Exonuclease activity associated with a multiprotein form of HeLa cell DNA polymerase alpha. Purification and properties of the exonuclease

The DNase that is associated with a multiprotein form of HeLa cell DNA polymerase alpha (polymerase alpha 2) has two distinct exonuclease activities: the major activity initiates hydrolysis from the 3' terminus and the other from the 5' terminus of single-stranded DNA. The two exonuclease activities show identical rates of thermal inactivation and coincidental migration during chromatofocusing, glycerol gradient centrifugation, and nondenaturing polyacrylamide gel electrophoresis of the DNase. Moreover, the purified DNase shows a single protein band of Mr 69,000 following nondenaturing polyacrylamide and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 3'----5' exonuclease activity hydrolyzes only single-stranded DNA substrates and the products are 5' mononucleotides. This activity recognizes and excizes mismatched bases at the 3' terminus of double-stranded DNA substrates. The 3'----5' exonuclease does not hydrolyze 3' phosphoryl terminated single-stranded DNA substrates. The 5'----3' exonuclease activity also only hydrolyzes single-stranded DNA substrates. The rate of hydrolysis, however is only about 1/25th the rate of the 3'----5' exonuclease. This exonuclease activity requires a 5' single-stranded terminus in order to initiate hydrolysis and does not proceed into double-stranded regions. The products of hydrolysis by 5'----3' exonuclease are also 5' nucleoside monophosphates.     

DNA polymerase gamma from Xenopus laevis. II. A 3'----5' exonuclease is tightly associated with the DNA polymerase activity

Xenopus laevis DNA polymerase gamma co-purifies with a tightly associated 3'----5' exonuclease. The purified enzyme lacks 5'----3' exonuclease and endonuclease activity. The ratio of the 3'----5' exonuclease activity to DNA polymerase gamma activity remains constant over the final three chromatographic procedures. In addition, these activities co-sediment under partially denaturing conditions in the presence of ethylene glycol. The associated 3'----5' exonuclease activity removes a terminally mismatched nucleotide more rapidly than a correctly base-paired 3'-terminal residue, as expected if this exonuclease has a proofreading function. The 3'----5' exonuclease has the ability to release a terminal phosphorothioated nucleotide, a property shared with T4 DNA polymerase, but not with Escherichia coli DNA polymerase I.     

Bacteriophage N4-coded 5'----3' exonuclease. Purification and characterization

Bacteriophage N4 DNA replication requires the activity of a phage-induced exonuclease. We show here that the activity is phage coded. We have purified this enzyme to apparent homogeneity. It has a denatured molecular weight of 45,000 and exists in solution as a dimer. Duplex DNA is the preferred substrate which it degrades in a 5'----3' direction to 5' mononucleotides by a distributive mechanism. The enzyme does not act at a nick or a gap; indeed, it requires an end for activity. A possible role for this exonuclease in N4 replication is discussed.     

Mechanism of exonuclease action of BAL 31 nuclease

Two kinetically and molecularly distinct forms ('fast' (F) and 'slow' (S] of nuclease BAL 31 from Alteromonas espejiana effect the length reduction of linear duplex DNAs through a 3'----5'-directed exonuclease activity in conjunction with an endonuclease activity against the 5'-terminated single-stranded tails generated by the exonuclease activity. No evidence for a 5'----3' mode of exonuclease action was seen. Single-stranded DNA is degraded predominantly by the 3'----5' exonuclease action. There is a pronounced decrease, to roughly constant values, of the average lengths of the tails in partially digested duplexes at a constant extent of digestion with increasing nuclease concentration. This decrease correlates with an increasing extent of ligatability, in the absence of repair, under conditions favoring the joining of fully base-paired ends. The exonuclease action, at least against duplex substrates, is quasi-processive and removes approx. 18 and 28 nucleotides per productive enzyme-substrate encounter for the S and F species, respectively. The dependence on Ca2+ and Mg2+ concentrations of the activities has been determined.     

Characterization of a 3''----5'' exonuclease activity in the phage phi 29-encoded DNA polymerase.

Purified protein p2 of phage phi 29, characterized as a specific DNA polymerase involved in the initiation and elongation of phi 29 DNA replication, contains a 3'----5' exonuclease active on single-stranded DNA, but not on double-stranded DNA. No 5'----3' exonuclease activity was found. The 3'----5' exonuclease activity was shown to be associated with the DNA polymerase since 1) the two activities were heat-inactivated with identical kinetics and 2) both activities, present in purified protein p2, cosedimented in a glycerol gradient.     

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.     

Characterization of 3''----5'' exonuclease associated with DNA polymerase of silkworm nuclear polyhedrosis virus.

3'----5' Exonuclease specific for single-stranded DNA copurified with DNA polymerase of nuclear polyhedrosis virus of silkworm Bombyx mori (BmNPV Pol). BmNPV Pol has no detectable 5'----3' exonuclease activity on single-stranded or duplex DNA. Analysis of the products of 3'----5' exonucleolytic reaction showed that deoxynucleoside monophosphates were released during the hydrolysis of single-stranded DNA. The exonuclease activity cosedimented with the polymerase activity during ultracentrifugation of BmNPV Pol in glycerol gradient. The polymerase and the exonuclease activities of BmNPV Pol were inactivated by heat with nearly identical kinetics. The mode of the hydrolysis of single-stranded DNA by BmNPV Pol-associated exonuclease was strictly distributive. The enzyme dissociated from single-stranded DNA after the release of a single dNMP and then reassociated with a next polynucleotide being degradated.     

The role of exonucleolytic processing and polymerase-DNA association in bypass of lesions during replication in vitro. Significance for SOS-targeted mutagenesis

The role of exonuclease activity in trans-lesion DNA replication with Escherichia coli DNA polymerase III holoenzyme was investigated. RecA protein inhibited the 3'----5' exonuclease activity of the polymerase 2-fold when assayed in the absence of replication and had no effect on turnover of dNTPs into dNMPs. In contrast, single-stranded DNA-binding protein, which had no effect on the exonuclease activity in the absence of replication, showed a pronounced 7-fold suppression of the 3'----5' exonuclease activity during replication. The excision of incorporated dNMP alpha S residues from DNA by the 3'----5' exonuclease activity of DNA polymerase III holoenzyme was inhibited 10-20-fold; still no increase in bypass of pyrimidine photodimers was observed. Thus, in agreement with our previous results in which the exonuclease activity was inhibited at the protein level (Livneh, Z. (1986) J. Biol. Chem. 261, 9526-9533), inhibition at the DNA level also did not increase bypass of photodimers. Fractionation of the replication mixture after termination of DNA synthesis on a Bio-Gel A-5m column under conditions which favor polymerase-DNA binding yielded a termination complex which could perform turnover of dNTPs into dNMPs. Adding challenge-primed single-stranded DNA to the complex yielded a burst of DNA synthesis which was promoted most likely by DNA polymerase III holoenzyme molecules transferred from the termination complex to the challenge DNA thus demonstrating the instability of the polymerase-DNA association. Addition of a fresh sample of DNA polymerase III holoenzyme to purified termination products, which consist primarily of partially replicated molecules with nascent chains terminated at UV lesions, did not result in any net DNA synthesis as expected. However, reactivation of lesion-terminated primers was achieved by pretreatment with a 3'----5' exonuclease which excised 200 nucleotides or more, generating new 3'-OH termini located away from the UV lesions. When these exonuclease-treated products were subjected to a second round of replication, an increased level of DNA synthesis was observed including additional bypass of photodimers. These results suggest the possibility that 3'----5' exonuclease processing might be required at least transiently during one of the stages of trans-lesion DNA replication, which is believed to be the mechanism of SOS-targeted mutagenesis.     

Complexes of nuclear DNA-polymerases with 3'----5'-exonucleases from the rat liver

"Editing" 3'----5' exonuclease activity of DNA polymerases corrects replication errors. This activity associated with procaryotic DNA polymerases is not intrinsic to purified mammalian DNA polymerases. By means of extraction and subsequent gel filtration, several subspecies of complexes of 3'----5' exonuclease (E.C. 3.1.4.26) with DNA polymerases alpha, beta (E.C. 2.7.7.7) and some other proteins were isolated from chromatin, nucleoplasm, nuclear membrane, and cytosol. Complexes containing 3'----5' exonuclease manifest from 40 to 70% of total DNA polymerase activity revealed in different compartments of a hepatocyte. Molecular masses of the complexes amount from 250 to 1500 kDa They dissociate as a result of solution hydrophobization. DNA polymerase alpha activity enhances 5--8 folds during cell transition from G0 to S-period. The value of the ratio of 3'----5' exonuclease activity of different complexes to their DNA polymerase activity varies from 0.5 to 12. Other cases of discovery of the complexes of DNA polymerases with 3'----5' exonucleases are discussed. It is suggested that the absence of 3'----5' exonuclease active site in the DNA polymerase polypeptide is compensated by the complex formation of the corresponding enzymes.     

Purification and properties of 2-aminoadipate: 2-oxoglutarate aminotransferase from bovine kidney.

Escherichia coli endonuclease IV hydrolyses the C(3')-O-P bond 5' to a 3'-terminal base-free deoxyribose. It also hydrolyses the C(3')-O-P bond 5' to a 3'-terminal base-free 2',3'-unsaturated sugar produced by nicking 3' to an AP (apurinic or apyrimidinic) site by beta-elimination; this explains why the unproductive end produced by beta-elimination is converted by the enzyme into a 3'-OH end able to prime DNA synthesis. The action of E. coli endonuclease IV on an internal AP site is more complex: in a first step the C(3')-O-P bond 5' to the AP site is hydrolysed, but in a second step the 5'-terminal base-free deoxyribose 5'-phosphate is lost. This loss is due to a spontaneous beta-elimination reaction in which the enzyme plays no role. The extreme lability of the C(3')-O-P bond 3' to a 5'-terminal AP site contrasts with the relative stability of the same bond 3' to an internal AP site; in the absence of beta-elimination catalysts, at 37 degrees C the half-life of the former is about 2 h and that of the latter 200 h. The extreme lability of a 5'-terminal AP site means that, after nicking 5' to an AP site with an AP endonuclease, in principle no 5'----3' exonuclease is needed to excise the AP site: it falls off spontaneously. We have repaired DNA containing AP sites with an AP endonuclease (E. coli endonuclease IV or the chromatin AP endonuclease from rat liver), a DNA polymerase devoid of 5'----3' exonuclease activity (Klenow polymerase or rat liver DNA polymerase beta) and a DNA ligase. Catalysts of beta-elimination, such as spermine, can drastically shorten the already brief half-life of a 5'-terminal AP site; it is what very probably happens in the chromatin of eukaryotic cells. E. coli endonuclease IV also probably participates in the repair of strand breaks produced by ionizing radiations: as E. coli endonuclease VI/exonuclease III, it is a 3'-phosphoglycollatase and also a 3'-phosphatase. The 3'-phosphatase activity of E. coli endonuclease VI/exonuclease III and E. coli endonuclease IV can also be useful when the AP site has been excised by a beta delta-elimination reaction.     

Linear plasmidlike DNA in the plant pathogenic fungus Fusarium oxysporum f. sp. conglutinans.

Double-stranded, 1.9-kilobase-pair (kbp) DNA molecules were found in 18 strains representing three pathogenic races of Fusarium oxysporum f. sp. conglutinans. The DNA element (pFOXC1) from a race 1 strain and the DNA element (pFOXC2) from a race 2 strain were shown by restriction endonuclease mapping to be linear. pFOXC2 was found in mitochondrial preparations and appears to have blocked 5' termini, as it was sensitive to 3'----5' exonuclease III but insensitive to 5'----3' lambda exonuclease. The major 1.8-kbp BglII restriction endonuclease fragment of pFOXC2 was cloned in plasmid pUC12. The recombinant plasmid (pCK1) was not homologous to the mitochondrial or nuclear genomes from F. oxysporum f. sp. conglutinans. This suggests that pFOXC2 is self-replicating. pCK1 was homologous to all 1.9-kbp DNA elements of race 2 but was not homologous to those of race 1 or race 5. All race 1 and 5 elements were also shown to share common DNA sequences.     

Human AP endonuclease possesses a significant activity as major 3'-5' exonuclease in human leukemia cells

Apurinic/apyrimidinic (AP) endonuclease (Ape1) is the major cellular enzyme responsible for repairing AP-sites in DNA. It can cleave the DNA phosphodiester backbone immediately 5(') to an AP-site. Ape1 also shows 3(')-phosphodiesterase activity, a 3(')-phosphatase activity, and an RNaseH activity. However, regarding its exonuclease activity, it remains controversial whether human Ape1 may possess a 3(')-5(') exonuclease activity. During the course of study to search for the major nuclease activity to double-stranded DNA in human leukemia cells, we purified a 37 kDa Mg(2+)-dependent exonuclease from cytosolic fraction of human leukemia U937 cells. Surprisingly, this exonuclease is Ape1. We demonstrated for the first time that Ape1 possesses a significant activity as major 3(')-5(') exonuclease in human leukemia cells. In addition, we also observed that translocation of cytoplasmic Ape1 into nucleus occurs during DNA damage.     

Binding of captan to DNA polymerase I from Escherichia coli and the concomitant effect on 5'----3' exonuclease activity

Captan (N-[(trichloromethyl)thio]-4-cyclohexene-1,2-dicarboximide) was shown to bind to DNA polymerase I from Escherichia coli. The ratio of [14C] captan bound to DNA pol I was 1:1 as measured by filter binding studies and sucrose gradient analysis. Preincubation of enzyme with polynucleotide prevented the binding of captan, but preincubation of enzyme with dGTP did not. Conversely, when the enzyme was preincubated with captan, neither polynucleotide nor dGTP binding was blocked. The modification of the enzyme by captan was described by an irreversible second-order rate process with a rate of 68 +/- 0.7 M-1 s-1. The interaction of captan with DNA pol I altered each of the three catalytic functions. The 3'----5' exonuclease and polymerase activities were inhibited, and the 5'----3' exonuclease activity was enhanced. In order to study the 5'----3' exonuclease activity more closely, [3H]hpBR322 (DNA-[3H]RNA hybrid) was prepared from pBR322 plasmid DNA and used as a specific substrate for 5'----3' exonuclease activity. When either DNA pol I or polynucleotide was preincubated with 100 microM captan, 5'----3' exonuclease activity exhibited a doubling of reaction rate as compared to the untreated sample. When 100 microM captan was added to the reaction in progress, 5'----3' exonuclease activity was enhanced to 150% of the control value. Collectively, these data support the hypothesis that captan acts on DNA pol I by irreversibly binding in the template-primer binding site associated with polymerase and 3'----5' exonuclease activities. It is also shown that the chemical reaction between DNA pol I and a single captan molecule proceeds through a Michaelis complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coprecipitation of 3'----5' exonuclease and DNA polymerase gamma with anti-DNA polymerase gamma antibody

Highly purified preparations of chick embryo DNA polymerase gamma contained 3'----5' exonuclease activity which might be responsible for the exonucleolytic proofreading during DNA synthesis [Kunkel, T.A. & Soni, A. (1988) J. Biol. Chem. 262, 4450-4459]. A rabbit antibody produced against highly purified chick DNA polymerase gamma precipitated 3'----5' exonuclease activity to the same extent as DNA polymerase gamma activity. Furthermore, the antibody neutralized the two enzyme activities to an equal extent. However, the exonuclease activity was more resistant than DNA polymerase gamma activity to thermal treatment at 50 degrees C, although both activities were partially protected with polynucleotides. The results obtained suggest that these two enzymes are associated as a single enzyme complex or that the two activities reside in a single molecule, and the active site of DNA polymerase gamma and 3'----5' exonuclease are, although not identical, closely correlated.     

Biochemical characterization of bacteriophage T4 Mre11-Rad50 complex

The Mre11-Rad50 complex (MR) from bacteriophage T4 (gp46/47) is involved in the processing of DNA double-strand breaks. Here, we describe the activities of the T4 MR complex and its modulation by proteins involved in homologous recombination. T4 Mre11 is a Rad50- and Mn(2+)-dependent dsDNA exonuclease and ssDNA endonuclease. ATP hydrolysis is required for the removal of multiple nucleotides via dsDNA exonuclease activity but not for the removal of the first nucleotide or for ssDNA endonuclease activity, indicating ATP hydrolysis is only required for repetitive nucleotide removal. By itself, Rad50 is a relatively inefficient ATPase, but the presence of Mre11 and dsDNA increases ATP hydrolysis by 20-fold. The ATP hydrolysis reaction exhibits positive cooperativity with Hill coefficients ranging from 1.4 for Rad50 alone to 2.4 for the Rad50-Mre11-DNA complex. Kinetic assays suggest that approximately four nucleotides are removed per ATP hydrolyzed. Directionality assays indicate that the prevailing activity is a 3' to 5' dsDNA exonuclease, which is incompatible with the proposed role of MR in the production of 3' ssDNA ends. Interestingly, we found that in the presence of a recombination mediator protein (UvsY) and ssDNA-binding protein (gp32), Mre11 is capable of using Mg(2+) as a cofactor for its nuclease activity. Additionally, the Mg(2+)-dependent nuclease activity, activated by UvsY and gp32, results in the formation of endonuclease reaction products. These results suggest that gp32 and UvsY may alter divalent cation preference and facilitate the formation of a 3' ssDNA overhang, which is a necessary intermediate for recombination-mediated double-strand break repair.     

Restoration of the primer activity of gamma-irradiated DNA by nucleases from rat liver chromatin

gamma-Irradiation of DNA results in a several-fold decrease of its primer activity measured as one substrate synthesis catalyzed by DNA polymerase beta. However, the combined treatment of injured DNA with 3'----5' exonuclease and endonuclease I from rat liver chromatin almost normalizes primer activity of DNA. Therefore the above-mentioned nucleases are capable of excising the gamma-injured nucleotides from 3'-OH ends of DNA.     

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.     

Mn2+-dependent endonuclease activity of chromatin

The endogenous endonuclease activity of chromatin in isolated rat liver nuclei in the presence of Mn2+, Mg2+ and Ca2+ + Mg2+ was studied. The existence of a Mn2+-dependent endonuclease activity not coupled with the Ca2+, Mg2+-dependent endonuclease was demonstrated, which was weaker than the former one in isolated cell nuclei but higher than in the preparation of Ca2+, Mg2+-dependent nuclease obtained by gel filtration through Toyopearl HW 60F. The Mn2+-dependent splitting of chromatin predominantly occurs at linker DNA of distal parts of chromatin loops. A split-off of purified DNA was more universal than in the presence of Ca2+, Mg2+-dependent endonuclease; the hydrolysis rate of native and denaturated DNA appeared to be the same.     

Action of Escherichia coli and human 5'----3' exonuclease functions at incised apurinic/apyrimidinic sites in DNA.

The 5'----3' exonuclease activity of E. coli DNA polymerase I and a related enzyme activity in mammalian cell nuclei, DNase IV, are unable to catalyse the excision of free deoxyribose-phosphate from apurinic/apyrimidinic (AP) sites incised by an AP endonuclease. Instead, the sugar phosphate residue is slowly released as part of a short oligonucleotide. These products have been characterised as dimers and trimers by comparison of their retention time on reverse-phase HPLC with reference compounds prepared by acid depurination of a dinucleotide, trinucleotide and tetranucleotide containing a 5'-terminal dAMP residue. The similar mode of action of these enzymes at 5'-incised AP sites provides an explanation for the minority of repair patches larger than one nucleotide observed when AP sites are repaired by E. coli and mammalian cell extracts in vitro and strengthens the functional analogy between the two activities.