1 Cod-X

Endonuclease

Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain such as Deoxyribonuclease I which cuts DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases or restriction enzymes, cleave only at very specific nucleotide sequences (Meselson and Yuan, 1968).

From: Nano-Inspired Biosensors for Protein Assay with Clinical Applications, 2019

Related terms:

Nuclease

Exonuclease

CRISPR

Base Excision Repair

MicroRNA

Apoptosis

Nested Gene

Cas9

Mutation

View all Topics

Advances in Radiation Biology

Malcolm C. Paterson, in Advances in Radiation Biology, 1978

A Ultraviolet Endonucleases

Endonucleases selectively active on pyrimidine dimer-distorted sites in UV-irradiated DNA have been highly purified from three prokaryotic sources: bacteriophage T4 (Yasuda and Sekiguchi, 1970; Friedberg and King, 1971), M. luteus (Kaplan et al., 1969; Carrier and Setlow, 1970; Nakayama et al., 1971), and E. coli (Braun and Grossman, 1974; Braun et al., 1975). Highly purified preparations of M. luteus UV endonuclease recognize sites altered by dimers only in native double-stranded DNA (Kaplan et al., 1969; Carrier and Setlow, 1970), whereas T4 endonuclease V is also effective toward dimer-containing single-stranded DNA (Friedberg, 1975).

The M. luteus UV endonuclease displays, at each dimer-containing site, a high degree of specificity for the dimer-containing strand (Carrier and Setlow, 1970), seldom erring and incising the complementary dimer-free strand (i.e., only 1 incorrect incision per 70 dimers in the opposite strand of an irradiated-unirradiated heteroduplex; Paribok and Tomilin, 1971).

Preincubation of UV-damaged DNA with photolyase under photoreactivating light precludes subsequent endonucleolytic cleavage by the M. luteus UV endonuclease; this finding singles out pyrimidine dimers as the sole class of UV photoproducts providing substrate sites for this enzyme (Kaplan et al., 1969; Carrier and Setlow, 1970; Patrick and Harm, 1973; Paterson et al., 1973). As indicated above, dimer-containing sites would seem to be the exclusive substrate of T4 endonuclease V (Friedberg, 1975) and may also constitute the sole UV-induced substrate of the E. coli UV endonuclease (Braun and Grossman, 1974).

Although all three types of dimers (cytosine–thymine heterodimer and cytosine and thymine homodimers) are acted upon by UV endonucleases, there is some evidence favoring differential rates of incision for the different dimers: the preferred substrate is the thymine homodimer, at least compared to its cytosine counterpart (Kushner et al., 1971; Patrick and Harm, 1973).

An endonuclease has been purified to apparent homogeneity from rat liver (Van Lancker and Tomura, 1974). The enzyme recognizes sites altered by pyrimidine dimers and acetylaminofluorene adducts in double-stranded DNA. Although its substrate specificity is not well-defined, this mammalian enzyme has many of the hallmarks of an UV endonuclease.

To date attempts to purify the dimer-recognizing endonuclease from human cells have been unsuccessful, probably foiled in part by the freezing-lability of the presumed enzyme (Duncan et al., 1975). Recently, however, endonucleolytic activities exhibiting atttributes expected of a true UV endonuclease have been detected (Lytle et al., 1973; Duncan et al., 1975), raising hope that an enzyme preparation of high purity will be forthcoming in the near future.

Radman (1975) has purified from E. coli an endonuclease, designated endonuclease III, having activity toward UV-irradiated DNA. Although both its physiological function and substrate are unknown, it is apparent that the activity of endonuclease III is independent of other known lesion-recognizing endonucleases, including UV endonuclease, endonuclease II, and apurinic site endonuclease.

View chapterPurchase book

DNA Restriction and Modification

G.W. Blakely, N.E. Murray, in Encyclopedia of Microbiology (Third Edition), 2009

In Vitro Assays for DNA Fragmentation

Endonuclease activities yielding discrete fragments of DNA are commonly detected in crude extracts of bacterial cells. More than one substrate may be used to increase the chance of providing DNA that includes appropriate target sequences. DNA fragments diagnostic of endonuclease activity are separated according to their size by electrophoresis through a matrix, usually an agarose gel, and are visualized by the use of autoradiography or a fluorescent dye, ethidium bromide, that intercalates between stacked base pairs.

Extensive screening of many bacteria, often obscure species for which there is no genetic test, has produced a wealth of endonucleases with different target sequence specificities. These endonucleases are referred to as restriction enzymes, even in the absence of biological experiments to indicate their role as a barrier to the transfer of DNA. Many of these enzymes are among the commercially available endonucleases that serve molecular biologists in the analysis of DNA (Table 1; see 'Applications and commercial relevance'). In vitro screens are applicable to all organisms, but to date R-M systems have not been found in eukaryotes, although some algal viruses encode them.

Table 1. Some Type II restriction endonucleases and their cleavage sitesa

Bacterial sourceEnzyme abbreviationSequences 5′ → 3′3′ ← 5′Noteb

aThe cleavage site for each enzyme is shown by the arrows.b1, produces blunt ends; 2, produces cohesive ends with 5′ single-stranded overhangs; 3, cohesive ends of Sau3AI and BamHI are identical; 4, produces cohesive ends with 3′ single-stranded overhangs; 5, Pu is any purine (A or G), and Py is any pyrimidine (C or T).

View chapterPurchase book

Renal Toxicology

L.H. Lash, B.S. Cummings, in Comprehensive Toxicology, 2010

7.04.6.2.8 Endonucleases

Endonucleases cleave DNA at internucleosomal linker regions, resulting in a characteristic 'ladder' pattern after agarose gel electrophoresis (Arends et al. 1990; Cohen et al. 1992; Compton 1992; McConkey et al. 1989; Nicotera et al. 1992). These enzymes are activated by both Ca2+ and Mg2+. Nephrotoxicants known to activate endonucleases include oxidants such as H2O2 and chemotherapeutics such as cisplatin (Kaushal et al. 2004; Li et al. 2004a; Ueda and Shah 1992a). Endonuclease-mediated DNA damage also occurs in chemical hypoxia induced by antimycin A in LLC-PK1 cells (Hagar et al. 1997).

The mechanism of endonuclease activation during cell death may involve mitochondria. Studies demonstrate that cisplatin induces the activation of endonuclease G in mitochondria of treated mouse proximal tubule cells, and that this nuclease is responsible for the DNA cleavage during apoptosis (Kaushal et al. 2004; Li et al. 2004a). However, the role of mitochondria in nephrotoxicant-induced endonuclease activation may be toxicant- and cell-dependent, as endonucleases were not activated in rabbit proximal tubule cells after exposure to the mitochondrial respiration inhibitors antimycin A, p-(trifluoromethoxy) phenylhydrazone, the oxidant tert-butyl hydroperoxide, or the Ca2+ ionophore A23187 (Schnellmann et al. 1993). Studies in postischemic rat kidney and rat proximal tubules isolated from hypoxic rats showed that minimal DNA laddering was present (Enright et al. 1994; Iwata et al. 1994). Therefore, the role of endonucleases in nephrotoxicity remains unresolved.

View chapterPurchase book

Advances in Radiation Biology

J.J. Roberts, in Advances in Radiation Biology, 1978

D Endonucleases Involved in Excision Repair

Extracts from both bacterial and mammalian cells have been shown to contain enzymes that recognize damage in DNA caused either by UV irradiation (Tables X and XI), x-irradiation, or γ-irradiation (Table XII) or by treatment with a number of chemical agents (Table XIII). In some instances the enzyme mixture has been found to recognize lesions introduced by more than one of these DNA-damaging agents. However, it does not necessarily follow that a particular enzyme can recognize more than one type of damage. Crude extracts used for such studies probably contain many enzymes. Alternatively, several different types of agent may in fact lead to the same final damage in DNA, as in the production of apurinic sites for both UV and x-irradiation and some alkylating agents. Nevertheless, it is apparent that there are enzymes in both bacterial and mammalian cells that recognize essentially four types of damage in DNA.

Table X. ENDONUCLEASE ACTIVITY AGAINST UV-IRRADIATED DNA IN MICROORGANISMS

SourceSubstrateAssaysConclusions/commentsAuthorsMicrococcus luteus32P Escherichia coli DNARelease of phosphomonoester groups by endonucleolytic incision. Formation of acid-soluble nucleotides from denatured DNA by exonuclease.Two enzyme activities isolated. Endonuclease, mol. wt. 15000 acts on UV-irradiated but not on native or single-stranded DNA. Photoproduct excision is dependent on the second enzyme, a magnesiun-requiring exonuclease. Five nucleotides released per single-strand incision.Kaplan et al. (1969)Native UVirradiated DNAProduction of degraded DNA.(i)

Breaks produced equal in number to thymine dimers that are not excised.

(ii)

Breaks occur in strand containing dimers.

Carrier and Setlow (1970)UV-irradiated single- or double-stranded DNADegradation of 32P labeled E. coli DNA; production of acid-soluble nucleo- and mol. wt. changes.(i)

Mol. wt. 13000–14000.

(ii)

It produces single-strand breaks.

(iii)

Active in presence of EDTA and not stimulated by Mg2+.

(iv)

Endonuclease activity against MMS-treated DNA in crude extract may be attributable to another class of enzyme different from UV endonuclease.

Nakayama et al. (1971); Okubo et al. (1971)DNA made after UV irradiation of normal human and XP cellsProduction of breaks in newly synthesized DNA.Repair endonuclease sites could result from (a) DNA exchanges between parental and daughter strands, (b) insertion of abnormal bases into the DNA made from irradiated templates, (c) recognition of dimer and incision in opposite strand.Buhl and Regan (1973a)Mutant and transformed strains of M. luteusUV-irradiated cellular DNACellular DNA breakdown; excision of pyrimidine dimers; production of single-strand breaks in cellular DNA and UV-induced delay in DNA synthesis.Mutants lacking the UV endonuclease are incapable of excising thymine-containing dimers.Mahler et al. (1971)T4-infected E. coli (T4 endonuclease V)Labeled UV-irradiated DNAFormation of phosphomonoesterase-sensitive forms(i)

No effect of enzyme on native or denatured DNA.

(ii)

Enzymes cleaves on 5′-side of a pyrimide dimer.

(iii)

Optimal activity at pH 7.5 with no requirement for divalent ions.

(iv)

A purified T4 endonuclease V, the v-gene product, does not degrade MMS-alkylated DNA and is not involved in repair or abortive repair of MMS-alkylated T4 DNA. The x and y genes of T4 and the polA and uvrD genes of E. coli are involved in repair of MMS-damaged T4 DNA. Two pathways of repair are involved.

Yasuda and Sekiguchi (1970)Yasuda and Sekiguchi (1976); Nishida et al. (1976); (see also Ebisuzaki et al., 1975)E. coliϕX 174 RF1 (3H) DNANicking of replicative form 1 ofϕX 174 DNAActivity absent in uvrA and uvrB excision-defective mutants but present in uvrC and uvrD mutants.Braun and Grossman (1974)Bacillus subtilisUV-irradiated, 14C-labeled DNASingle-strand breaksActivity similar to T4 endonuclease V, M. luteus and E. coli endonucleases.Hayase et al. (1975)

Table XI. ENDONUCLEASE ACTIVITY AGAINST UV-IRRADIATED DNA IN MAMMALIAN CELLS

SourceSubstrateAssayConclusions/commentsAuthorsHeLa[3H]TdR-labeled Escherichia coli UV-irradiated double-stranded DNALiberation of acid-soluble productsEnzyme showed Mg dependency similar to, but pH dependency different from, nonspecific DNase (see Slor and Lev, 1973).Burt and Brent (1971)UV-irradiated superhelical DNA (PM2)Conversion to circular form after nicking (isolated in alkaline sucrose)(i)

Heat-sensitive activity independent of divalent cations.

(ii)

Activity present in normal and XP cells

Brent (1972a)HeLa; SV40-transformed primary XP fibroblasts; untransformed XP cells (deficient UV repair); heterozygotic XP cells; (normal UV repair)(i)

UV-irradiated labeled adenovirus 5 DNA

(ii)

32P-labeled RF DNA fromϕX174

Production of single-strand breaks in DNA(i)

Reduction in mol. wt. of UV-irradiated DNA by extracts of all four cell lines.

(ii)

Activity not stimulated by prior UV irradiation.

(iii)

Similar activities in stationary phase cells.

(iv)

No low mol. wt. material formed.

(v)

No loss of activity when UV-irradiated DNA treated with photoreactivating enzyme.

Bacchetti et al. (1972)HeLa; normal and XP fibroblasts and lymphocytesLabeled E. coli UV-irradiated DNALiberation of acid-soluble products(i)

Same activity in normal and XP cells.

(ii)

Activity found to be inhibited by heavily irradiated or single-stranded (SS) DNA. The SS DNA degraded faster than UV-irradiated DNA by enzyme extract.

(iii)

DNA irradiated with low dose of UV was not attacked by crude enzyme extract.

(iv)

Authors concluded that the enzyme is an exonuclease-specific for SS DNA.

Slor and Lev (1973)HeLa; adult primary, fetal, and XP skin fibroblastsUV-irradiated supercoiled PM2 DNAConversion to circular form (isolated in neutral sucrose; see Brent, 1972).No differences between several XP lines, primary fetal or adult normal skin cells, but higher activity in HeLa and WI 38 cells; results suggest that a mixture of endonuclease enzymes is present in cells.Duker and Teebor (1975)Human lymphoblastsUV-irradiated supercoiled PM2 DNAConversion to nicked circlesThree different endonucleases characterized. Activity against apurinic and apyrimidine sites (AP) after UV, which increased with time, apparently due to spontaneous loss of damaged bases. Another endonuclease acts on non-AP sites present with 10 times the frequency of AP sites.Brent (1975,1976)Calf thymusUV- and γ-irradiated PM2 or adenovirus 5 DNABreaks in DNAPurified enzyme acts on both UV- and γ-irradiated DNA. Site of action is a photoproduct other than pyrimidine dimer.Bacchetti and Benne (1975)

Table XII. ENDONUCLEASE ACTIVITY AGAINST γ-IRRADIATED DNA IN MICROORGANISMS AND MAMMALIAN CELLS

SourceSubstrateAssayConclusions/commentsAuthorsMicrococcus luteus, partially purified extractγ-Ray-induced damage in plasmid DNA of E. coli minicells.Introduction of nicks in superhelical, covalently closed, circular λdv DNA to give relaxed circles(i)

Nuclease-sensitive sites disappear on incubation after irradiation.

(ii)

The endonuclease also acts on UV-irradiated DNA as indicated by competition studies.

Paterson and Setlow (1972)Crude extractγ-Irradiated 3H- or 14C-labeled E. coli DNAProduction of single-strand breaks(i)

Endonuclease-sensitive sites are lesions other than cyclobutane pyrimidine dimers.

(ii)

UV-irradiated DNA competes for endonuclease activity toward both UV- and γ-irradiated DNAs.

(iii)

The two activities are separable on DEAE cellulose columns.

(iv)

The extract was also active against MMS-treated DNA and UV-irradiated DNA.

Setlow and Carrier (1973)ExtractsE. coli DNA isolated from 14C- and 3H-labeled cells various times after γ-irradiationProduction of single-strand breaks(i)

Disappearance with time of endonuclease-sensitive sites.

(ii)

As few as two sites for 108 daltons can be detected after γ-irradiation.

(iii)

Thymine dimer excision-deficient uvr – strain also removed endonuclease-sensitive sites after γ-irradiation but not after UV irradiation.

(iv)

uvrA-Gene product not required for loss of γ-ray damage.

Wilkins (1973a,b)Escherichia coli endo I−, E. coli endo I –uvrA6-extractsγ-Irradiated or osmium tetroxide oxidized poly-(dA-dT)Removal of products of 5,6-dihydroxydihydrothymine type (t ')uvrA-Gene product not required for excision of t'products. On average 8–16 nucleotides removed per ring-damaged base excised. Excision accomplished in absence of radiation-induced strand breaks.Hariharen and Cerutti (1974)E. coli B and K12X-irradiated supercoiled, circularϕX-174 RF DNA or PM2 DNAConversion to relaxed formFor every single-strand break induced by x-rays under anerobic condition, there is one induced site sensitive to the enzyme. The same amount of enzyme is present in mutant deficient in endonuclease II or mutants lacking DNA polymerase I.Strniste and Wallace (1975)Crude HeLa sonicateγ-Irradiated superhelical PM2 DNAConversion to nicked circular relaxed formExtract also active against UV-irradiated DNA.Brent (1973)Calf thymus extractγ-Irradiated superhelical PM2 DNAConversion to nicked circular relaxed formExtract also active against UV-irradiated DNA.Bacchetti and Benne (1975)Rat liver extractDNA from irradiated rats or irradiated rat liver DNABreaks in DNA 32P release after endonuclease and alkaline phosphatase. Stimulation of priming ability.Extract also active against UV- and γ-irradiated and carcinogen-bound DNA. Damaged bases are presumed target in irradiated DNA.Tomura and Van Lancker (1975)Whole cell and nuclear sonicates of normal and Fanconis anemia cellsIrradiated labeled T7 or λ-phage DNALoss of photoproducts of 5,6-dihydroxydihydrothymine type (t')Whole-cell sonicates demonstrate a partial deficiency for excision of γ-ray-induced thymine damage for two of four FA skin fibroblast lines. Deficiency more marked in nuclear sonicates.Remsen and Cerutti (1976)Human lymphoblastoidDepurinated PM2 DNAConversion to nicked circlesNumber of endonuclease susceptible (apurinic/apyrimidinic) sites reached maximum immediately after irradiation and did not increase further.Brent (1976)

Endonucleases that recognize thymine dimers in DNA might also recognize cross-links in DNA introduced by difunctional alkylating agents, mitomycin C or Psoralen plus light. Possibly the same endonuclease or if not the same then a closely related endonuclease(s) may also recognize bulky chemical substituents, such as those introduced into DNA by 2-acetylaminofluorene (Van Lancker and Tomura, 1974) or 7-bromomethylbenz[a]anthracene (Maher et al., 1974), and produce "nicks" in the DNA.5 Endonuclease II isolated from E. coli possesses the properties of glycosidase that can remove alkylpurines by scission of the purine ribose linkage. O 6-Alkylguanine and 3-alkyladenine moieties in methylated and ethylated DNA are probably removed by this enzyme (Kirtikar and Goldthwait, 1974; Lawley and Orr, 1970; Lawley and Warren, 1975). The organ specificity of the carcinogenic alkylnitrosoureas, ENU and MNU, for nervous tissue is probably accounted for by difference in the tissue distribution of this enzyme (Goth and Rajewsky, 1974a,b; Kleihues and Margison, 1974). More recently endonuclease activity that recognizes apurinic sites produced in DNA following either UV or x-irradiation and a variety of alkylating agents has been found in extracts of bacterial (Verly and Paquette, 1972), rat liver (Verly and Paquette, 1973), calf thymus (Ljungquist and Lindahl, 1974), and plant (Verly et al., 1973) cells (Table XIV). Previously reported endonucleolytic activity against UV- or x-irradiated DNA could have been due to the existence of such lesions in DNA. Characterization of enzymes specific for particular substituents on chemically modified DNAs require an analysis of the excision of identified products on the DNA. Such studies are now possible for a variety of DNA-damaging agents including alkylating agents, 2-acetylaminofluorene, aromatic hydrocarbons, 7-bromomethylbenzanthracene, 4-nitroquinoline-l-oxide, where the extent and nature of the reactions occurring with DNA (sometimes following metabolism) have been largely characterized (see earlier). For a fuller discussion of the enzymes involved in the repair of DNA, see reviews by Grossman (1974) and Grossman et al. (1975). In the latter review, the term correctional endonuclease or correndonuclease has been introduced to describe an endonuclease that acts specifically on damaged DNA resulting in correctional pathways in vivo. A correndonuclease I-type endonuclease is defined as one that is specific for damaged DNAs possessing monoadduct derivatives. Those correctional endonucleases that are specific for diadductmodified regions of DNA are referred to as correndonuclease II.

Table XIV. ENDONUCLEASE ACTIVITY AGAINST APURINIC ACID SITES IN DNA

SourceSubstrateAssayConclusions/commentsAuthorsEscherichia coli endonucleaseDepurinated T4Velocity sedimentation and viscometry of DNASingle- and double-strand breaks in depurinated and depurinated-reduced DNA: enzyme hydrolyzes phosphodiester bonds at site of depurination.Hadi and Goldthwait (1971)Labeled, T7 depurinated DNA (acid-treated)Velocity sedimentation changes of DNAApurinic sites disappear during incubation of the DNA with E. coli endonuclease, DNA polymerase I, and T4 ligase. It is suggested that the endonuclease is a repair enzyme involved in the maintenance of DNA in all cells (animal, plant, and bacterial; see above ref.)Verly et al. (1974a,b)Purified E. coli endonuclease3H-labeled E. coli depurinated DNA (heated, MMS-alkylated DNA)Release of acid-soluble radioactivityPossibly the same enzyme as endonuclease II (see Table XIII). No activity against native or alkylated DNA.Verly and Paquette (1972), Paquette et al. (1972)Phasaeolus aureus3H-labeled E. coli depurinated DNA (heated, MMS-alkylated DNA)Release of acid-soluble radioactivityLittle effect on normal DNA. Effect on alkylated DNA greatly increased if some sites depurinated.Verly et al. (1973)Rat liver endonuclease3H-labeled E. coli depurinated DNA (heated, MMS-alkylated DNA)Release of acid-soluble radioactivityNo activity against normal DNA or alkylated DNA, i.e., same properties as the E. coli endonuclease for depurinated DNA (see above).Verly and Paquette (1973)Calf thymus extractsHeat-induced apurinic sites in E. coli or phage PM2 DNA.Changes in transforming activity of Bacillus subtilis DNA; nicks in covalently closed PM2 DNA circlesMol. wt. 30,000–35,000. Strongly stimulated by Mg2+. Production of single-strand breaks in double-stranded DNA at apurinic acid sites. No cleavage of native or alkali-denatured DNA, or DNA-containing thymine dimers or alkylated sites.Ljungquist and Lindahl (1974)Calf thymus endonucleaseDouble-stranded, covalently closed, circular PM2 DNA either treated with MMS, irradiated with UV light, or irradiated with γ-raysFormation of single-strand interruptions at apurinic sites in double-stranded DNAThe authors argue that crude extracts from microbial or mammalian cells acting on UV-irradiated (Bacchetti et al., 1972; Brent, 1972a), x-irradiated (Brent, 1973; Setlow and Carrier, 1973), or alkylated DNA (Friedberg and Goldthwait, 1969) may be acting at apurinic sites formed as secondary lesions after irradiation or alkylation (see Brent, 1975).Ljungquist et al. (1974)Calf thymus endonucleaseApurinic sitesPurified preparation did not possess exonuclease or phosphatase activities—unlike corresponding E. coli enzyme, endonuclease II.Ljungquist et al. (1975)HeLa; W1-38; human skin fibroblasts, normal, progeria, and Fanconi's anemia cell extractsAcid-induced apurinic sites in PM2 DNAConversion of superhelical DNA to nicked formApurinic site endonuclease activity (different from UV-endonuclease activity) equal in all human cell lines. No deficiency in progeria or Fanconi's anemia cells.Teebor and Duker (1975)Human lymphoblastsApurinic sites in UV- or x-irradiated PM2 DNAConversion to nicked formPurified enzyme acted quantitatively on apurinic sites in DNA. Activity optimum over pH range 7–8.5 and absolutely dependent on Mg2+. No detectable activity against intact DNA.Brent (1976)

It should be noted that these terms are separate from the already established term endonuclease II for a preparation from E. coli which, in fact, appears to act primarily on monoadducts in DNA (see Table XIII).

View chapterPurchase book

Ribonucleases - Part B

Klaus Klumpp, ... Balraj Handa, in Methods in Enzymology, 2001

Use of Synthetic RNA for Study of Influenza Virus Endonuclease

Endonuclease Assay. In the standard assay, 0.1–0.5 nM cap-labeled RNA (specific activity, 10,000–20,000 cpm/fmol) is incubated with 1–5 nM RNP (according to RNP estimation using OD measurement at 260 nm) in buffer C in a total volume of 5 µl for 1–10 min at 30°. The reaction is stopped by addition of 5 µl of formamide loading buffer. The samples are incubated at 98° for 2 min and 1- to 2-µl aliquots are then immediately loaded onto a preheated 38 × 50 cm, 20% (w/v) acrylamide gel of 0.4-mm thickness. The gel is run at 100 W for 2–3 hr, and then fixed for 15 min in 10% (v/v) ethanol, 10% (v/v) acetic acid, dried under vacuum at 80°, and exposed to a PhosphorImager cassette (Molecular Dynamics, Sunnyvale, CA). Band intensities are quantified with ImageQuant 5.0 software. To analyze coupled endonuclease–transcription initiation reactions, CTP is added to the endonuclease assay. The samples are treated identically to the endonuclease assay described above. To measure transcription initiation independently of endonuclease, chemically synthesized, capped Gil primer RNA can be used in the assay.29 The apparent Km values for CTP are 10 and 12 nM, respectively, under the enzyme excess conditions of this assay.

Endoribonuclease Activity of Influenza Virus Ribonucleoprotein. Figure 5 shows time courses for the endonuclease and coupled endonuclease–transcription initiation reactions catalyzed by influenza virus RNP-associated polymerase complex. The endonuclease of RNP generates an 11-mer capped RNA (G11). In the presence of CTP, the polymerase of RNP elongates the G11 primer by 1 nucleotide to generate a 12-mer RNA (G11 + 1nt). Under the reaction conditions, the substrate is fully depleted. Up to 80–90% of the radioactivity is converted into specific product bands G11 and G11 + 1nt, the residual 10–20% is spread into evenly distributed background degradation products of various lengths. The relative rates of the endonuclease and the coupled nuclease–initiation reactions are identical, indicating that the rate–limiting step under these conditions occurs before transcription initiation. The good fit to a single exponential equation is consistent with enzyme excess conditions in the assay. This can be confirmed by enzyme and RNA titrations. Figure 6b shows that with increasing concentrations of RNA the reaction kinetics can be shifted from single exponential toward biphasic curves. Burst kinetics have been observed previously for this reaction and suggest that product dissociation is rate limiting under substrate excess conditions.28 The extent of the burst is directly dependent on the concentration of active sites. Figure 6a shows a doubling of burst size with double enzyme concentration in the assay. The estimated concentrations of endonuclease active sites in the experiment shown in Fig. 6 are 0.1 and 0.2 nM, corresponding to about 10% according to the amount of RNP estimated from OD measurement at 260 nm. At RNA concentrations below the concentration of active sites, the product formation kinetics can be fitted to single exponential curves. As expected, under enzyme excess conditions, at a constant concentration of RNA, the relative rates of substrate cleavage are not changed at different protein concentrations (Fig. 6c).

Fig. 5. Single turnover endonuclease and coupled endonuclease–transcription initiation reactions. RNPs (4 nM) were incubated with 0.32 nM 32P-labeled G20 RNA for various times up to 10 min at 30°. One set of reactions contained 10 µM CTP as an additional substrate. Left: Analytical gel electrophoresis of the reaction products. The positions of RNA substrate (G20) and products of endonuclease (G11) and transcription initiation (G11 + 1nt) are indicated on the left. The asterisk indicates an additional, low-level endonuclease cleavage site at nucleotide 16 of G20. Right: The relative band intensities as determined from the gel could be fitted to a single exponential progress curve as shown. Endonuclease (RNP, G20 RNA), solid squares; transcription initiation (RNP, G20 RNA, CTP), open squares.

View chapterPurchase book

RNA Editing

Jason Carnes, Kenneth D. Stuart, in Methods in Enzymology, 2007

2.1.4 Examination of endonuclease activity

Endonuclease activity appears to be inefficient when assessing full‐round editing, but the standard full‐round protocol outlined below can be modified to enhance cleavage product abundance. The master mix for both insertion and deletion assays includes ATP, which is a cofactor for RNA ligase activity. However, adenosine nts also affect RNA editing endonuclease activities. Adenosine nts stimulate deletion cleavage activity, but conversely inhibit insertion cleavage activity (Cruz‐Reyes et al., 1998). Thus, if observing robust ligase activity is not desired, the adenosine nucleotide added to each master mix can be changed to enhance cleavage. We substitute 30 mM ADP for 30 mM ATP to enhance cleavage in deletion assays, and remove the 200 μM ATP entirely to enhance cleavage in insertion assays. Some residual ligase activity is often observed in these assays, presumably due to ligase that is already adenylated.

View chapterPurchase book

Advances in Radiation Biology

Lawrence Grossman, in Advances in Radiation Biology, 1974

b Ionizing radiation

An endonuclease-containing extract isolated from M. luteus can incise DNA exposed to γ-irradiation under anaerobic conditions. This reaction seemed to be specific, and the activity proved to be resolvable from the UV-endonuclease activity found in the same organism (Paterson and Setlow, 1972). DNA modified by γ-radiation appears to be refractory to the action of the homogeneous UV-dependent endonuclease. It is conceivable, therefore, that the distortions imposed by pyrimidine dimerization are considerably different from those observed with ionizing or x-radiation. The lack of activity of the pure enzyme on γ-irradiation DNA and the resolution of a separable γ-irradiation specific activity by Paterson and Setlow (1972) indicate that the enzymes involved in the early stages of repair are distortion-specific.

View chapterPurchase book

Mechanisms of DNA Repair

Alba Guarné, in Progress in Molecular Biology and Translational Science, 2012

A The Endonuclease Site

The endonuclease site of MutL is a composite active site where the DQHAX2EX4E, [A/S]C[K/R], and C[P/N]HGRP motifs are provided by one MutL protomer, while the FXR motif is contributed by the other protomer of the dimer (Fig. 6).96 Accordingly, this motif is absent in eukaryotic MutL homologs bearing endonuclease activity, but present in yeast and human MLH1.99 Except for the [A/S]C[K/R] motif, contributed by the external subdomain, all the motifs that define the endonuclease site reside in the dimerization subdomain of the protein (Fig. 6).

Fig. 6. Organization of the endonuclease site of B. subtilis MutL. Ribbon diagram of the C-terminal domain of B. subtilis MutL-CTD (PDB ID: 3KDK) shown in light gray with the C-terminal helix of the neighboring protomer of the dimer shown in dark gray. The conserved motifs related to the endonuclease activity of the protein are shown in pink (443GQ), yellow (462DQHAX2EX4E), cyan (572SCK), green (604CPHRGP), and red (623FXR), with the strictly conserved residues shown as color-coded sticks. The disordered loop connecting the external and dimerization domains is shown as a dotted line. The N- and C-terminal ends of the domain are labeled N and C. The conserved motif responsible for the interaction with the β-sliding clamp (487QXXIXP) is shown in orange with the key residues shown as color-coded sticks and labeled.

Aquifex aeolicus MutL, which lacks the external subdomain, is a proficient endonuclease, suggesting that this subdomain of the protein has minimal effect on the endonuclease activity of MutL (Fig. 4).93,116A. aeolicus MutL has the conserved [A/S]C[K/R] motif at the linker connecting the αA and αE helices; however, it is unlikely that it will adopt a similar conformation to that seen in the structures of B. subtilis and N. gonorrhoeae MutL. Therefore, the [A/S]C[K/R] motif presumably affects the endonuclease activity of MutL only marginally. The role of the conserved GQ motif is also unclear. In contrast to the [A/S]C[K/R], C[P/N]HGRP, and FXR motifs that cluster around the endonuclease motif (DQHAX2EX4E), the GQ motif resides at the dimerization interface (Figs. 4 and 6). However, it is close to the αD–αE loop and hence may indirectly contribute to the overall stability of the endonuclease site.

Not all the conserved residues in the endonuclease motif are equally important either (Fig. 6). For instance, mutation of the Gln463 or Glu473 (DQHAX2EX4E) does not disrupt mismatch repair activity in B. subtilis.96 Similarly, mutation of the equivalent glutamic acid in hPMS2 (Glu710, DQHAX2EX4E) does not affect mismatch repair activity.99 Conversely, mutation of Asp462, His464, or Glu468 in B. subtilis MutL (DQHAX2EX4E) or the corresponding residues in hPMS2 (Asp699, His701, and Glu705) completely abrogates mismatch repair function.67,96,99

View chapterPurchase book

Advances in Radiation Biology

Roger R. Hewitt, Raymond E. Meyn, in Advances in Radiation Biology, 1978

C Enzymatic Activities

Endonuclease activities that appear to be specific for pyrimidine dimers in UV-irradiated DNA have been purified from E. coli infected with T4 bacteriophage, M. luteus, and uninfected E. coli (see reviews by Grossman, 1974; Grossman et al., 1975; Friedberg, 1975). The activity is absent in some UV-sensitive bacterial mutants and it is not induced by infection with UV-sensitive T4v− phage, thus providing direct evidence for the involvement of UV endonucleases in the initiation of excision repair associated with recovery. However, the presence or absence of UV endonuclease in cells is not always diagnostic for UV resistance, since some UV-sensitive mutants possess the activity.

Other endonuclease activities (reviewed by Grossman et al., 1975) that recognize structural or chemical changes in DNA produced by x-rays, chemicals, or treatments that cause elimination of bases from DNA have also been detected and in some cases purified from bacterial or mammalian sources. Ultraviolet-irradiated DNA can also serve as substrate for some of these activities. The relationship between damage-specific endonucleases and the recovery of mammalian cells is not presently clear because deficiencies in appropriate enzymes have not been identified in UV-sensitive mutants. This probably represents the complexity of DNA repair, in which similar types of damage may serve as substrates for more than one repair pathway and each pathway may require the coordinated activity of several proteins. In addition, the substrate specificities of repair endo- and exonucleases may reside in proteins that function in recognizing and stabilizing substrates for an appropriate pathway. Thus, the specificities and efficiencies of activities examined in subcellular or purified preparations may differ from those in vivo.

Recent studies of enzymatic activities potentially associated with repair and recovery of UV-irradiated mammalian cells are of two general types: (1) those in which purified enzymes from bacterial sources have been used as tools to examine retention or elimination of photochemical damage (see Section III,A; see also review by Paterson, this volume) and (2) those in which the identity and characteristics of enzymes from mammalian sources have been examined as potential diagnostic indicators of repair proficiency and for their photochemical substrates.

Endonuclease activities on UV-irradiated DNA have been detected in extracts from human cells (Burt and Brent, 1971; Brent, 1972, 1975; Bacchetti et al., 1972). However, the identity of the substrates recognized in the irradiated DNA is not clear. For example, the activity detected by Bacchetti et al. (1972) remained unaffected by treatment with purified photoreactivating enzyme. Thus, the sites for this activity appear to be photochemical lesions other than pyrimidine dimers. In addition, many attempts to identify an activity that represents the initial step in DNA repair associated with recovery from UV exposure have been unsuccessful, since similar amounts of activity have been detected in extracts from both normal human and XP cells (Bacchetti et al., 1972; Slor and Lev, 1973; Duker and Teebor, 1975). However, recently Duncan et al. (1975) and Mortlemans et al. (1976) have demonstrated that cell-free extracts of human cells are able to excise thymine dimers from both exogenous and endogenous DNA, indicating the presence of both endonucleolytic and exonucleolytic activities. The endonucleolytic activity was shown to be inactivated by freezing. In their comparative studies of extracts of normal and XP cells, Mortlemans et al. (1976) found no differences in excising abilities when purified irradiated DNA was used as substrate. However, when excision from cellular DNA presumably in chromatin was examined, normal cell extracts were proficient and an XP extract was deficient in the process.

Endonucleases specific for damaged DNA have been purified from rat liver (Van Lancker and Tomura, 1974), from calf thymus (Bacchetti and Benne, 1975), and partially purified from human lymphoblasts (Brent, 1975). The rat liver activity introduces single-strand breaks in UV-irradiated and acetylaminofluorene (AAF)-treated double-stranded DNA. This endonuclease appears to yield 3′-PO4 termini, as indicated by the requirement for alkaline phosphatase to exhibit enhanced priming activity for polymerization by E. coli polymerase I. Sequential treatment with the endonuclease, alkaline phosphatase, and polymerase I releases acid-soluble thymine dimers or AAF-base complexes from appropriate substrates, indicating that the endonucleolytic incisions are in proximity to the dimers or AAF-base complexes.

The activity from calf thymus (Bacchetti and Benne, 1975) introduces single-strand breaks in double-stranded DNA irradiated with UV or γ-rays. No activity was detected in unirradiated double-stranded or single-stranded DNA. The activities on UV- or γ-irradiated DNA retained similar properties with respect to pH optima, independence of divalent cation, and inhibition by heat, NaCl, and transfer RNA throughout purification, and they cochromatographed in each fractionation system. Thus, the activities appear to be associated with the same protein. The photochemical substrate in UV-irradiated DNA is not a pyrimidine dimer, since it was not eliminated by treatment with purified photoreactivating enzyme and it is produced with a yield of one endonuclease-sensitive site per thirty-five thymine dimers.

During the course of purifying an endonuclease active on UV-irradiated DNA, Brent (1975) resolved an activity that was apparently specific for depurinated DNA from a nonspecific endonuclease. A third activity was identified that was several times more active on UV-irradiated than on untreated DNA, but its lability prevented further purification or detailed characterization of its substrate specificity.

Another finding of significance to DNA repair in mammalian cells has recently been reported by Huang et al. (1975). It had previously been found that lymphocytes from patients with chronic lymphocytic leukemia (CLL) exhibited a greater than normal capacity to repair DNA damage induced by UV-irradiation and x-rays (Huang et al., 1972). However, a subsequent search for increases in the activities of repair-related endonucleases and polymerases that could account for the enhanced repair in CLL cells was unsuccessful (Huang, unpublished results, cited in Huang et al., 1975). However, a purified DNA-binding protein that binds to UV-irradiated or single-stranded DNA has been purified from CLL cells. This protein was not detected in normal lymphocytes (Huang et al., 1975). The binding protein has no endonucleolytic activity, but it enhances the rate of UV endonucleolytic activity of purified M. luteus enzyme. Thus, the binding protein may contribute to the enhanced DNA repair in CLL cells by stabilizing photoproducts and thereby increase the efficiency of cellular repair endonucleases.

The ultimate success of surveys for repair-related enzymatic activities in crude or partially purified extracts of cells from different species and/or from cell lines of differing UV sensitivity depends on utilizing well-defined substrates and assay conditions that minimize interference by nonspecific activities. The possibility of confusing repair and degradative enzymes was suggested by Slor and Lev (1973) who demonstrated that heavily irradiated DNA becomes a substrate for exonuclease activity that acts on single-stranded DNA. Such confusion is avoided in part by utilizing irradiated covalent circular DNA as substrate and limiting UV exposure to produce only a few substrate sites per molecule. However, even covalent circular DNA is a potential substrate for aspecific activities under certain conditions. Single-strand specific endonucleases can break superhelical covalent circular DNA (Beard et al., 1973; Kato et al., 1973; Wang, 1974; Gray et al., 1975) and exposure to high UV fluences can enhance the activities of these endonucleases (Kato and Fraser, 1973; Sishido and Ando, 1974; Hewitt and Gray, unpublished results), presumably by altering the conformation of the irradiated substrate (Denhardt and Kato, 1973; Gupta and Mitra, 1974) and producing denatured regions associated with photoproducts.

Photoenzymatic repair of pyrimidine dimers has been detected in a marsupial cell line P. tridactylis by Krishnan and Painter (1972), in human leukocytes (Sutherland, 1974), and in normal human fibroblasts (Sutherland et al., 1975; Sutherland, 1975). Comparative studies of the activity in normal human and XP-variant cells (Sutherland and Oliver, 1975) or excision-deficient XP cells (Sutherland et al., 1976) demonstrated that both types of XP cells have reduced amounts of PHR enzyme. However, since photoreactivation of mammalian cell survival has not yet been demonstrated, it is not known whether any causal relationship exists between photoenzymatic repair and cellular recovery of UV-irradiated cells.

The properties of purified PHR enzyme from human leukocytes have been reported by Sutherland (1974). In contrast to PHR enzyme from E. coli, the human enzyme is inhibited by high ionic strength, which may account for previous failures to detect the activity in human cells. Sutherland et al. (1976) have shown that the photoenzymatic repair observed in cells, cell extracts, or with purified PHR enzyme have very similar action spectra for the reactivating light.

In summary, the status of enzymatic activities in mammalian cells that may account for cellular recovery appears to be in an early, but rapidly accelerating state of development.

View chapterPurchase book

Advances in Radiation Biology

Jerry R. Williams, in Advances in Radiation Biology, 1976

a Repair endonuclease activities detected in mammalian cells.

Repair endonuclease activities have been detected in mammalian cells by exposing DNA irradiated with ionizing radiation or UV light to cell extracts or partially purified fractions. This has been demonstrated with DNA extracted from mammalian cells as well as supercoiled DNA from phage or virus. The latter technique is more sensitive because endonuclease conversion of supercoiled DNA to linear DNA is more easily and accurately detected. Endonuclease activity for γ-irradiated DNA has been demonstrated in HeLa cell extracts by Brent (1973) and in calf thymus extracts by Lindahl (1974). Similar activity for UV-irradiated DNA has been observed by Bacchetti et al. (1972) in HeLa extracts; by Van Lancker and Tomura (1972, 1974) in rat liver extracts; by Duncan et al. (1975) in human lymphocytes; and by Duker and Teebor (1975) in HeLa and WI-38 cell extracts.

View chapterPurchase book

Recommended publications:

Molecular Cell

Journal

Journal of Molecular Biology

Journal

Cell

Journal

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis

Journal

Browse Journals & Books

About ScienceDirect

Remote access

Shopping cart

Advertise

Contact and support

Terms and conditions

To be continued

Rate this book