Human endonuclease V as a repair enzyme for DNA deamination

The human endonuclease V gene is located in chromosome 17q25.3 and encodes a 282 amino acid protein that shares about 30% sequence identity with bacterial endonuclease V. This study reports biochemical properties of human endonuclease V with respect to repair of deaminated base lesions. Using soluble proteins fused to thioredoxin at the N-terminus, we determined repair activities of human endonuclease V on deoxyinosine (I)-, deoxyxanthosine (X)-, deoxyoxanosine (O)- and deoxyuridine (U)-containing DNA. Human endonuclease V is most active with deoxyinosine-containing DNA but with minor activity on deoxyxanthosine-containing DNA. Endonuclease activities on deoxyuridine and deoxyoxanosine were not detected. The endonuclease activity on deoxyinosine-containing DNA follows the order of single-stranded I>G/I>T/I>A/I>C/I. The preference of the catalytic activity correlates with the binding affinity of these deoxyinosine-containing DNAs. Mg(2+) and to a much less extent, Mn(2+), Ni(2+), Co(2+) can support the endonuclease activity. Introduction of human endonuclease V into Escherichia coli cells deficient in nfi, mug and ung genes caused three-fold reduction in mutation frequency. This is the first report of deaminated base repair activity for human endonuclease V. The relationship between the endonuclease activity and deaminated deoxyadenosine (deoxyinosine) repair is discussed.     

Active site plasticity of endonuclease V from Salmonella typhimurium

Base deamination is a major type of DNA damage under nitrosative stress. Endonuclease V initiates repair of deaminated base damage by making a nucleolytic incision one nucleotide away from the 3' side of the lesion. Within the endonuclease V family, the substrate specificities are different from one enzyme to another. In this study, we investigated deamination lesion cleavage activities of endonuclease V from the macrophage-residing pathogen, Salmonella typhimurium. Salmonella endonuclease V exhibits limited turnover on cleavage of deoxyinosine- and xanthosine-containing DNA. Binding analysis indicates that this single-turnover property is caused by tight binding to nicked products. The nicking activity is similar between the double-stranded deoxyinosine- and deoxyxanthosine-containing DNA. Cleavage rates are not affected by bases opposite the deoxyinosine or deoxyxanthosine lesions. The enzyme is also active on single-stranded deoxyinosine- and deoxyxanthosine-containing DNA. Unlike endonuclease V from Thermotoga maritima, Salmonella endonucleae V can only turnover deoxyuridine-containing DNA to a limited extent when substrate is in excess. Binding analysis indicates that Salmonella endonuclease V achieves tight binding to deoxyuridine-containing DNA, a property that distinguishes it from Thermotoga endonuclease V. Cleavage analysis on mismatch-containing DNA also indicates that the active site of Salmonella endonuclease V can accommodate pyrimidine-containing mismatches, resulting in more comparable cleavage of pyrimidine- and purine-containing mismatches. This comprehensive DNA cleavage and binding analysis reveals the plastic nature in the active site of Salmonella endonuclease V, which allows the enzyme to enfold both purine and pyrimidine deaminated lesions or base pair mismatches.     

Endonuclease V protects Escherichia coli against specific mutations caused by nitrous acid

Endonuclease V (deoxyinosine 3'-endonuclease) of Escherichia coli K-12 is a putative DNA repair enzyme that cleaves DNA's containing hypoxanthine, uracil, or mismatched bases. An endonuclease V (nfi) mutation was tested for specific mutator effects on a battery of trp and lac mutant alleles. No marked differences were seen in frequencies of spontaneous reversion. However, when nfi mutants were treated with nitrous acid at a level that was not noticeably mutagenic for nfi(+) strains, they displayed a high frequency of A:T-->G:C, and G:C-->A:T transition mutations. Nitrous acid can deaminate guanine in DNA to xanthine, cytosine to uracil, and adenine to hypoxanthine. The nitrous acid-induced A:T-->G:C transitions were consistent with a role for endonuclease V in the repair of deaminated adenine residues. A confirmatory finding was that the mutagenesis was depressed at a locus containing N(6)-methyladenine, which is known to be relatively resistant to nitrosative deamination. An alkA mutation did not significantly enhance the frequency of A:T-->G:C mutations in an nfi mutant, even though AlkA (3-methyladenine-DNA glycosylase II) has hypoxanthine-DNA glycosylase activity. The nfi mutants also displayed high frequencies of nitrous acid-induced G:C-->A:T transitions. These mutations could not be explained by cytosine deamination because an ung (uracil-DNA N-glycosylase) mutant was not similarly affected. However, these findings are consistent with a role for endonuclease V in the removal of deaminated guanine, i.e., xanthine, from DNA. The results suggest that endonuclease V helps to protect the cell against the mutagenic effects of nitrosative deamination.     

A deoxyinosine specific endonuclease from hyperthermophile, Archaeoglobus fulgidus: a homolog of Escherichia coli endonuclease V

Deoxyadenosine undergoes spontaneous deamination to deoxyinosine in DNA. Based on amino acids sequence homology, putative homologs of endonuclease V were identified in several organisms including archaebacteria, eubacteria as well as eukaryotes. The translated amino acid sequence of the Archaeoglobus fulgidus nfi gene shows 39% identity and 55% similarity to the E. coli nfi gene. A. fulgidus endonuclease V was cloned and expressed in E. coli as a C-terminal hexa-histidine fusion protein. The C-terminal fusion protein was purified to apparent homogeneity by a combination of Ni(++) affinity and MonoS cation exchange liquid chromatography. The purified C-terminal fusion protein has a molecular weight of about 25kDa and showed endonuclease activity towards DNA containing deoxyinosine. A. fulgidus endonuclease V has an absolute requirement for Mg(2+) and an optimum reaction temperature at 85 degrees C. However, in contrast to E. coli endonuclease V, which has a wide substrate spectrum, endonuclease V from A. fulgidus recognized only deoxyinosine. These data suggest that the deoxyinosine cleavage activity is a primordial activity of endonuclease V and that multiple enzymatic activities of E. coli endonuclease V were acquired later during evolution.     

Modeling of Escherichia coli Endonuclease V structure in complex with DNA

Endonuclease V (EndoV) is a metal-dependent DNA repair enzyme involved in removal of deaminated bases (e.g., deoxyuridine, deoxyinosine, and deoxyxanthosine), with pairing specificities different from the original bases. Homologs of EndoV are present in all major phyla from bacteria to humans and their function is quite well analyzed. EndoV has been combined with DNA ligase to develop an enzymatic method for mutation scanning and has been engineered to obtain variants with different substrate specificities that serve as improved tools in mutation recognition and cancer mutation scanning. However, little is known about the structure and mechanism of substrate DNA binding by EndoV. Here, we present the results of a bioinformatic analysis and a structural model of EndoV from Escherichia coli in complex with DNA. The structure was obtained by a combination of fold-recognition, comparative modeling, de novo modeling and docking methods. The modeled structure provides a convenient tool to study protein sequence-structure-function relationships in EndoV and to engineer its further variants.     

A novel endonuclease that may be responsible for damaged DNA base repair in Pyrococcus furiosus

DNA is constantly damaged by endogenous and environmental influences. Deaminated adenine (hypoxanthine) tends to pair with cytosine and leads to the A:T→G:C transition mutation during DNA replication. Endonuclease V (EndoV) hydrolyzes the second phosphodiester bond 3′ from deoxyinosine in the DNA strand, and was considered to be responsible for hypoxanthine excision repair. However, the downstream pathway after EndoV cleavage remained unclear. The activity to cleave the phosphodiester bond 5′ from deoxyinosine was detected in a Pyrococcus furiosus cell extract. The protein encoded by PF1551, obtained from the mass spectrometry analysis of the purified fraction, exhibited the corresponding cleavage activity. A putative homolog from Thermococcus kodakarensis (TK0887) showed the same activity. Further biochemical analyses revealed that the purified PF1551 and TK0887 proteins recognize uracil, xanthine and the AP site, in addition to hypoxanthine. We named this endonuclease Endonuclease Q (EndoQ), as it may be involved in damaged base repair in the Thermococcals of Archaea.     

Arabidopsis thaliana endonuclease V is a ribonuclease specific for inosine-containing single-stranded RNA

Endonuclease V is highly conserved, both structurally and functionally, from bacteria to humans, and it cleaves the deoxyinosine-containing double-stranded DNA in Escherichia coli, whereas in Homo sapiens it catalyses the inosine-containing single-stranded RNA. Thus, deoxyinosine and inosine are unexpectedly produced by the deamination reactions of adenine in DNA and RNA, respectively. Moreover, adenosine-to-inosine (A-to-I) RNA editing is carried out by adenosine deaminase acting on dsRNA (ADARs). We focused on Arabidopsis thaliana endonuclease V (AtEndoV) activity exhibiting variations in DNA or RNA substrate specificities. Since no ADAR was observed for A-to-I editing in A. thaliana, the possibility of inosine generation by A-to-I editing can be ruled out. Purified AtEndoV protein cleaved the second and third phosphodiester bonds, 3′ to inosine in single-strand RNA, at a low reaction temperature of 20–25°C, whereas the AtEndoV (Y100A) protein bearing a mutation in substrate recognition sites did not cleave these bonds. Furthermore, AtEndoV, similar to human EndoV, prefers RNA substrates over DNA substrates, and it could not cleave the inosine-containing double-stranded RNA. Thus, we propose the possibility that AtEndoV functions as an RNA substrate containing inosine induced by RNA damage, and not by A-to-I RNA editing in vivo.     

Repair of deaminated bases in DNA

Deamination of DNA bases can occur spontaneously, generating highly mutagenic lesions such as uracil, hypoxanthine, and xanthine. When cells are under oxidative stress that is induced either by oxidizing agents or by mitochondrial dysfunction, additional deamination products such as 5-hydroxymethyluracil (5-HMU) and 5-hydroxyuracil (5-OH-Ura) are formed. The cellular level of these highly mutagenic lesions is increased substantially when cells are exposed to DNA damaging agent, such as ionizing radiation, redox reagents, nitric oxide, and others. The cellular repair of deamination products is predominantly through the base excision repair (BER) pathway, a major cellular repair pathway that is initiated by lesion specific DNA glycosylases. In BER, the lesions are removed by the combined action of a DNA glycosylase and an AP endonuclease, leaving behind a one-base gap. The gapped product is then further repaired by the sequential action of DNA polymerase and DNA ligase. DNA glycosylases that recognize uracil, 5-OH-Ura, 5-HMU (derived from 5-methylcytosine) and a T/G mismatch (derived from a 5-methylcytosine/G pair) are present in most cells. Many of these glycosylases have been cloned and well characterized. In yeast and mammalian cells, hypoxanthine is efficiently removed by methylpurine N-glycosylase, and it is thought that BER might be an important pathway for the repair of hypoxanthine. In contrast, no glycosylase that can recognize xanthine has been identified in either yeast or mammalian cells. In Escherichia coli, the major enzyme activity that initiates the repair of hypoxanthine and xanthine is endonuclease V. Endonuclease V is an endonuclease that hydrolyzes the second phosphodiester bond 3' to the lesion. It is hypothesized that the cleaved DNA is further repaired through an alternative excision repair (AER) pathway that requires the participation of either a 5' endonuclease or a 3'-5' exonuclease to remove the damaged base. The repair process is then completed by the sequential actions of DNA polymerase and DNA ligase. Endonuclease V sequence homologs are present in all kingdoms, and it is conceivable that endonuclease V might also be a major enzyme that initiates the repair of hypoxanthine and xanthine in mammalian cells.     

Lesion recognition and cleavage by endonuclease V: a single-molecule study

Endonuclease V (endo V) recognizes and cleaves deoxyinosine in deaminated DNA. These enzymatic activities are precursors of DNA repair and are fueled by metal ions such as Ca2+ and Mg2+, with the former being associated with protein binding and the latter with DNA cleavage. Using the technique of fluorescence resonance energy transfer (FRET), we determined the single-molecule kinetics of endo V in a catalytic cycle using a substrate of deoxyinosine-containing single-stranded DNA (ssDNA). The ssDNA was labeled with TAMRA, a fluorescence donor, while the endo V was labeled with Cy5, a fluorescence acceptor. The time lapses of FRET, resulting from the sequential association, recognition, and dissociation of the deoxyinosine by the endo V, were determined at 5.9, 14.5, and 9.1 s, respectively, in the presence of Mg2+. In contrast, the process of deoxyinosine recognition appeared little affected by the metal type. The prolonged association and dissociation events in the presence of the Ca2+-Mg2+ combination, as compared to that of Mg2+ alone, support the hypothesis that endo V has two metal binding sites to regulate its enzymatic activities.     

Two new site-specific endonucleases from Staphylococcus species strain D5

Staphylococcus species strain D5 containing two site-specific endonucleases, SspD5 I and SspD5 II, was found during screening of a bacterial strain collection from soil. These endonucleases were purified to functional homogeneity by sequential chromatography. Site-specific endonuclease SspD5 I recognizes sequence 5;-GGTGA(8N/8N) downward arrow-3; on DNA. Unlike Hph I, it cleaves DNA at a distance of 8 nucleotides from the recognized sequence on both chains producing blunt-end DNA fragments, while endonuclease Hph I cleaves DNA forming mononucleotide 3;-OH protruding ends. Thus, endonuclease SspD5 I is a new type II site-specific endonuclease and a neoschizomer of endonuclease Hph I. The advantage of this new endonuclease is that the blunt-end DNA products of this enzyme can be inserted without additional treatment into vector DNAs cleaved with endonucleases yielding DNA blunt-ends. Endonuclease SspD5 II recognizes site 5'-ATGCA T-3' and thus is an isoschizomer of endonuclease Nsi I. The molecular mass of SspD5 I is about 35 kD and that of SspD5 II is 40 kD. The enzymes exhibit maximal activity at 37 degrees C. The optimal buffer for the reaction is HRB (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 100 mM NaCl, and 1 mM dithiothreitol).     

Removal of deoxyinosine from the Escherichia coli chromosome as studied by oligonucleotide transformation

Deoxyinosine (dI) is produced in DNA by the hydrolytic or nitrosative deamination of deoxyadenosine. It is excised in a repair pathway that is initiated by endonuclease V, the product of the nfi gene. The repair was studied in vivo using high-efficiency oligonucleotide transformation mediated by the Beta protein of bacteriophage lambda in a mismatch repair-deficient host. Escherichia coli was transformed with oligonucleotides containing a selectable A-G base substitution mutation. When the mutagenic dG was replaced by a dI in the oligonucleotide, it lost 93-99% of its transforming ability in an nfi(+) cell, but it remained fully functional in an nfi mutant. Therefore, endonuclease V is responsible for most of the removal of deoxyinosine from DNA. New nfi mutants were isolated based on the strong selection provided by their tolerance for transformation by dI-containing DNA. The repair patch for dI was then measured by determining how close to the transforming dG residue a dI could be placed in the oligonucleotide before it interferes with transformation. At the endonuclease V cleavage site, three nucleotides were preferentially removed from the 3' end and two nucleotides were removed from the 5' end. dI:dT and dI:dC base pairs gave the same results. Caveats include possible interference by Beta protein and by mispaired bases. Thus, oligonucleotide transformation can be used to determine the relative importance of redundant repair pathways, to isolate new DNA repair mutants, and to determine with high precision the sizes of repair tracts in intact cells.     

Endonuclease V (nfi) Mutant of Escherichia coli K-12

Endonuclease V (deoxyinosine 3′ endonuclease), the product of the nfi gene, has a specificity that encompasses DNAs containing dIMP, abasic sites, base mismatches, uracil, and even untreated single-stranded DNA. To determine its importance in DNA repair pathways, nfi insertion mutants and overproducers (strains bearing nfi plasmids) were constructed. The mutants displayed a twofold increase in spontaneous mutations for several markers and an increased sensitivity to killing by bleomycin and nitrofurantoin. An nfi mutation increased both cellular resistance to and mutability by nitrous acid. This agent should generate potential cleavage sites for the enzyme by deaminating dAMP and dCMP in DNA to dIMP and dUMP, respectively. Relative to that of a wild-type strain, an nfi mutant displayed a 12- to 1,000-fold increase in the frequency of nitrite-induced mutations to streptomycin resistance, which are known to occur in A · T base pairs. An nfi mutation also enhanced the lethality caused by a combined deficiency of exonuclease III and dUTPase, which has been attributed to unrepaired abasic sites. However, neither the deficiency nor the overproduction of endonuclease V affected the growth of the single-stranded DNA phages M13 or X174 nor of Uracil-containing bacteriophage λ. These results suggest that endonuclease V has a significant role in the repair of deaminated deoxyadenosine (deoxyinosine) and abasic sites in DNA, but there was no evidence for its cleavage in vivo of single-stranded or uracil-containing DNA.     

Characterization of a wide range base-damage-endonuclease activity of mammalian rpS3

Mammalian rpS3, a ribosomal protein S3 with a DNA repair endonuclease activity, nicks heavily UV-irradiated DNA and DNA containing AP sites. RpS3 calls for a novel endonucleolytic activity on AP sites generated from pyrimidine dimers by T4 pyrimidine dimer glycosylase activity. This study revealed that rpS3 cleaves the lesions including AP sites, thymine glycols, and other UV damaged lesions such as pyrimidine dimers. This enzyme does not have a glycosylase activity as predicted from its amino acid sequence. However, it has an endonuclease activity on DNA containing thymine glycol, which is exactly overlapped with UV-irradiated or AP DNAs, indicating that rpS3 cleaves phosphodiester bonds of DNAs containing altered bases with broad specificity acting as a base-damage-endonuclease. RpS3 cleaves supercoiled UV damaged DNA more efficiently than the relaxed counterpart, and the endonuclease activity of rpS3 was inhibited by MgCl2 on AP DNA but not on UV-irradiated DNA.     

Endonuclease V-mediated deoxyinosine excision repair in vitro

Deoxyinosine (dI) in DNA can arise from hydrolytic or nitrosative deamination of deoxyadenosine. It is excised in a repair pathway that is initiated by endonuclease V, the nfi gene product, in Escherichia coli. Repair was studied in vitro using M13mp18 derived heteroduplexes containing a site-specific deoxyinosine. Unpaired dI/G mismatch resides within the recognition site for XhoI restriction endonucleases, permitting evaluation of repair occurring on deoxyinosine-containing DNA strand. Our results show that dI lesions were efficiently repaired in nfi⁺ E. coli extracts but the repair level was much reduced in nfi mutant extracts. We subjected the deoxyinosine-containing heteroduplex to a purified system consisting of soluble endonuclease V fusion protein, DNA polymerase I, and DNA ligase, along with the four deoxynucleoside triphosphates. Interestingly we found these three proteins alone are sufficient to process the dI lesion efficiently. We also found that the 3′-exonuclease activity of DNA polymerase I is sufficient to remove the dI lesion in this minimum reconstituted assay.     

Binding of T4 endonuclease V to deoxyribonucleic acid irradiated with ultraviolet light

Endonuclease V of bacteriophage T4 binds to UV-irradiated deoxyribonucleic acid (DNA) but not to unirradiated DNA. We have developed an assay to detect this binding, based on the retention of enzyme--DNA complexes on nitrocellulose filters. The amount of complex retained, ascertained by using radioactive DNA, is a measure of T4 endonuclease V activity. The assay is simple, rapid, and specific, which makes it useful for detecting T4 endonuclease V activity both in crude lysates and in purified preparations. We have used it to monitor enzyme activity during purification and to study binding of the enzyme to DNA under conditions that minimize the ability of the enzyme to nick DNA. From our data we conclude that (1) T4 endonuclease V binds to UV-irradiated DNA but not to DNA that has been previously incised by the endonuclease, (2) equilibrium between the free and complexed form of the enzyme is attained under our reaction conditions, (3) dissociation of enzyme--DNA complexes is retarded by sodium cyanide, and (4) retention of enzyme--DNA complexes on nitrocellulose filters is enhanced by high concentrations of saline--citrate.     

Multiple cleavage activities of endonuclease V from Thermotoga maritima: recognition and strand nicking mechanism

Endonuclease V is a deoxyinosine 3'-endonuclease which initiates removal of inosine from damaged DNA. A thermostable endonuclease V from the hyperthermophilic bacterium Thermotoga maritima has been cloned and expressed in Escherichia coli. The DNA recognition and reaction mechanisms were probed with both double-stranded and single-stranded oligonucleotide substrates which contained inosine, abasic site (AP site), uracil, or mismatches. Gel mobility shift and kinetic analyses indicate that the enzyme remains bound to the cleaved inosine product. This slow product release may be required in vivo to ensure an orderly process of repairing deaminated DNA. When the enzyme is in excess, the primary nicked products experience a second nicking event on the complementary strand, leading to a double-stranded break. Cleavage at AP sites suggests that the enzyme may use a combination of base contacts and local distortion for recognition. The weak binding to uracil sites may preclude the enzyme from playing a significant role in repair of such sites, which may be occupied by uracil-specific DNA glycosylases. Analysis of cleavage patterns of all 12 natural mismatched base pairs suggests that purine bases are preferrentially cleaved, showing a general hierarchy of A = G > T > C. A model accounting for the recognition and strand nicking mechanism of endonuclease V is presented.     

The excision of 3′ penultimate errors by DNA polymerase I and its role in endonuclease V-mediated DNA repair

Deamination of adenine can occur spontaneously under physiological conditions, and is enhanced by exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat, generating the highly mutagenic lesion of deoxyinosine in DNA. Such DNA lesions tends to generate A:T to G:C transition mutations if unrepaired. In Escherichia coli, deoxyinosine is primarily removed through a repair pathway initiated by endonuclease V (endo V). In this study, we compared the repair of three mutagenic deoxyinosine lesions of A-I, G-I, and T-I using E. coli cell-free extracts as well as reconstituted protein system. We found that 3′-5′ exonuclease activity of DNA polymerase I (pol I) was very important for processing all deoxyinosine lesions. To understand the nature of pol I in removing damaged nucleotides, we systemically analyzed its proofreading to 12 possible mismatches 3′-penultimate of a nick, a configuration that represents a repair intermediate generated by endo V. The results showed all mismatches as well as deoxyinosine at the 3′ penultimate site were corrected with similar efficiency. This study strongly supports for the idea that the 3′-5′ exonuclease activity of E. coli pol I is the primary exonuclease activity for removing 3′-penultimate deoxyinosines derived from endo V nicking reaction.     

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.     

Mechanism of action of Micrococcus luteus gamma-endonuclease

Micrococcus luteus extracts contain gamma-endonuclease, a Mg2+-independent endonuclease that cleaves gamma-irradiated DNA. This enzyme has been purified approximately 1000-fold, and the purified enzyme was used to study its substrate specificity and mechanism of action. gamma-Endonuclease cleaves DNA containing either thymine glycols, urea residues, or apurinic sites but not undamaged DNA or DNA containing reduced apurinic sites. The enzyme has both N-glycosylase activity that releases thymine glycol residues from OsO4-treated DNA and an associated apurinic endonuclease activity. The location and nature of the cleavage site produced has been determined with DNA sequencing techniques. gamma-Endonuclease cleaves DNA containing thymine glycols or apurinic sites immediately 3' to the damaged or missing base. Cleavage results in a 5'-phosphate terminus and a 3' baseless sugar residue. Cleavage sites can be converted to primers for DNA polymerase I by subsequent treatment with Escherichia coli exonuclease III. The mechanism of action of gamma-endonuclease and its substrate specificity are very similar to those identified for E. coli endonuclease III.     

Endonuclease G: a (dG)n X (dC)n-specific DNase from higher eukaryotes.

An endonuclease activity (termed endonuclease G) that selectively cleaves DNA at (dG)n X (dC)n tracts has been partially purified from immature chicken erythrocyte nuclei. Sites where n greater than or equal to 9 are cleaved in a manner that resembles types II and III restriction nucleases. The nicking rate of the G-strand is 4- to 10-fold higher than that of the C-strand depending on the length of the (dG)n X (dC)n tract and/or nucleotide composition of the flanking sequences. Endonuclease G hydrolyzes (dG)24 X (dC)24 of supercoiled DNA in a bimodal way every 9-11 nucleotides, the maxima in one strand corresponding to minima in the opposite, suggesting that it binds preferentially to one side of the double helix. The nuclease produces 5' phosphomonoester ends and its activity is dependent on Mg2+ or Mn2+. The wide distribution and high relative activity of endonuclease G in a variety of tissues and species argues for a general role of the enzyme. The striking correlation between genetic instability and poly(dG) X poly(dC) tracts in DNA suggests that these sequences and endonuclease G are involved in recombination processes.