Excision of apurinic sites from DNA with enzymes isolated from rat-liver chromatin

Apurinic sites were excised from phi X174 RF DNA with two enzymes isolated from rat liver chromatin: an apurinic/apyrimidinic endodeoxyribonuclease and a 5'-3'-exonuclease; the resulting gap was filled with DNA polymerase beta also prepared from rat liver chromatin and the repair was fully terminated with T4 ligase.     

Excision of apurinic and/or apyrimidinic sites from DNA by nucleolytical enzymes from rat brain

Apurinic and/or apyrimidinic (AP) sites were excised from PM2 phage DNA by two enzymes: an AP endodeoxyribonuclease isolated from rat neocortex chromatin and a rat brain exodeoxyribonuclease, DNase B III. The resulting gap was filled with DNA polymerase beta prepared from rat liver and finally ligated by Escherichia coli DNA ligase.     

Cellular localization of the apurinic/apyrimidinic endodeoxyribonucleases in rat liver

A method has been developed to purify rat liver nuclei; the isolated nuclei keep both nuclear membranes and retain more than 90% of the cell apurinic/apyrimidinic (AP) endodeoxyribonuclease activity. The nuclear enzyme is located mostly in chromatin non-histones; there is also an important amount of activity in the nuclear sap and some in the nuclear membranes. The cytoplasmic AP endodeoxyribonuclease activity is shared between mitochondria, cytosol and membranes. Different cell compartments appear to contain different AP endodeoxyribonuclease species: the membrane enzyme is activated by Triton whereas the other enzymes are rather inhibited; the nuclear sap enzyme has a higher molecular weight and a higher thermal resistance than the chromatin enzyme. A hypothesis is formulated according to which: (1) the chromatin enzyme is the only species important for nuclear DNA repair; (2) the species present in the other cell compartments might be precursors of the chromatin AP endodeoxyribonuclease.     

Localization of the phosphoester bond hydrolyzed by the major apurinic/apyrmidinic endodeoxyribonuclease from rat-liver chromatin

The major apurinic/apyrimidinic (AP) endodeoxyribonuclease from rat liver chromatin, an enzyme specific for AP sites in DNA, cleaves the phosphodiester bridge which is the immediate neighbour of the AP site on its 5' side leaving 3'-hydroxyl and 5'-phosphate ends. In contrast with Escherichia coli endonuclease VI, this chromatin enzyme is inactive on reduced AP sites.     

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.     

Possible roles of beta-elimination and delta-elimination reactions in the repair of DNA containing AP (apurinic/apyrimidinic) sites in mammalian cells.

Histones and polyamines nick the phosphodiester bond 3' to AP (apurinic/apyrimidinic) sites in DNA by inducing a beta-elimination reaction, which can be followed by delta-elimination. These beta- and delta-elimination reactions might be important for the repair of AP sites in chromatin DNA in either of two ways. In one pathway, after the phosphodiester bond 5' to the AP site has been hydrolysed with an AP endonuclease, the 5'-terminal base-free sugar 5'-phosphate is released by beta-elimination. The one-nucleotide gap limited by 3'-OH and 5'-phosphate ends is then closed by DNA polymerase-beta and DNA ligase. We have shown in vitro that such a repair is possible. In the other pathway, the nicking 3' to the AP site by beta-elimination occurs first. We have shown that the 3'-terminal base-free sugar so produced cannot be released by the chromatin AP endonuclease from rat liver. But it can be released by delta-elimination, leaving a gap limited by 3'-phosphate and 5'-phosphate. After conversion of the 3'-phosphate into a 3'-OH group by the chromatin 3'-phosphatase, there will be the same one-nucleotide gap, limited by 3'-OH and 5'-phosphate, as that formed by the successive actions of the AP endonuclease and the beta-elimination catalyst in the first pathway.     

Excision repair of pyrimidine dimers from simian virus 40 minichromosomes in vitro

The ability of DNA repair enzymes to carry out excision repair of pyrimidine dimers in SV40 minichromosomes irradiated with 16 to 64 J/m2 of UV light was examined. Half of the dimers were substrate for the DNA glycosylase activity of phage T4 UV endonuclease immediately after irradiation, but this limit decreased to 27% after 2 h at 0 degrees C. Moreover, the apyrimidinic (AP) endonuclease activity of the enzyme did not incise all of the AP sites created by glycosylase activity, although all AP sites were substrate for HeLa AP endonuclease II. The initial rate of the glycosylase was 40% that upon DNA. After incision by the T4 enzyme, excision was mediated by HeLa DNase V (acting with an exonuclease present in the chromatin preparation). Under physiological salt conditions, excision did not proceed appreciably beyond the damaged nucleotides in DNA or chromatin. With chromatin, about 70% of the accessible dimers were removed, but at a rate slower than for DNA. Finally, HeLa DNA polymerase beta was able to fill the short gaps created after dimer excision, and these patches were sealed by T4 DNA ligase. Overall, roughly 30% of the sites incised by the endonuclease were ultimately sealed by the ligase. The resistance of some sites was due to interference with the ligase by the chromatin structure, as only 30-40% of the nicks created in chromatin by pancreatic DNase could be sealed by T4 or HeLa DNA ligases. The overall excision repair process did not detectably disrupt the chromatin structure, since the repair label was recovered in Form I DNA present in 75 S condensed minichromosomes. Although other factors might stimulate the rate of this repair process, it appears that the enzymes utilized could carry out excision repair of chromatin to a limit near that observed at the initial rate in mammalian cells in vivo.     

Base-excision repair in carrot cells. Partial purification and characterization of uracil-DNA glycosylase and apurinic/apyrimidinic endodeoxyribonuclease

Uracil-DNA glycosylase and apurinic/apyrimidinic (AP) endodeoxyribonuclease have been purified from cultured carrot cells. The two enzymes, separated by affinity chromatography on Sepharose-poly(rU), were found to have properties similar to those of the homologous bacterial and mammalian enzymes. The action of AP endodeoxyribonuclease on (dA)230 . (dT, dU)230 partially depyrimidinated by uracil-DNA glycosylase suggests that these two enzymes might act successively to initiate the repair of uracil-containing DNA.     

The phosphonomethyl analogue of 3-phosphoglycerate is a potent competitive inhibitor of phosphoglycerate mutases.

Deoxyribonuclease IV, a 5'-3' exonuclease degrading double-stranded DNA from intra-strand nicks, has been purified from the chromatin of rat liver cells. The enzyme, which has an Mr of 58000, excises the apurinic (AP) sites from a depurinated DNA nicked 5' to these AP sites with the chromatin AP endonuclease. The excision is not the result of hydrolysis of the phosphodiester bond 3' to the AP sites since the excision product does not behave as deoxyribose 5-phosphate but as its 2,3-unsaturated derivative. This result suggests that, to remove the AP sites from the DNA nicked by an AP endonuclease, the chromatin deoxyribonuclease IV rather acts as a catalyst of beta-elimination.     

Chromatin 3''-phosphatase/5''-OH kinase cannot transfer phosphate from 3'' to 5'' across a strand nick in DNA.

Rat liver chromatin contains a 3'-phosphatase/5'-OH kinase which may be involved in the repair of DNA strand breaks limited by 3'-phosphate/5'-OH ends. In order to determine whether the phosphate group can be transferred directly from the 3' to the 5' position, a polynucleotide duplex was synthesized between poly (dA) and oligo (dT) segments which had 3'-[32P]phosphate and 5'-OH ends. The oligo (dT) segments were separated by simple nicks as shown by the ability of T4 DNA ligase to seal the nick after the 3'-phosphate was removed by a phosphatase and the 5' end was phosphorylated with a kinase. The chromatin 3'-phosphatase/5'-OH kinase was unable to transfer phosphate directly from the 3' to the 5' end of the oligo (dT) segments in the original duplex; ATP was needed to phosphorylate the 5'-OH end. It is concluded that the chromatin 3'-phosphatase/5'-OH kinase is unable to convert a 3'-phosphate/5'-OH nick which cannot be repaired by DNA ligase directly into a 3'-OH/5'-phosphate nick which can be repaired by DNA ligase; the chromatin enzyme rather acts in two steps: hydrolysis of the 3'-phosphate followed by ATP-mediated phosphorylation of the 5'-OH end.     

Intranucleosomal localization of the AP endodeoxyribonuclease of rat liver chromatin

The chromatin AP endodeoxyribonuclease of rat liver cells is located in the cores as well as in the linkers of the nucleosomes.     

The mechanism of base excision repair in Chlamydiophila pneumoniae

Repair of damaged DNA is of great importance in maintaining genome integrity, and there are several pathways for repair of damaged DNA in almost all organisms. Base excision repair (BER) is a main process for repairing DNA carrying slightly damaged bases. Several proteins are required for BER; these include DNA glycosylases, AP endonuclease, DNA polymerase, and DNA ligase. In some bacteria the single-stranded specific exonuclease, RecJ, is also involved in BER. In this research, six Chlamydiophila pneumoniae (C. pneumoniae) genes, encoding uracil DNA glycosylase (CpUDG), endonuclease IV (CpEndoIV), DNA polymerase I (CpDNApolI), endonuclease III (CpEndoIII), single-stranded specific exonuclease RecJ (CpRecJ), and DNA ligase (CpDNALig), were inserted into the expression vector pET28a. All proteins, except for CpDNALig, were successfully expressed in E. coli, and purified proteins were characterized in vitro. C. pneumoniae BER was reconstituted in vitro with CpUDG, CpEndoIV, CpDNApolI and E. coli DNA ligase (EcDNALig). After uracil removal by CpUDG, the AP site could be repaired by two BER pathways that involved in the replacement of either one (short patch BER) or multiple nucleotides (long patch BER) at the lesion site. CpEndoIII promoted short patch BER via its 5'-deoxyribophosphodiesterase (5'-dRPase) activity, while CpRecJ had little effect on short patch BER. The flap structure generated during DNA extension could be removed by the 5'-exonuclease activity of CpDNApolI. Based on these observations, we propose a probable mechanism for BER in C. pneumoniae.     

Mode of inhibition of short-patch base excision repair by thymine glycol within clustered DNA lesions

Clustered DNA damage, where two or more lesions are located proximally to each other, is frequently induced by ionizing radiation. Individual base lesions within a cluster are repaired by base excision repair. In this study we addressed the question of how thymine glycol (Tg) within a cluster would affect the repair of opposing lesions by human cell extracts. We have found that Tg located opposite to an abasic site does not affect cleavage of this site by apurinic/apyrimidinic (AP) endonuclease. However, Tg significantly compromised the next step of the repair. Although purified DNA polymerase beta was able to incorporate the correct nucleotide (dAMP) opposite to Tg, the rate of incorporation was reduced by 3-fold. Tg does not affect 5'-sugar phosphate removal by the 2-deoxyribose-5-phosphate (dRP) lyase activity of DNA polymerase beta, but further processing of the strand break by purified DNA ligase III was slightly diminished. In agreement with these findings, although an AP site located opposite to Tg was efficiently incised in human cell extract, only a limited amount of fully repaired product was observed, suggesting that such clustered DNA lesions may have a significantly increased lifetime in human cells compared with similar single-standing lesions.     

Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1).

Two forms of DNA base excision-repair (BER) have been observed: a 'short-patch' BER pathway involving replacement of one nucleotide and a 'long-patch' BER pathway with gap-filling of several nucleotides. The latter mode of repair has been investigated using human cell-free extracts or purified proteins. Correction of a regular abasic site in DNA mainly involves incorporation of a single nucleotide, whereas repair patches of two to six nucleotides in length were found after repair of a reduced or oxidized abasic site. Human AP endonuclease, DNA polymerase beta and a DNA ligase (either III or I) were sufficient for the repair of a regular AP site. In contrast, the structure-specific nuclease DNase IV (FEN1) was essential for repair of a reduced AP site, which occurred through the long-patch BER pathway. DNase IV was required for cleavage of a reaction intermediate generated by template strand displacement during gap-filling. XPG, a related nuclease, could not substitute for DNase IV. The long-patch BER pathway was largely dependent on DNA polymerase beta in cell extracts, but the reaction could be reconstituted with either DNA polymerase beta or delta. Efficient repair of gamma-ray-induced oxidized AP sites in plasmid DNA also required DNase IV. PCNA could promote the Pol beta-dependent long-patch pathway by stimulation of DNase IV.     

Formation, detection and repair of AP sites

The paper is an outline review of the main aspects concerning the formation and repair of AP (apurinic/apyrimidinic) sites in DNA as well as some of the chemical properties allowing their quantitative determination. A new method for the measurement of AP sites based on their reaction with [14C]methoxyamine is described. It has been applied to the measurement of AP sites produced in DNA either by physical (gamma-rays) or chemical (methyl methanesulphonate, osmium tetroxide) agents. The method has also been used to quantify the excision of abnormal bases from DNA under the action of specific DNA glycosylases and to prevent the chemical or enzymatic degradation of DNA containing AP sites. The paper contains data about the purification and characterization of uracil-DNA glycosylase and AP endodeoxyribonuclease from carrot cells, two enzymes involved in the first steps of base excision repair through AP site intermediates. The biological effects of unrepaired AP sites are also discussed.     

Selective inhibition by methoxyamine of the apurinic/apyrimidinic endonuclease activity associated with pyrimidine dimer-DNA glycosylases from Micrococcus luteus and bacteriophage T4

The UV endonucleases [endodeoxyribonuclease (pyrimidine dimer), EC 3.1.25.1] from Micrococcus luteus and bacteriophage T4 possess two catalytic activities specific for the site of cyclobutane pyrimidine dimers in UV-irradiated DNA: a DNA glycosylase that cleaves the 5'-glycosyl bond of the dimerized pyrimidines and an apurinic/apyrimidinic (AP) endonuclease that thereupon incises the phosphodiester bond 3' to the resulting apyrimidinic site. We have explored the potential use of methoxyamine, a chemical that reacts at neutral pH with AP sites in DNA, as a selective inhibitor of the AP endonuclease activities residing in the M. luteus and T4 enzymes. The presence of 50 mM methoxyamine during incubation of UV- (4 kJ/m2, 254 nm) treated, [3H]thymine-labeled poly(dA).poly(dT) with either enzyme preparation was found to protect completely the irradiated copolymer from endonucleolytic attack at dimer sites, as assayed by yield of acid-soluble radioactivity. In contrast, the dimer-DNA glycosylase activity of each enzyme remained fully functional, as monitored retrospectively by release of free thymine after either photochemical- (5 kJ/m2, 254 nm) or photoenzymic- (Escherichia coli photolyase plus visible light) induced reversal of pyrimidine dimers in the UV-damaged substrate. Our data demonstrate that the inhibition of the strand-incision reaction arises because of chemical modification of the AP sites and is not due to inactivation of the enzyme by methoxyamine. Our results, combined with earlier findings for 5'-acting AP endonucleases, strongly suggest that methoxyamine is a highly specific inhibitor of virtually all AP endonucleases, irrespective of their modes of action, and may therefore prove useful in a wide variety of DNA repair studies.     

Five repair pathways in one context: chromatin modification during DNA repair.

The eukaryotic cell is faced with more than 10 000 various kinds of DNA lesions per day. Failure to repair such lesions can lead to mutations, genomic instability, or cell death. Therefore, cells have developed 5 major repair pathways in which different kinds of DNA damage can be detected and repaired: homologous recombination, nonhomologous end joining, nucleotide excision repair, base excision repair, and mismatch repair. However, the efficient repair of DNA damage is complicated by the fact that the genomic DNA is packaged through histone and nonhistone proteins into chromatin, a highly condensed structure that hinders DNA accessibility and its subsequent repair. Therefore, the cellular repair machinery has to circumvent this natural barrier to gain access to the damaged site in a timely manner. Repair of DNA lesions in the context of chromatin occurs with the assistance of ATP-dependent chromatin-remodeling enzymes and histone-modifying enzymes, which allow access of the necessary repair factors to the lesion. Here we review recent studies that elucidate the interplay between chromatin modifiers / remodelers and the major DNA repair pathways.     

Repair of abasic sites in DNA

Repair of both normal and reduced AP sites is activated by AP endonuclease, which recognizes and cleaves a phosphodiester bond 5' to the AP site. For a short period of time an incised AP site is occupied by poly(ADP-ribose) polymerase and then DNA polymerase beta adds one nucleotide into the repair gap and simultaneously removes the 5'-sugar phosphate. Finally, the DNA ligase III/XRCC1 complex accomplishes repair by sealing disrupted DNA ends. However, long-patch BER pathway, which is involved in the removal of reduced abasic sites, requires further DNA synthesis resulting in strand displacement and the generation of a damage-containing flap that is later removed by the flap endonuclease. Strand-displacement DNA synthesis is accomplished by DNA polymerase delta/epsilon and DNA ligase I restores DNA integrity. DNA synthesis by DNA polymerase delta/epsilon is dependent on proliferating cell nuclear antigen, which also stimulates the DNA ligase I and flap endonuclease. These repair events are supported by multiple protein-protein interactions.     

Abasic site recognition by two apurinic/apyrimidinic endonuclease families in DNA base excision repair: the 3' ends justify the means

DNA damage occurs unceasingly in all cells. Spontaneous DNA base loss, as well as the removal of damaged DNA bases by specific enzymes targeted to distinct base lesions, creates non-coding and lethal apurinic/apyrimidinic (AP) sites. AP sites are the central intermediate in DNA base excision repair (BER) and must be processed by 5' AP endonucleases. These pivotal enzymes detect, recognize, and cleave the DNA phosphodiester backbone 5' of, AP sites to create a free 3'-OH end for DNA polymerase repair synthesis. In humans, AP sites are processed by APE1, whereas in yeast the primary AP endonuclease is termed APN1, and these enzymes are the major constitutively expressed AP endonucleases in these organisms and are homologous to the Escherichia coli enzymes Exonuclease III (Exo III) and Endonuclease IV (Endo IV), respectively. These enzymes represent both of the conserved 5' AP endonuclease enzyme families that exist in biology. Crystal structures of APE1 and Endo IV, both bound to AP site-containing DNA reveal how abasic sites are recognized and the DNA phosphodiester backbone cleaved by these two structurally unrelated enzymes with distinct chemical mechanisms. Both enzymes orient the AP-DNA via positively charged complementary surfaces and insert loops into the DNA base stack, bending and kinking the DNA to promote flipping of the AP site into a sequestered enzyme pocket that excludes undamaged nucleotides. Each enzyme-DNA complex exhibits distinctly different DNA conformations, which may impact upon the biological functions of each enzyme within BER signal-transduction pathways.     

DNA synthesis and dRPase activities of polymerase beta are both essential for single-nucleotide patch base excision repair in mammalian cell extracts

In mammalian cells the majority of altered bases in DNA are processed through a single-nucleotide patch base excision repair mechanism. Base excision repair is initiated by a DNA glycosylase that removes a damaged base and generates an abasic site (AP site). This AP site is further processed by an AP endonuclease activity that incises the phosphodiester bond adjacent to the AP site and generates a strand break containing 3'-OH and 5'-sugar phosphate ends. In mammalian cells, the 5'-sugar phosphate is removed by the AP lyase activity of DNA polymerase beta (Pol beta). The same enzyme also fills the gap, and the DNA ends are finally rejoined by DNA ligase. We measured repair of oligonucleotide substrates containing a single AP site in cell extracts prepared from normal and Pol beta-null mouse cells and show that the reduced repair in Pol beta-null extracts can be complemented by addition of purified Pol beta. Using this complementation assay, we demonstrate that mutated Pol beta without dRPase activity is able to stimulate long patch BER. Mutant Pol beta deficient in DNA synthesis, but with normal dRPase activity, does not stimulate repair in Pol beta-null cells. However, under conditions where we measure base excision repair accomplished exclusively through a single-nucleotide patch BER, neither dRPase nor DNA synthesis mutants of Pol beta alone, or the two together, were able to complement the repair defect. These data suggest that the dRPase and DNA synthesis activities of Pol beta are coupled and that both of these Pol beta functions are essential during short patch BER and cannot be efficiently substituted by other cellular enzymes.