The lethal and mutagenic action of 3H incorporated into the 2' position of the deoxyribose in the DNA of the extracellular phage lambda]

The lethal and mutagenic effects of 3H decay in 2' position of deoxyribose residues in DNA of extracellular lambda phage were studied, [2'-3H]-deoxyadenosine (3H-dA) or [2'-3H]-thymidine (3H-dT) being used as labelled DNA precursors. As estimated by the efficiency of the lethal and mutagenic actions of 3H decay in position 2' was significantly lower than that of the decay in the incorporated 3H-pyrimidines. The genetic effects of 3H decay in 2' position may be attributed to the radiation effect of beta-particles on DNA. In UV-irradiated E. coli cells, with the induced SOS repair, the mutagenic effect of 3H-dA in phage lambda is significantly higher than that of 3H-dT. This is perhaps related to the formation in DNA of AP-sites, resulting from 3H-decay in 2' position, and to the predominant incorporation of adenosine residues opposite to AP-sites during SOS repair.     

Multiplicity of strand incision at G:T base mismatches in DNA by human cell extracts

Cell extract from the HT29 human colon carcinoma cell line (lacking mutator phenotype) was used to study the ATP-dependent G:T mismatch repair. We found that when a 45-bp (model) DNA with a single CpG/TpG mispair was incubated with the cell extract and ATP, it was incised immediately 5' and 3' to the mismatched T, and we noted that the actual 5'- and 3'-labeled fragments were similar to the cleaved products of thymine DNA glycosylase (TDG). This TDG-like cleavage product was enhanced (5-fold) with stimulation of several novel fragments, as inferred from the effect on incision at CpG/TpG site of the addition of G:U competitor DNA and ATP to the HT29 extract. The novel fragments were compatible with a strand incision on both sides of the mismatch (the third phosphodiester bond 5' and the second phosphodiester bond 3' to the mismatched T) and an incision 3' to the mismatched T, respectively. This suggests that while the ATP-dependent (TDG-like) incision activity, contrary to expectation, shows a lack of substrate competition, its catalytic property is likely modified by an interaction with G:U mispair. These multiple ATP-dependent incision events were not detected when extracts of the mismatch repair (MMR) defective HCT15 or HCT116 cell line were augmented with ATP and G:U. We postulate that these multiple ATP-dependent incision events possibly require the same MMR factors, and together they constitute a modified single ATP-dependent G:T incision activity. This activity toward the CpG/TpG was competitively inhibited by a 45-bp DNA with an ApG/TpT mispair; incision at a single site 5' to the latter mismatch compares with one of the multiple sites incised 5' to the former mismatch. These results suggest that one of several mismatch-incision factors is required by the human ATP-dependent G:T incision activity, in addition to MMR factors and ATP.     

DNA-substrate sequence specificity of human G:T mismatch repair activity.

G:T mispairs in DNA originate spontaneously via deamination of 5-methylcytosine. Such mispairs are restored to normal G:C pairs by both E. coli K strains and human cells. In this study we have analyzed the repair by human cell extracts of G:T mismatches in various DNA contexts. We performed two sets of experiments. In the first, repair was sequence specific in that G:T mispairs at CpG sites at four different CpG sites were repaired, but a G:T mismatch at a GpG site was not. Cytosine hemimethylation did not block repair of a substrate containing a CpG/GpT mismatch. In the second set of experiments, substrates with a G:T mismatch at a fixed position were constructed with an A, T, G, or C 5' to the mismatched G, and alterations in the complementary strand to allow otherwise perfect Watson-Crick pairing. All were incised just 5' to the mismatched T and competed for repair incision with a G:T substrate in which a C was 5' to the mismatched G. Thus human G:T mismatch activity shows sequence specificity, incising G:T mismatched pairs at some DNA sites, but not at others. At an incisable site, however, incision is little influenced by the base 5' to the mismatched G.     

Repair of a synthetic abasic site involves concerted reactions of DNA synthesis followed by excision and ligation.

A synthetic analog of an abasic site in DNA is efficiently repaired by a short-patch repair mechanism in soluble extracts of Xenopus laevis oocytes (Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 9:3750-3757, 1989). We present a detailed analysis of the repair mechanism, using extracts depleted of endogenous nucleotide pools. ATP was required for repair with a sharp optimal concentration of 5 mM. The initial rate of repair was increased by preincubation of the DNA in the extract in the presence of ATP. During this preincubation, the DNA was cleaved on the 5' side of the lesion by a class II apurinic-apyrimidinic endonuclease, but removal of the abasic sugar residue was not observed prior to addition of deoxynucleotides to the reaction. Immediately following DNA synthesis, excision and ligation proceeded in a coordinated manner to complete repair. DNA preincubated in the extract in the absence of deoxynucleotides remained associated with repair enzymes during gel filtration. These observations suggest that the enzymes involved in concerted repair of the abasic site form a complex on DNA.     

Delayed DNA joining at 3' mismatches by human DNA ligases.

Repair synthesis catalysed by DNA polymerase beta at 1 nt gaps occurs in the main pathway of mammalian base excision repair. DNA polymerase beta has no exonucleolytic proof-reading ability, and exhibits high error frequency during DNA synthesis. Consequently, continuous correction of endogenous DNA damage by short-patch repair synthesis might lead to a high spontaneous mutation rate, unless subsequent steps in the repair pathway allow for selective removal of incorporation errors. We show here that both human DNA ligase I and III discriminate strongly between a correctly paired versus a mispaired residue at the 3' position of a nick in DNA, when assayed in the presence of physiological concentrations of KCl. The resulting delay in joining after misincorporation by DNA polymerase beta during gap filling could allow for removal of the mismatched terminal residue by a distinct 3' exonuclease.     

Efficiency of repair of an abasic site within DNA clustered damage sites by mammalian cell nuclear extracts

Ionizing radiation induces clustered DNA damage sites which have been shown to challenge the repair mechanism(s) of the cell. Evidence demonstrating that base excision repair is compromised during the repair of an abasic (AP) site present within a clustered damage site is presented. Simple bistranded clustered damage sites, comprised of either an AP-site and 8-oxoG or two AP-sites, one or five bases 3' or 5' to each other, were synthesized in oligonucleotides, and repair was carried out in xrs5 nuclear extracts. The rate of repair of an AP-site when present opposite 8-oxoG is reduced by up to 2-fold relative to that when an AP-site is present as an isolated lesion. The mechanism of repair of the AP-site shows asymmetry, depending on its position relative to 8-oxoG on the opposite strand. The AP-site is rejoined by short-patch base excision repair when the lesions are 5' to each other, whereas when the lesions are 3' to one another, rejoining of the AP-site occurs by both long-patch and short-patch repair processes. The major stalling of repair occurs at the DNA ligase step. 8-OxoG and an AP-site present within a cluster are processed sequentially, limiting the formation of double-strand breaks to <4%. In contrast, when two AP-sites are contained within the clustered DNA damage site, both AP-sites are incised simultaneously, giving rise to double-strand breaks. This study provides new insight into understanding the processes that lead to the biological consequences of radiation-induced DNA damage and ultimately tumorigenesis.     

"Action-at-a distance" of a new DNA oxidative damage product 6-furfuryl-adenine (kinetin) on template properties of modified DNA

N(6)-furfuryladenine (kinetin, K) was shown to have cytokinin activity and antiageing effects. It also appears to protect DNA against oxidative damage mediated by the Fenton reaction. Kinetin was identified as a natural component of DNA in plant extract, calf thymus DNA, fresh DNA preparations from human cell culture, as well as in human urine. A proposed mechanism of kinetin synthesis includes furfural, the oxidative damage product of a 2-deoxyribose moiety of DNA, which reacts with an adenine residue to form N(6)-furfuryladenine at DNA level. The identification of kinetin in plant cell extracts, as well as human urine, suggests its excision from DNA by repair mechanisms. Since such a bulky modification as kinetin induces conformational changes of DNA, this could lead to mutations. Therefore, it was interesting to analyze an effect of kinetin on coding properties of DNA. Chemically synthesized oligodeoxynucleotide (20-mer) containing kinetin AAAACTGCCGTCCTGAKGAT was used as a primer. It was elongated in a polymerase chain reaction (PCR) on a template plasmid pEW1 harboring a 210-bp fragment of DNA derived from the 5' end of HIV mRNA. The PCR product of that length containing kinetin in position 17 from the 5' end was isolated and sequenced. Interestingly, DNA polymerase correctly incorporates thymine opposite of kinetin (an adenine derivative) on the complementary strand, but the misincorporations occur in a vicinity of the modified base.     

Repair of tandem base lesions in DNA by human cell extracts generates persisting single-strand breaks

Clustered DNA damage, where two or more lesions are located proximal to each other on the same or opposite DNA strands, is frequently produced as a result of exposure to ionising radiation. It has been suggested that such complex damaged sites pose problems for repair pathways. In this study, we addressed the question of how two 8-oxoguanine lesions, located two nucleotides apart on the same DNA strand, are repaired. We find that in human cell extracts repair of either of the 8-oxoguanine lesions within a tandem damaged site is initiated randomly and that the majority of the initiated repair proceeds to completion. However, a fraction of the initiated repair is delayed at the stage of an incised AP site and the rate of further processing of this incised AP site is dependent on the position of the remaining 8-oxoguanine. If the remaining 8-oxoguanine residue is located near the 5' terminus of the incised abasic site, repair continues as efficiently as repair of a single 8-oxoguanine residue. However, repair is delayed after the incision step when the remaining 8-oxoguanine residue is located near the 3' terminus. Although the presence of the 8-oxoguanine residue near the 3' terminus did not affect either DNA polymerase beta activity or poly(ADP)ribose polymerase-1 affinity and turnover on an incised AP site, we find that 8-oxoguanine-DNA glycosylase has reduced ability to remove an 8-oxoguanine residue located near the 3' terminus of the incised AP site. We find that binding of the 8-oxoguanine-DNA glycosylase to this 8-oxoguanine residue inhibits DNA repair synthesis by DNA polymerase beta, thus delaying repair. We propose that interference between a DNA glycosylase and DNA polymerase during the repair of tandem lesions may lead to accumulation of the intermediate products that contain persisting DNA strand breaks.     

Suppression of base excision repair reactions by apoptotic 24kDa-fragment of poly(ADP-ribose) polymerase 1 in bovine testis nuclear extract

In this study, we examined the interaction of PARP1 and its apoptotic 24kDa-fragment with DNA duplexes mimicking different stages/pathways of base excision repair (BER) using a photocross-linking technique combined to in vitro functional assay. We found that endogenous PARP1 was photocross-linked to the gapped, nicked and flap containing DNA structures and its apoptotic 24kDa-fragment (p24), like PARP1, can interact with the same BER DNA intermediates. Effects of exogenous p24 on the repair of DNA duplexes containing a one nucleotide gap with furan phosphate or phosphate group at the 5'-end of the downstream primer were studied in bovine testis nuclear extract. We showed that the interaction of p24 with DNA, as a whole, inhibited the BER reactions. However, gap filling and nick sealing catalyzed by the enzymes of the extract with DNA substrates characteristic for short patch (SP) BER pathway cannot be completely inhibited by p24. In contrast, binding of p24 to DNA duplex with a 5'-furan or a 5'-flap at the 5'-side of a nick inhibits strand-displacement DNA synthesis and activity of FEN1 in the repair of DNA via long patch (LP) BER pathway. Stimulation of the LP BER reactions induced by the addition of FEN1 or PCNA to the extract is suppressed by p24 thereby indicating that p24 can efficiently compete with these proteins of LP BER. Addition of pol beta to the extract can partially overcome the inhibitory effect of p24 and restore strand-displacement DNA synthesis. Thus, the apoptotic 24kDa-fragment of PARP1 may be considered as more efficient in inhibition of the LP than SP pathway and the effect may depend on the ratio of p24 to the repair enzymes catalyzing precise stages of BER.     

A nonuniform distribution of excision repair synthesis in nucleosome core DNA

We have investigated the distribution in nucleosome core DNA of nucleotides incorporated by excision repair synthesis occurring immediately after UV irradiation in human cells. We show that the differences previously observed for whole nuclei between the DNase I digestion profiles of repaired DNA (following its refolding into a nucleosome structure) and bulk DNA are obtained for isolated nucleosome core particles. Analysis of the differences obtained indicates that they could reflect a significant difference in the level of repair-incorporated nucleotides at different sites within the core DNA region. To test this possibility directly, we have used exonuclease III digestion of very homogeneous sized core particle DNA to "map" the distribution of repair synthesis in these regions. Our results indicate that in a significant fraction of the nucleosomes the 5' and 3' ends of the core DNA are markedly enhanced in repair-incorporated nucleotides relative to the central region of the core particle. A best fit analysis indicates that a good approximation of the data is obtained for a distribution where the core DNA is uniformly labeled from the 5' end to position 62 and from position 114 to the 3' end, with the 52-base central region being devoid of repair-incorporated nucleotides. This distribution accounts for all of the quantitative differences observed previously between repaired DNA and bulk DNA following the rapid phase of nucleosome rearrangement when it is assumed that linker DNA and the core DNA ends are repaired with equal efficiency and the nucleosome structure of newly repaired DNA is identical with that of bulk chromatin. Furthermore, the 52-base central region that is devoid of repair synthesis contains the lowest frequency cutting sites for DNase I in vitro, as well as the only "internal" locations where two (rather than one) histones interact with a 10-base segment of each DNA strand.     

Initiation of unidirectional ColE2 DNA replication by a unique priming mechanism.

The ColE2 DNA can be replicated in an in vitro system consisting of a crude extract of Escherichia coli cells. DNA synthesis requires a plasmid-coded protein (Rep) and host DNA polymerase I but not host RNA polymerase. Replication starts at a fixed region containing the origin and proceeds unidirectionally. The leading- and lagging-strand DNA fragments synthesized around the origin were identified from early replicative intermediates. The 5' end of the leading-strand DNA fragment was mapped at a unique position in the minimal origin and carried RNA of a few residues. The results suggested that the initiation of the leading-strand DNA synthesis does not require the host DnaG protein. Thus the Rep protein itself seems to be a primase. Synthesis of the primer RNA at a fixed site in the origin region on a double-stranded DNA template is a unique property of the ColE2 Rep protein among other known primases. The 3' end of the lagging-strand DNA fragment was mapped at a unique position just at the end of the minimal origin region. Termination of the lagging-strand DNA fragment at that position seems to be the mechanism of the unidirectional replication of ColE2 plasmid.     

9-amino-ellipticine inhibits the apurinic site-dependent base excision-repair pathway.

The aromatic amine 9-amino-ellipticine is a synthetic DNA intercalating compound derived from the antitumor agent ellipticine, which cleaves at very low doses DNA containing apurinic sites by beta-elimination through formation of a Schiff base. This compound has been shown to potentiate the cytotoxic effect of alkylating drugs, such as dimethyl sulfate, in E. coli through a mechanism involving apurinic sites. We have studied the ability of 9-amino-ellipticine to inhibit an enzymatic repair system mimicking base-excision repair, in which E. coli exonuclease III only presents an endonuclease for apurinic/apyrimidinic site activity. 10 microM of 9-amino-ellipticine inhibits 70% of apurinic site repair. Other intercalating agents with similar affinities for DNA do not induce any inhibition. In another system designed for the direct assay of the exonuclease III-induced incisions 5' to AP sites 10 microM of 9-amino-ellipticine inhibits 65% of the endonuclease for apurinic/apyrimidinic site activity of E. coli exonuclease III. The 9-amino-ellipticine-induced formation of a 2',3'-unsaturated deoxyribose and cleavage at the 3' side of the apurinic site, and possible creation of an adduct, as suggested by Bertrand and coworkers (1989), on the 3' position of the deoxyribose seem to strongly inhibit the endonuclease for apurinic/apyrimidinic site activity. 9-Amino-ellipticine appears therefore to be the first small ligand which can inhibit, by an irreversible modification of the substrate, the repair of apurinic sites through the base excision-repair pathway at a pharmacological concentration.     

Influence of GATC sequences on Escherichia coli DNA mismatch repair in vitro.

The effect of the number and position of DNA adenine methylation (dam) sites, i.e., d(GATC) sequences, on mismatch repair in Escherichia coli was investigated. The efficiency of repair was measured in an in vitro assay which used an f1 heteroduplex containing a G/T mismatch within the single EcoRI site. Both an increase in the number of dam sites and a shortened distance between dam site and mismatched site increased the efficiency of mismatch repair. The sequences adjacent to d(GATC) also affected the efficiency of methylation-directed mismatch repair. Furthermore, heteroduplexes with one extra dam site located close to either the 5' or 3' end of the excised base increased the repair efficiency to about the same extent. The findings suggest that the mismatch repair pathway has no preferred polarity.     

Use of crosslinking for revealing the DNA phosphate groups forming specific contacts with the E. coli Fpg protein

Specific contacts between DNA phosphate groups and positively charged nucleophilic amino acids from the Escherichia coli Fpg protein play a significant role in DNA-Fpg protein interaction. In order to identify these phosphate groups the chemical crosslinking procedure was carried out. The probing of the Fpg protein active center was performed using a series of reactive DNA duplexes containing both a single 7,8-dihydro-8-oxoguanosine (oxoG) residue and O-alkyl-substituted pyrophosphate internucleotide groups at the same time. Reactive internucleotide groups were introduced in dsDNA immediately 5' or 3' to the oxidative lesion and one or two nucleotides 5' or 3' away from it. We showed that the Fpg protein specifically binds to the modified DNA duplexes. The binding efficiency varied with the position of the reactive group and was higher for the duplexes containing substituted pyrophosphate groups at the ends of pentanucleotide with the oxoG in the center. The nicking efficiency of the DNA duplexes containing the reactive groups one or two nucleotides 5' away from the lesion was higher as compared to non-modified DNA duplex bearing only the oxidative damage. We found two novel non-hydrolizable substrate analogs for the Fpg protein containing pyrophosphate and substituted pyrophosphate groups 3' adjacent to the oxoG. Using crosslinking, we revealed the phosphate groups, 3' and 5' adjacent to the lesion, which have specific contacts with nucleophilic amino acids from the E. coli Fpg protein active center. The crosslinking efficiency achieved 30%. The approaches developed can be employed in the studies of pro- and eucaryotic homologs of the E. coli Fpg protein as well as other repair enzymes.     

Multifunctional human apurinic/apyrimidinic endonuclease 1: the role of additional functions

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is multifunctional enzyme. APEI is involved in the DNA base excision repair process (BER). APE1 participates in BER by cleaving the DNA adjacent to the 5' side of an AP site to produce a hydroxyl group at the 3' terminus of an unmodified nucleotide upstream of the nick and a 5' deoxyribose phosphate moiety downstream. In addition to its AP-endonucleolytic function, APE1 possesses 3' phosphodiesterase, 3'-5' exonuclease and 3' phosphatase activities. Independently of being characterized as DNA repair protein, APE1 was identified as redox-factor (Ref-1). Our own and literature data on the role of APE1 additional functions in cell metabolism and on interactions of APE1 with DNA and other proteins that participate in BER are analyzed in this review.     

Repair of an interstrand DNA cross-link initiated by ERCC1-XPF repair/recombination nuclease

Interstrand DNA cross-link damage is a severe challenge to genomic integrity. Nucleotide excision repair plays some role in the repair of DNA cross-links caused by psoralens and other agents. However, in mammalian cells there is evidence that the ERCC1-XPF nuclease has a specialized additional function during interstrand DNA cross-link repair, beyond its role in nucleotide excision repair. We placed a psoralen monoadduct or interstrand cross-link in a duplex, 4-6 bases from a junction with unpaired DNA. ERCC1-XPF endonucleolytically cleaved within the duplex on either side of the adduct, on the strand having an unpaired 3' tail. Cross-links that were cleaved only on the 5' side were purified and reincubated with ERCC1-XPF. A second cleavage was then observed on the 3' side. Relevant partially unwound structures near a cross-link may be expected to arise frequently, for example at stalled DNA replication forks. The results show that the single enzyme ERCC1-XPF can release one arm of a cross-link and suggest a novel mechanism for interstrand cross-link repair.     

Restriction endonucleases in Azospirillum

Azospirillum brasilense, A. amazonense, and A. lipoferum strains were screened for restriction endonucleases using phage lambda DNA. The extract of A. brasilense 29711 cleaved lambda DNA into specific fragments. It was concluded that this strain possesses a class II restriction endonuclease which was named AbrI. AbrI has a single recognition site on lambda DNA at position of approx. 33 500 bp. AbrI was characterized as an isoschizomer of XhoI, which cuts lambda DNA at 33 498 bp and cleaves double-stranded DNA at the sequence 5'-C TCGAG-3'. From other Azospirilla strains only A. amazonense QRZ42 extracts (AamI activity) cleaved DNA into specific fragments under certain conditions.     

Two pathways for removal of nonhomologous DNA ends during double-strand break repair in Saccharomyces cerevisiae.

During repair of a double-strand break (DSB) by gene conversion, one or both 3' ends of the DSB invade a homologous donor sequence and initiate new DNA synthesis. The use of the invading DNA strand as a primer for new DNA synthesis requires that any nonhomologous bases at the 3' end be removed. We have previously shown that removal of a 3' nonhomologous tail in Saccharomyces cerevisiae depends on the nucleotide excision repair endonuclease Rad1/Rad10, and also on the mismatch repair proteins Msh2 and Msh3. We now report that these four proteins are needed only when the nonhomologous ends of recombining DNA are 30 nucleotides (nt) long or longer. An additional protein, the helicase Srs2, is required for the RAD1-dependent removal of long 3' tails. We suggest that Srs2 acts to extend and stabilize the initial nascent joint between the invading single strand and its homolog. 3' tails shorter than 30 nt are removed by another mechanism that depends at least in part on the 3'-to-5' proofreading activity of DNA polymerase delta.     

Characterization of a DNA repair domain containing the dihydrofolate reductase gene in Chinese hamster ovary cells

The formation and removal of UV-induced pyrimidine dimers were measured in restriction fragments near and within the essential dihydrofolate reductase (DHFR) gene in Chinese hamster ovary cells in order to map the genomic fine structure of DNA repair. Dimer frequencies were determined at 0, 8, and 24 h after irradiating the cells with 20 J/m2 UV light (254 nm). Within 8 h, the cells had removed more than 40% of the dimers from sequences near the 5' end of the gene, somewhat fewer from the 3' end, but only 2% from the 3' flanking region and 10% from a region upstream from the gene. The corresponding extent of repair in the genome as a whole is 5-10% in the 8-h period. Isoschizomeric restriction enzyme analysis was used to detect the level of methylation in the fragments in which repair was measured. We found that the only hypomethylated sites in and around the DHFR gene were in the fragment near its 5' end, which displayed maximal DNA repair efficiency. The size of the region of preferential DNA repair at the DHFR locus appears to be in the range of 50-80 kilobases, and this finding is discussed in relation to genomic domains and the structure of mammalian chromatin.     

Identification of oligothymidylates as new simple substrates for Escherichia coli DNA photolyase and their use in a rapid spectrophotometric enzyme assay

Escherichia coli DNA photolyase exhibits the same turnover number (3.4 min-1) for the repair of dimers in oligothymidylates [oligo(dT)n] containing 4-18 thymine residues. This rate is identical with that observed with polythymidylate and with native DNA. The enzyme exhibits a similar high affinity with oligomers containing seven or more thymine residues. A decrease in affinity is detectable with oligo(dT)n when n = 4-6. The enzyme is active with oligo(dT)3, but no evidence for saturation was obtained at dimer concentrations up to 15 microM where the observed repair rate is 43% of the turnover number observed with the higher homologues. Nearly quantitative (90-100%) repair is observed with oligo(dT)n when n is greater than or equal to 9. Photolyase can repair internal dimers and dimers at a 5' end where the terminal ribose is phosphorylated but not at unphosphorylated 5' or 3' ends. The latter can explain a progressive decrease in the extent of repair observed with short-chain oligomers. The observed specificity can also explain why the enzyme is inactive with oligo(dT)2 [p(dT)2] since the only dimer possible in oligo(dT)2 involves an unphosphorylated 3' end. That the enzyme can repair dimers in short-chain, single-stranded analogues for DNA suggests that in catalysis with DNA recognition of the dimer itself is important as opposed to recognition of the deformation in DNA structure produced by the dimer. Dimer repair with oligo(dT)n is detected by the increase in absorbance at 260 nm, a feature which is used as the basis for a rapid spectrophotometric assay with a lower detection limit around 150 pmol of dimer repaired.