family recognizes and processes diverse DNA structures. hExo1 induces a sharp bend in the DNA at nicks or gaps. Frayed 5' ends of nicked duplexes resemble flap junctions, unifying the mechanisms of endo- and exonucleolytic processing. Conformational control of a mobile region in the catalytic site suggests a mechanism for allosteric regulation by binding to protein partners. The relative arrangement of substrate binding sites in these enzymes provides an elegant solution to a complex geometrical puzzle of substrate recognition and processing.     

Complete enzymatic synthesis of DNA containing the SV40 origin of replication

The replication of simian virus 40 origin-containing DNA has been reconstituted in vitro with SV40 large T antigen and purified proteins isolated from HeLa cells. Covalently closed circular DNA (RF I') daughter molecules are formed in the presence of T antigen, a single-stranded DNA binding protein and DNA polymerase alpha-primase complex, together with ribonuclease H, DNA ligase, topoisomerase II, and a double-stranded specific exonuclease that has been purified to homogeneity. The 44-kDa exonuclease-digested oligo(rA) annealed to poly(dT) in the 5'----3' direction. DNA ligase and the 5'----3' exonuclease were essential for RF I' formation. Covalently closed circular duplex DNA and full length linear single-stranded DNA were detected by alkaline gel electrophoresis as products of the complete system. DNA replication in the absence of either DNA ligase or the 5'----3' exonuclease yielded DNA products that were half length (approximately 1500 nucleotides) and smaller Okazaki-like fragments (approximately 200 nucleotides). Hybridization experiments showed that the longer chains were synthesized from the leading strand template, while the small products were synthesized from the lagging strand template. These results suggest that the RNA primers attached to 5' ends of replicated DNA are completely removed by the 5'----3' exonuclease, with the assistance of RNase H.     

Deoxyribonucleic acid polymerase of bacteriophage T7. Characterization of the exonuclease activities of the gene 5 protein and the reconstituted polymerase.

Homogeneous gene 5 protein of bacteriophage T7, a subunit of T7 DNA polymerase, catalyzes the stepwise hydrolysis of single-stranded DNA in a 3' leads to 5' direction to yield nucleoside 5'-monophosphates. The gene 5 protein itself does not hydrolyze duplex DNA. However, in the presence of Escherichia coli thioredoxin, the host-specified subunit of T7 DNA polymerase, duplex DNA is hydrolyzed in a 3' leads to 5' direction to yield nucleoside 5'-monophosphates. The apparent Km for thioredoxin in the reaction is 4.8 x 10(-8) M, a value similar to that for the apparent Km of thioredoxin in the complementation assay with gene 5 protein to restore T7 DNA polymerase activity. Both exonuclease activities require Mg2+ and a sulfhydryl reagent for optimal activity, and both activities are sensitive to salt concentration. Deoxyribonucleoside 5'-triphosphates inhibit hydrolysis by both exonuclease activities; hydrolysis of single-stranded DNA by the gene 5 protein is inhibited even in the absence of thioredoxin where there is less than 2% active T7 DNA polymerase. E. coli DNA binding protein (helix destabilizing protein) stimulates the hydrolysis of duplex DNA up to 9-fold under conditions where the hydrolysis of the single-stranded DNA is inhibited 4-fold.     

The roles of fission yeast exonuclease 5 in nuclear and mitochondrial genome stability

The Exo5 family consists of bi-directional, single-stranded DNA-specific exonucleases that contain an iron-sulfur cluster as a structural motif and have multiple roles in DNA metabolism. S. cerevisiae Exo5 is essential for mitochondrial genome maintenance, while the human ortholog is important for nuclear genome stability and DNA repair. Here, we identify the Exo5 ortholog in Schizosaccharomyes pombe (spExo5). The activity of spExo5 is highly similar to that of the human enzyme. When the single-stranded DNA is coated with single-stranded DNA binding protein RPA, spExo5 become a 5′-specific exonuclease. Exo5Δ mutants are sensitive to various DNA damaging agents, particularly interstrand crosslinking agents. An epistasis analysis places exo5⁺ in the Fanconi pathway for interstrand crosslink repair. Exo5⁺ is in a redundant pathway with rad2⁺, which encodes the flap endonuclease FEN1, for mitochondrial genome maintenance. Deletion of both genes lead to severe depletion of the mitochondrial genome, and defects in respiration, indicating that either spExo5 or spFEN1 is necessary for mitochondrial DNA metabolism.     

Simian virus 40 (SV40) T antigen binds specifically to double-stranded DNA but not to single-stranded DNA or DNA/RNA hybrids containing the SV40 regulatory sequences.

Simian virus 40 T antigen has been shown previously to bind specifically with high affinity to sites within the regulatory region of double-stranded simian virus 40 DNA. Using competition filter binding and the DNA-binding immunoassay, we show that T antigen did not bind specifically to either early or late single-stranded DNA containing these binding sites. Moreover, T antigen did not bind these sequences present in single-stranded RNA, RNA/RNA duplexes, or RNA/DNA hybrids. T antigen did, however, bind as efficiently to single-stranded DNA-cellulose as to double-stranded DNA-cellulose. This binding was nonspecific because it was independent of the presence of T-antigen-binding sites. The implications of these observations are discussed.     

Wheat (Triticum vulgare) chloroplast nuclease ChSI exhibits 5' flap structure-specific endonuclease activity

The structure-specific ChSI nuclease from wheat (Triticum vulgare) chloroplast stroma has been previously purified and characterized in our laboratory. It is a single-strand-specific DNA and RNA endonuclease. Although the enzyme has been initially characterized and used as a structural probe, its biological function is still unknown. Localization of the ChSI enzyme inside chloroplasts, possessing their own DNA that is generally highly exposed to UV light and often affected by numerous redox reactions and electron transfer processes, might suggest, however, that this enzyme could be involved in DNA repair. The repair of some types of DNA damage has been shown to proceed through branched DNA intermediates which are substrates for the structure-specific DNA endonucleases. Thus we tested the substrate specificity of ChSI endonuclease toward various branched DNAs containing 5' flap, 5' pseudoflap, 3' pseudoflap, or single-stranded bulged structural motifs. It appears that ChSI has a high 5' flap structure-specific endonucleolytic activity. The catalytic efficiency (k(cat)/K(M)) of the enzyme is significantly higher for the 5' flap substrate than for single-stranded DNA. The ChSI 5' flap activity was inhibited by high concentrations of Mg(2+), Mn(2+), Zn(2+), or Ca(2+). However, low concentrations of divalent cations could restore the loss of ChSI activity as a consequence of EDTA pretreatment. In contrast to other known 5' flap nucleases, the chloroplast enzyme ChSI does not possess any 5'-->3' exonuclease activity on double-stranded DNA. Therefore, we conclude that ChSI is a 5' flap structure-specific endonuclease with nucleolytic activity toward single-stranded substrates.     

Human herpesvirus 6B origin-binding protein: DNA-binding domain and consensus binding sequence.

We previously demonstrated by a DNA-binding assay that the human herpesvirus 6B (HHV-6B) replication origin has a structure similar to those of alphaherpesviruses, although the HHV-6B and herpes simplex virus type 1 (HSV-1) origin-binding proteins (OBPs) and origins are not interchangeable. Here we describe additional properties of the interaction between HHV-6B OBP and the HHV-6B origin. Competitive electrophoretic mobility shift assays (EMSAs) with DNA duplexes containing single-base alterations allowed deduction of a consensus DNA sequence for HHV-6B-specific OBP binding, YGWYCWCCY, where Y is T or C and W is T or A, while that for HSV-1-specific binding was reported to be YGYTCGCACT. By EMSA, the HHV-6B OBP DNA-binding domain was mapped to a segment containing amino acids 482 to 770. However, in Southwestern (protein-DNA) blotting, the region sufficient for the DNA binding encompassed only amino acids 657 to 770. Similarly, Southwestern blotting showed that amino acids 689 to 851 of HSV-1 OBP had HSV-1 origin-binding activity, although this region was insufficient for origin binding in the EMSA. Although the longer DNA-binding domains identified by EMSA have marginal overall homology among HHV-6B and alphaherpesvirus OBP homologs, the smaller regions sufficient for the binding observed by Southwestern blotting have significant similarity. From these results, we propose a hypothesis that the DNA-binding domain of herpesvirus OBPs consists of two subdomains, one containing a conserved motif that contacts DNA directly, and another, less well conserved, that may modulate either the conformation or accessibility of the binding domain.     

Comparison of the catalytic parameters and reaction specificities of a phage and an archaeal flap endonuclease

Flap endonucleases (FENs) catalyse the exonucleolytic hydrolysis of blunt-ended duplex DNA substrates and the endonucleolytic cleavage of 5'-bifurcated nucleic acids at the junction formed between single and double-stranded DNA. The specificity and catalytic parameters of FENs derived from T5 bacteriophage and Archaeoglobus fulgidus were studied with a range of single oligonucleotide DNA substrates. These substrates contained one or more hairpin turns and mimic duplex, 5'-overhanging duplex, pseudo-Y, nicked DNA, and flap structures. The FEN-catalysed reaction properties of nicked DNA and flap structures possessing an extrahelical 3'-nucleotide (nt) were also characterised. The phage enzyme produced multiple reaction products of differing length with all the substrates tested, except when the length of duplex DNA downstream of the reaction site was truncated. Only larger DNAs containing two duplex regions are effective substrates for the archaeal enzyme and undergo reaction at multiple sites when they lack a 3'-extrahelical nucleotide. However, a single product corresponding to reaction 1 nt into the double-stranded region occurred with A. fulgidus FEN when substrates possessed a 3'-extrahelical nt. Steady-state and pre-steady-state catalytic parameters reveal that the phage enzyme is rate-limited by product release with all the substrates tested. Single-turnover maximal rates of reaction are similar with most substrates. In contrast, turnover numbers for T5FEN decrease as the size of the DNA substrate is increased. Comparison of the catalytic parameters of the A. fulgidus FEN employing flap and double-flap substrates indicates that binding interactions with the 3'-extrahelical nucleotide stabilise the ground state FEN-DNA interaction, leading to stimulation of comparative reactions at DNA concentrations below saturation with the single flap substrate. Maximal multiple turnover rates of the archaeal enzyme with flap and double flap substrates are similar. A model is proposed to account for the varying specificities of the two enzymes with regard to cleavage patterns and substrate preferences.