Effect of ATP on the Friend Murine leukemia virus-associated endonuclease activity and the endonuclease activity of the avian myeloblastosis virus RNA-directed DNA polymerase

The Mn2+-dependent endonuclease activity associated with the avian myeloblastosis virus RNA-directed DNA polymerase has been shown to be activated by ATP in the presence of Mg2+. In the presence of Mn2+ the endonucleolytic activity was stimulated about 3-fold by the addition of ATP. The earlier identified Mr = 40,000 Friend murine leukemia virus (F-MuLV)-associated endonuclease which functions in the presence of both Mg2+ and Mn2+ has also been shown to be similarly stimulated by ATP. For both endonuclease activities stimulation was only observed at ATP concentrations above 0.5 mM, and it did not increase upon elevating the ATP concentration above 2.5 mM. ADP and dATP also stimulated both activities, although not to the same extent as ATP. GTP had no apparent effect and AMP seemed to inhibit both activities. The effect ATP analogs had on the F-MuLV associated endonuclease activity could suggest that the endonuclease reaction in the presence of ATP might involve the cleavage of beta-gamma phosphate bonds in ATP. Neither adenyl-5'-yl imidodiphosphate nor (beta, gamma-methylene)adenosine 5'-triphosphate stimulated the activity, whereas significant stimulation was observed in the presence of (alpha, beta-methylene)adenosine 5'-triphosphate. Although no ATPase activity could be detected in the purified F-MuLV endonuclease preparation, the data do not exclude the possibility that ATP may be cleaved in amounts which are equivalent to the number of nicks introduced into DNA by the virus-associated endonuclease. In the presence of ATP and Mg2+ the F-MuLV-associated endonuclease nicked both supercoiled and linear DNA duplexes extensively, although the former was nicked more readily than the latter. Single-stranded DNA functioned poorly as a substrate. The nicks introduced by the enzyme contained a 5'-phosphoryl terminus and a 3'-hydroxyl group.     

Characterization of an endonuclease activity associated with Friend-murine leukemia virus

An endonuclease activity shown to be associated with Friend leukemia virus has been characterized using double-stranded phi X174 DNA as substrate. In the presence of Mg2+, the endonuclease activity was able to convert supercoiled circular DNA duplexes to the relaxed form by introducing single-stranded nicks into the DNA. Most of the nicked DNA duplexes contained only one nick per strand, since unit length DNA was the predominant species obtained when the nicked DNA was analyzed by alkaline sucrose gradient centrifugation. The regions into which the nick could be introduced were evenly distributed around the circular DNA molecule. When Mn2+ was substituted for Mg2+ in the reaction mixture, the number of nicks introduced into circular DNA duplexes by the virus associated endonuclease was greatly increased. In contrast to circular duplexes, linear duplexes and single-stranded DNA functioned poorly as substrates for the virus-associated enzyme. The Friend leukemia virus-associated endonuclease activity is with respect to these characteristics very similar to the endonuclease activity associated with the p32 protein of the avian myeloblastosis virus [1]. The molecular weight of the Friend leukemia virus endonuclease was estimated by gel filtration on a Sephacryl S-200 column to be about 45 000.     

Structural and enzymological characterization of a deoxyribonucleic acid dependent adenosine triphosphatase from KB cell nuclei

We have purified to near homogeneity the single DNA-dependent ATPase activity that we have identified in extracts of KB cell nuclei. The protein structure of the enzyme was defined by sodium dodecyl sulfate gel electrophoresis, which revealed a single protein band of 75000 daltons that was coincident with the profile of ATPase activity resolved by the final step of agarose-ATP chromatography or by isoelectric focusing. The enzyme has a pI of 8.5, a Stokes' radius by gel filtration of 3.8 nm, and a sedimentation coefficient in high salt of 5.3 S. At low ionic strength the enzyme activity sediments at 7.0 S, suggesting that it may dimerize under these conditions. The purified enzyme has a specific activity of 5.9 X 10(5) nmol of ATP hydrolyzed per h per mg of protein and is devoid of endonuclease, exonuclease, RNA or DNA polymerase, nicking-closing, and gyrase activities at exclusion limits of 10(-6)-10(-8) of the ATPase activity. The enzyme can hydrolyze only ATP or dATP, to generate ADP or dADP plus Pi, but the other NTPs and dNTPs are competitive inhibitors of the enzyme with respect to ATP. A divalent cation (Mg2+ greater than Mn2+ greater than Ca2+) as well as a nucleic acid cofactor is required for activity. Single-stranded DNA or deoxyhomopolymers are most effective, but blunt-ended linear and nicked circular duplex DNA molecules are also used at Vmax values approximately 20% of that obtained with single-stranded DNA. Intact duplex DNA and polyribonucleotides are unable to support ATP hydrolysis. Velocity gradient sedimentation studies corroborate the interpretations of the kinetic analyses and demonstrate enzyme binding to single-stranded DNA and nicked duplex DNA but not to intact duplex DNA. Although we have not succeeded directly in demonstrating DNA unwinding by this protein, preliminary results suggest that in the presence of ATP, the ATPase can stimulate the reactivity of homogeneous human DNA polymerases alpha and beta on nicked duplex DNA substrates.     

Purification and properties of DNA endonucleases associated with Friend leukemia virus.

An endonuclease associated with the core of Friend leukemia virus (FLV) has been purified more than 10(3)-fold by ion exchange chromatography and gel filtration. Its molecular weight was determined by gel filtration to be about 40,000. Divalent cations were required for the endonuclease to function and KCl concentrations above 50 mM inhibited the enzyme activity. In the presence of Mg++ the purified enzyme nicked preferentially supercoiled circular DNA duplexes and in most of these molecules only one single-stranded nick was introduced per strand. The regions into which the nick could be introduced appeared to be randomly distributed on the circular molecule. When Mn++ was substituted for Mg++ the number of nicks introduced into DNA by the purified enzyme was greatly increased, and both relaxed circular and linear DNA duplexes were nicked as well as supercoiled circular DNA duplexes. Prior to its purification, however, in the presence of Mn++ the endonuclease activity in the virus extract was able to differentiate between circular and linear DNA duplexes, since both supercoiled and relaxed circular duplexes were nicked much more readily than linear duplexes. Single-stranded DNA functioned poorly as a substrate for the purified enzyme.     

AP endonuclease 1 has no biologically significant 3(')-->5(')-exonuclease activity

The 3(')-->5(')-exonucleolytic activity of human apurinic/apyrimidinic endonuclease 1 (APE1) on mispaired DNA at the 3(')-termini of recessed, nicked or gapped DNA molecules was analyzed and compared with the primary endonucleolytic activity. We found that under reaction conditions optimal for AP endonuclease activity the 3(')-->5(')-exonuclease activity of APE1 manifests only at enzyme concentration elevated by 6-7 orders of magnitude. This activity does not show a preference to mismatched compared to matched DNA structures as well as to nicked or gapped DNA substrates in comparison to recessed ones. Therefore, the 3(')-->5(')-exonuclease activity associated with APE1 can hardly be considered as key mechanism that improves fidelity of DNA repair.     

Nuclease TT1 from Thermus thermophilus HB8 has an endonuclease activity preferential to circular DNAs.

Homogeneously purified nuclease TT1 from Thermus thermophilus HB8 is known as an exonuclease to produce 5'-mononucleotides. Besides the exonuclease activity, nuclease TT1 also possesses endonuclease activity preferential to superhelical (form I) and single-stranded circular DNA. Although the rate of cleavage is slower than that of form I, covalently closed circular DNA (form I') is also cleaved. Form I DNA was nicked to yield relaxed circles (form II) first, and was then nicked at the opposite site to yield unit length linear DNA (form III) which was subsequently hydrolyzed to 5'-mononucleotides exonucleolytically. Both endo- and exo-nuclease activities co-migrate on polyacrylamide gels. The general properties of the endonuclease activity are very similar to those of the exonuclease activity. The temperature optimum for endonuclease activity was 85 degrees C. The pH-optimum was in pH-range from 7.5-9.1. The enzyme was active over a wide range of Mg2+ concentrations (2.5-125 mM), and was inhibited by EDTA. A linear substrate such as (dT)8 was a competitive inhibitor for this endonuclease activity.     

Mycobacterium tuberculosis RecA intein possesses a novel ATP-dependent site-specific double-stranded DNA endonuclease activity

Mycobacterium tuberculosis recA harbors an intervening sequence in its open reading frame, presumed to encode an endonuclease (PI-MtuI) required for intein homing in inteinless recA allele. Although the protein-splicing ability of PI-MtuI has been characterized, the identification of its putative endonuclease activity has remained elusive. To investigate whether PI-MtuI possesses endonuclease activity, recA intervening sequence was cloned, overexpressed, and purified to homogeneity. Here we show that PI-MtuI bound both single- and double-stranded DNA with similar affinity but failed to cleave DNA in the absence of cofactors. Significantly, PI-MtuI nicked supercoiled DNA in the presence of alternative cofactors but required both Mn(2+) and ATP to generate linear double-stranded DNA. We observed that PI-MtuI was able to inflict a staggered double-strand break 24 bp upstream of the insertion site in the inteinless recA allele. Similar to a few homing endonucleases, DNA cleavage by PI-MtuI was specific with an exceptionally long cleavage site spanning 22 bp. The kinetic mechanism of PI-MtuI promoted cleavage supports a sequential rather than concerted pathway of strand cleavage with the formation of nicked double-stranded DNA as an intermediate. Together, these results reveal that RecA intein is a novel Mn(2+)-ATP-dependent double-strand specific endonuclease, which is likely to be important for homing process in vivo.     

Properties of a DNA repair endonuclease from mouse plasmacytoma cells

The properties of a DNA-repair endonuclease isolated from mouse plasmacytoma cells have been further studied. It acted on ultraviolet-light-irradiated supercoiled DNA, and the requirement for a supercoiled substrate was absolute at ultraviolet light doses below 1.5 kJ m-2. At higher doses relaxed DNA could also serve as a substrate, but the activity on this DNA was due mostly to hydrolysis of ultraviolet-light-induced apurinic/apyrimidinic (AP) sites by the AP-endonuclease activity associated with the enzyme. The latter enzyme activity did not require a supercoiled form of the DNA. The enzyme also introduced nicks in unirradiated d(A-T)n. The nicked ultraviolet-light-irradiated DNA served as a substrate for DNA polymerase I, showing that the nicks contained free 3'-OH ends. Treatment of the nicked ultraviolet-light-irradiated DNA with bacterial alkaline phosphatase followed by T4 polynucleotide kinase, resulted in the phosphorylation of the 5' ends of the nicks, indicating that the nicks possessed a 5'-phosphate group; 5'- and 3'-mononucleotide analyses of the labelled DNA suggested that the enzyme introduced breaks primarily between G and T residues. The enzyme did not act on any specific region on the supercoiled DNA molecule; it produced random nicks in ultraviolet-light-modified phi X 174 replicative form I DNA. Antibodies raised against ultraviolet-light-irradiated DNA inhibited the activity. DNA adducts such as N-acetoxy-2-acetylaminofluorene and psoralen were not recognized by the enzyme. It is suggested that the enzyme has a specificity directed toward helical distortions.     

Cooperation of the N-terminal Helicase and C-terminal endonuclease activities of Archaeal Hef protein in processing stalled replication forks

Blockage of replication fork progression often occurs during DNA replication, and repairing and restarting stalled replication forks are essential events in all organisms for the maintenance of genome integrity. The repair system employs processing enzymes to restore the stalled fork. In Archaea Hef is a well conserved protein that specifically cleaves nicked, flapped, and fork-structured DNAs. This enzyme contains two distinct domains that are similar to the DEAH helicase family and XPF nuclease superfamily proteins. Analyses of truncated mutant proteins consisting of each domain revealed that the C-terminal nuclease domain independently recognized and incised fork-structured DNA. The N-terminal helicase domain also specifically unwound fork-structured DNA and Holliday junction DNA in the presence of ATP. Moreover, the endonuclease activity of the whole Hef protein was clearly stimulated by ATP hydrolysis catalyzed by the N-terminal domain. These enzymatic properties suggest that Hef efficiently resolves stalled replication forks by two steps, which are branch point transfer to the 5'-end of the nascent lagging strand by the N-terminal helicase followed by template strand incision for leading strand synthesis by the C-terminal endonuclease.     

Isolation and purification of calcium and magnesium dependent endonuclease from rat liver nuclei

An endonuclease associated with rat liver chromatin was extracted with 0.6 M NaCl and purified by ammonium sulfate fractionation and Sephadex G-100 gel filtration. The enzyme produces single strand scissions on native DNA at neutral pH in the presence of 1 mM CaCl₂ and 5 mM MgCl₂. Alkali-denatured DNA was not nicked by the enzyme. Omission of Ca²⁺ reduced the enzyme activity to about one seventh. Without Ca²⁺, however, Mn²⁺ was more effective than Mg²⁺. The molecular weight of the enzyme is about 27,000.     

An endonucleolytic activity of nuclease TT1 specific for superhelical DNA.

Highly purified nuclease TT1 from T. thermophilus HB8 acts on a linear single- and double-stranded DNA as an exonuclease and produces 5'-mononucleotides either from the 5'- or 3'-terminus. It was found that the enzyme also possesses an endonuclease activity specific for superhelical (form I) and single-stranded circular DNA. Form I of various kinds of DNA (phi X174, PM2, Co1E1 and RF 1010 etc.) is nicked to yield first relaxed circles (form II) and then nicked at the opposite site to yield unit length linear DNA (form III), which is subsequently hydrolyzed from the 5'- or 3'-terminus. A single cleavage of the form I of phi X174 DNA seemed to occur at a limited number of unique sites. Both endonuclease and the known exonuclease activities co-migrate on polyacrylmide gels, show the same pH and temperature optima, are stimulated by Mg2+ and are inactivated by EDTA similarly.     

The time-resolved kinetics of superhelical DNA cleavage by BamHI restriction endonuclease

The kinetics of the cleavage of superhelical plasmid DNA (pBR322) by the restriction endonuclease, BamHI, have been analyzed in terms a compartmental model consistent with the chemistry first proposed by Rubin and Modrich (Rubin, R. A., and Modrich, P. (1978) Nucleic Acids Res. 5, 2991-2997) for analysis of the kinetics of the restriction endonuclease, EcoRI. The model was defined in terms of two compartments representing DNA substrate (bound and free), two compartments representing nicked intermediate (bound and free), one compartment representing linear product, and one compartment for free enzyme. A simultaneous analysis of concentration changes over time of the three DNA forms (superhelical, nicked, and linear) at six different enzyme concentrations was undertaken employing this compartmental model using SAAM (Simulation Analysis And Modeling) software. Results showed that rate constants characterizing the association of enzyme with superhelical DNA (6.0 x 10(5) M-1 s-1) and nicked DNA (2.8 x 10(5) M-1 s-1) were similar in magnitude and rate constants characterizing cleavage of the first (1.2 x 10(-2) s-1) and second phosphodiester bonds (3.1 x 10(-2) s-1) were also similar. The analysis yields a kinetically determined equilibrium constant of 12.9 nM for the dissociation of nicked intermediate from the enzyme. The rate constant describing the release of the nicked intermediate from the enzyme has a value of 3.7 x 10(-3) s-1. By comparing the value of this release rate constant to the value of the constant describing the second cleavage event, it can be determined that only 10% of the nicked intermediate bound to the enzyme is released as free nicked DNA and that 90% of the nicked intermediate is processed to the linear form without being released. Hence, most of the DNA is cleaved as the result of a single enzyme-DNA recognition event. No steady state assumptions were made in the analysis. The approach was to directly solve the differential equations which described the kinetic processes using an interactive method. This study demonstrates the usefulness of this approach for the analysis of kinetics of protein-DNA interactions for the restriction endonucleases.     

Photoalkylated DNA and ultraviolet-irradiated DNA are incised at cytosines by endonuclease III.

Photoalkylation, the ultraviolet irradiation of DNA with isopropanol and di-tert-butylperoxide, causes a variety of base alterations. These include 8-(2-hydroxy-2-propyl)guanines, 8-(2-hydroxy-2-propyl)adenines and thymine dimers. An E. coli endonuclease against photoalkylated DNA was assayed by conversion of superhelical PM2 phage DNA to the nicked form. Enzyme activities were compared between extracts of strain BW9109 (xth-), lacking exonuclease III activity, and strain BW434 (xth-,nth-), deficient in both exonuclease III and endonuclease III. The endonuclease level in the double mutant against substrate photoalkylated DNA was under 20% of the activity in the mutant lacking only exonuclease III. Irradiation of the DNA substrate in the absence of isopropanol did not affect the activity in either strain. Analysis by polyacrylamide gel electrophoresis identified the sites of DNA cleavage by purified E. coli endonuclease III as cytosines, both in DNA irradiated at biologically significant wavelengths and in photoalkylated DNA. Neither 8-(2-hydroxy-2-propyl)purines, pyrimidine dimers, uracils nor 6-4'-(pyrimidin-2'-one)pyrimidines were substrates for the enzyme.     

A cellular single-stranded DNA-dependent ATPase associated with simian virus 40 chromatin

A single-stranded DNA-dependent ATPase from monkey kidney tissue culture cells (CV-1) has been found associated with SV40 chromatin. This ATPase activity is distinguishable from the ATPase activity of T-antigen by the following properties: the Km for ATP, elution from phosphocellulose, and stimulation of the ATPase activity by single-stranded DNA but not by double-stranded DNA. The ATPase has been isolated and characterized from the nuclei of uninfected cells. ATP hydrolysis is dependent on single-stranded DNA and a divalent cation. The km values for ATP and single-stranded DNA are 0.024 mM and 0.09 microgram/ml, respectively. The affinity of the ATPase for single-stranded DNA is sufficiently high that the enzyme co-sediments with single-stranded DNA in glycerol gradients. The binding of single-stranded DNA is independent of ATP and MgCl2; however, ATP hydrolysis increases the exchange of enzyme between different DNA molecules. Form I (superhelical) SV40 DNA is also a substrate for ATPase binding, but relaxed Form I, Form II (nicked circular), and double-stranded linear SV40 DNAs are not substrates. Because the DNA helix within chromatin is not under the same kind of tortional strain as Form I DNA, we hypothesize that the ATPase is bound to the single-stranded regions of replication forks in the SV40 chromatin.     

DNase Induced After Infection of KB Cells by Herpes Simplex Virus Type 1 or Type 2 II. Characterization of an Associated Endonuclease Activity

Purified preparations of the "exonuclease" specified by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) possess an endonuclease activity. The exonuclease and endonuclease activities copurify and cosediment in a sucrose density gradient. Endonuclease activity is only observed in the presence of a divalent cation, and Mg(2+) or Mn(2+) is equally effective as a cofactor with an optimal concentration of 2 mM. A slight amount of endonuclease activity is observed in the presence of Ca(2+), whereas no activity occurs in the presence of Zn(2+). In the presence of Mg(2+), Ca(2+) and Zn(2+) are inhibitory. Comparison of exonuclease and endonuclease activity in the presence of various divalent cations revealed that, at concentrations of Mn(2+) greater than 1 mM, only endonuclease activity occurs whereas endonuclease and exonuclease activity occur at all concentrations of Mg(2+). The endonuclease was affected by putrescine and spermidine to the same extent as the exonuclease activity, but in marked contrast the endonuclease was inhibited by a 10-fold-lower concentration of spermine compared to the exonuclease. The activity specified by HSV-1 and HSV-2 has very similar properties. HSV-1 and HSV-2 endonuclease cleave covalently closed circular DNA to yield, firstly, nicked circles and then linear DNA which is subsequently hydrolyzed to small oligonucleotides. Cleavage does not appear to be base sequence specific. Conversion of nicked circles to linear DNA and subsequent degradation of linear DNA occurs more rapidly in the presence of Mg(2+) than Mn(2+) presumably by virtue of the presence of the exonuclease activity. Nonsuperhelical covalently closed circular duplex DNA is cleaved by the endonucleases at a rate 60 times slower than the rate observed on the supercoiled form. These data indicate that the HSV-1 and HSV-2 endonuclease preferentially recognize single-stranded DNA regions.     

den V gene of bacteriophage T4 codes for both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities.

Recent studies have shown purified preparations of phage T4 UV DNA-incising activity (T4 UV endonuclease or endonuclease V of phage T4) contain a pyrimidine dimer-DNA glycosylase activity that catalyzes hydrolysis of the 5' glycosyl bond of dimerized pyrimidines in UV-irradiated DNA. Such enzyme preparations have also been shown to catalyze the hydrolysis of phosphodiester bonds in UV-irradiated DNA at a neutral pH, presumably reflecting the action of an apurinic/apyrimidinic endonuclease at the apyrimidinic sites created by the pyrimidine dimer-DNA glycosylase. In this study we found that preparations of T4 UV DNA-incising activity contained apurinic/apyrimidinic endonuclease activity that nicked depurinated form I simian virus 40 DNA. Apurinic/apyrimidinic endonuclease activity was also found in extracts of Escherichia coli infected with T4 denV+ phage. Extracts of cells infected with T4 denV mutants contained significantly lower levels of apurinic/apyrimidinic endonuclease activity; these levels were no greater than the levels present in extracts of uninfected cells. Furthermore, the addition of DNA containing apurinic or apyrimidinic sites to reactions containing UV-irradiated DNA and T4 enzyme resulted in competition for pyrimidine dimer-DNA glycosylase activity against the UV-irradiated DNA. On the basis of these results, we concluded that apurinic/apyrimidinic endonuclease activity is encoded by the denV gene of phage T4, the same gene that codes for pyrimidine dimer-DNA glycosylase activity.     

The HsdR subunit of R.EcoR124II: cloning and over-expression of the gene and unexpected properties of the subunit.

Type I restriction endonucleases are composed of three subunits, HsdR, HsdM and HsdS. The HsdR subunit is absolutely required for restriction activity; while an independent methylase is composed of HsdM and HsdS subunits. DNA cleavage is associated with a powerful ATPase activity during which DNA is translocated by the enzyme prior to cleavage. The presence of a Walker type I ATP-binding site within the HsdR subunit suggested that the subunit may be capable of independent enzymatic activity. Therefore, we have, for the first time, cloned and over-expressed the hsdRgene of the type IC restriction endonuclease EcoR124II. The purified HsdR subunit was found to be a soluble monomeric protein capable of DNA- and Mg2+-dependent ATP hydrolysis. The subunit was found to have a weak nuclease activity both in vivo and in vitro, and to bind plasmid DNA; although was not capable of binding a DNA oligoduplex. We were also able to reconstitute the fully active endonuclease from purified M. EcoR124I and HsdR. This is the first clear demonstration that the HsdR subunit of a type I restriction endonuclease is capable of independent enzyme activity, and suggests a mechanism for the evolution of the endonuclease from the independent methylase.     

An ATP-inhibited endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence: purification and characterization

In our studies on the role of enzymes in plant DNA replication, recombination, and repair, we isolated from cauliflower (Brassica oleracea L. var. botrytis) inflorescences a single-stranded DNA-specific endonuclease that was inhibited by ATP. The endonuclease, designated cauliflower nuclease II, was purified to near homogeneity through six successive column chromatographies. The enzyme is a single polypeptide with a molecular mass of 70 kDa as judged by the results of sodium dodecyl sulfate-polyacry amide gel electrophoresis, activity gel, and gel-filtration column chromatography. The enzyme can cleave a linear or a circular single-stranded DNA but cannot cut or nick a double-stranded DNA. The mode of activity of the nuclease is endonucleolytic and non-processive. Interestingly, the endonuclease activity is strongly inhibited by less than 0.1 mM ATP, although the role of this inhibition is thus far unclear. While ATPγS and GTP can also inhibit the activity, other ribonucleoside triphosphates are much less effective. The optimum pH of the enzyme is 5.6. The enzyme requires an exceptionally high ionic strength, 0.2 M KCI for optimum activity, and without these ions no activity can be detected. The endonuclease activity is stimulated by Ca²⁺, which cannot be replaced by Mg²⁺ or Mn²⁺. The features of the enzyme and its relation to plant DNA metabolism are discussed. Received: 26 March 1998 / Accepted: 4 June 1998     

Novel endonuclease in Archaea cleaving DNA with various branched structure

We identified a novel structure-specific endonuclease in Pyrococcus furiosus. This nuclease contains two distinct domains, which are similar to the DEAH helicase family at the N-terminal two-third and the XPF endonuclease superfamily at the C-terminal one-third of the protein, respectively. The C-terminal domain has an endonuclease activity cleaving the DNA strand at the 5'-side of nicked or flapped positions in the duplex DNA. The nuclease also incises in the proximity of the 5'-side of a branch point in the template strand for leading synthesis in the fork-structured DNA. The N-terminal helicase may work cooperatively to change the fork structure suitable for cleavage by the C-terminal endonuclease. This protein, designated as Hef (helicase-associated endonuclease for fork-structured DNA), may be a prototypical enzyme for resolving stalled forks during DNA replication, as well as working at nucleotide excision repair.