Fidelity of uracil-initiated base excision DNA repair in Escherichia coli cell extracts

The error frequency and mutational specificity associated with Escherichia coli uracil-initiated base excision repair were measured using an M13mp2 lacZalpha DNA-based reversion assay. Repair was detected in cell-free extracts utilizing a form I DNA substrate containing a site-specific uracil residue. The rate and extent of complete uracil-DNA repair were measured using uracil-DNA glycosylase (Ung)- or double-strand uracil-DNA glycosylase (Dug)-proficient and -deficient isogenic E. coli cells. In reactions utilizing E. coli NR8051 (ung(+) dug(+)), approximately 80% of the uracil-DNA was repaired, whereas about 20% repair was observed using NR8052 (ung(-) dug(+)) cells. The Ung-deficient reaction was insensitive to inhibition by the PBS2 uracil-DNA glycosylase inhibitor protein, implying the involvement of Dug activity. Under both conditions, repaired form I DNA accumulated in conjunction with limited DNA synthesis associated with a repair patch size of 1-20 nucleotides. Reactions conducted with E. coli BH156 (ung(-) dug(+)), BH157 (ung(+) dug(-)), and BH158 (ung(-) dug(-)) cells provided direct evidence for the involvement of Dug in uracil-DNA repair. The rate of repair was 5-fold greater in the Ung-proficient than in the Ung-deficient reactions, while repair was not detected in reactions deficient in both Ung and Dug. The base substitution reversion frequency associated with uracil-DNA repair was determined to be approximately 5.5 x 10(-)(4) with transversion mutations dominating the mutational spectrum. In the presence of Dug, inactivation of Ung resulted in up to a 7.3-fold increase in mutation frequency without a dramatic change in mutational specificity.     

DNA polymerase lambda mediates a back-up base excision repair activity in extracts of mouse embryonic fibroblasts

Mammalian DNA polymerase (pol) lambda is a member of the X-family of DNA polymerases and has striking enzymatic and structural similarities to mammalian DNA pol beta. Because pol beta provides two important enzymatic activities for base excision repair (BER), we examined whether pol lambda might also contribute to BER. We used extracts from mouse embryonic fibroblasts representing wild-type and null genotypes for pol beta and pol lambda. In combination with neutralizing antibodies against pol beta and pol lambda, our results show a BER deficiency in the pol lambda -/- cell extract compared with extract from isogenic wild-type cells. In addition, the pol lambda antibody strongly reduced in vitro BER in the pol beta -/- cell extract. These data indicate that pol lambda is able to contribute to BER in mouse fibroblast cell extract.     

Aphidicolin-resistant and -sensitive base excision repair in wild-type and DNA polymerase beta-defective mouse cells

Several DNA polymerases (Pols) can add complementary bases at the gap created during the base excision repair (BER). To characterize the BER resynthesis step, the repair of a single abasic site by wild-type and Pol beta-defective mouse cell extracts was analysed in the presence of aphidicolin, a specific inhibitor of replicative Pols. We show that there is a competition between distributive and processive Pols for the nucleotide addition at the primer terminus. In wild-type cell extracts, the initial nucleotide insertion involves mainly Pol beta but the elongation step is carried out by a replicative Pol. Conversely, in Pol beta-null cell extracts the synthesis step is carried out by a replicative Pol without any switching to an auxiliary polymerase. We present evidence that short-patch repair synthesis occurs even in the absence of both Pol beta and replicative Pols. Exogeneously added purified human Pol lambda was unable to stimulate this back-up synthesis.     

DNA excision repair in mammalian cell extracts.

The many genetic complementation groups of DNA excision-repair defective mammalian cells indicate the considerable complexity of the excision repair process. The cloning of several repair genes is taking the field a step closer to mechanistic studies of the actions and interactions of repair proteins. Early biochemical studies of mammalian DNA repair in vitro are now at hand. Repair synthesis in damaged DNA can be monitored by following the incorporation of radiolabelled nucleotides. Synthesis is carried out by mammalian cell extracts and is defective in extracts from cell lines derived from individuals with the excision-repair disorder xeroderma pigmentosum. Biochemical complementation of the defective extracts can be used to purify repair proteins. Repair of damage caused by agents including ultraviolet irradiation, psoralens, and platinating compounds has been observed. Neutralising antibodies against the human single-stranded DNA binding protein (HSSB) have demonstrated a requirement for this protein in DNA excision repair as well as in DNA replication.     

REV1 mediated mutagenesis in base excision repair deficient mouse fibroblast

The DNA polymerase beta (Pol beta) null background renders mouse embryonic fibroblast (MEF) cells base excision repair deficient and hyper-mutagenic upon treatment with the monofunctional alkylating agent, methyl methanesulfonate (MMS). This effect involves an increase in all types of base substitutions, with a modest predominance of G to A transitions. In the present study, we examined the hypothesis that the MMS-induced mutagenesis in the Pol beta null MEF system is due to a lesion bypass mechanism. We studied the effect of RNAi mediated down-regulation of the lesion bypass factor REV1. The steady-state level of REV1 protein was reduced by more than 95% using stable expression of a siRNA construct in a Pol beta null cell line. We found that REV1 expression is required for the MMS-induced mutagenesis phenotype of Pol beta null MEF cells. In contrast, cell survival after MMS treatment is not reduced in the absence of REV1.     

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.     

The base substitution fidelity of DNA polymerase beta-dependent single nucleotide base excision repair

Damaged DNA bases are removed from mammalian genomes by base excision repair (BER). Single nucleotide BER requires several enzymatic activities, including DNA polymerase and 5',2'-deoxyribose-5-phosphate lyase. Both activities are intrinsic to four human DNA polymerases whose base substitution error rate during gap-filling DNA synthesis varies by more than 10,000-fold. This suggests that BER fidelity could vary over a wide range in an enzyme dependent manner. To investigate this possibility, here we describe an assay to measure the fidelity of BER reactions reconstituted with purified enzymes. When human uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and DNA ligase 1 replace uracil opposite template A or G, base substitution error rates are 相似文献   

Characterization of the DNA polymerase requirement of human base excision repair.

Base excision repair is one of the major mechanisms by which cells correct damaged DNA. We have developed an in vitro assay for base excision repair which is dependent on a uracil-containing DNA template. In this report, we demonstrate the fractionation of a human cell extract into two required components. One fraction was extensively purified and by several criteria shown to be identical to DNA polymerase beta (Polbeta). Purified, recombinant Polbeta efficiently substituted for this fraction. Escherichia coli PolI, mammalian Poldelta and to a lesser extent Polalpha and epsilon also functioned in this assay. We provide evidence that multiple polymerases function in base excision repair in human cell extracts. A neutralizing antibody to Polbeta, which inhibited repair synthesis catalyzed by pure Polbeta by approximately 90%, only suppressed repair in crude extracts by a maximum of approximately 70%. An inhibitor of Polbeta, ddCTP, decreased base excision repair in crude extracts by approximately 50%, whereas the Polalpha/delta/epsilon inhibitor, aphidicolin, reduced the reaction by approximately 20%. A combination of these chemical inhibitors almost completely abolished repair synthesis. These data suggest that Polbeta is the major base excision repair polymerase in human cells, but that other polymerases also contribute to a significant extent.     

Changing capacity for DNA excision repair in mouse embryonic cells in vitro

Capacity for excision repair of ultraviolet radiation damage to DNA in primary cultures of mouse embryonic cells is dependent on the gestational stage and the duration of in vitro growth. Fibroblasts of mouse embryos at 13–15 days gestation excise thymine dimers and perform unscheduled DNA synthesis after ultraviolet radiation. After several successive transfers in vitro, concomitantly with a pronounced reduction in growth rate, ability for excision repair decreases. DNA repair capacity is impaired in cells obtained from embryos at late stages of development (17–19 days gestation). Experiments with epithelial kidney cells from 5-day-old mice indicate that capacity for excision repair may depend on cell type and its origin.     

Mixed spermatogenic germ cell nuclear extracts exhibit high base excision repair activity

Spermatogenic cells exhibit a lower spontaneous mutation frequency than somatic tissues in a lacI transgene and many base excision repair (BER) genes display the highest observed level of expression in the testis. In this study, uracil-DNA glycosylase-initiated BER activity was measured in nuclear extracts prepared from tissues obtained from each of three mouse strains. Extracts from mixed spermatogenic germ cells displayed the greatest activity followed by liver then brain for all three strains, and the activity for a given tissue was consistent among the three strains. Levels of various BER proteins were examined by western blot analyses and found to be consistent with activity levels. Nuclear extracts prepared from purified Sertoli cells, a somatic component of the seminiferous epithelium, exhibited significantly lower activity than mixed spermatogenic cell-type nuclear extracts, thereby suggesting that the high BER activity observed in mixed germ cell nuclear extracts was not a characteristic of all testicular cell types. Nuclear extracts from thymocytes and small intestines were assayed to assess activity in a mitotically active cell type and tissue. Overall, the order of tissues/cells exhibiting the greatest to lowest activity was mixed germ cells > Sertoli cells > thymocytes > small intestine > liver > brain.     

Single-nucleotide patch base excision repair of uracil in DNA by mitochondrial protein extracts.

Mammalian mitochondria contain several 16.5 kb circular DNAs (mtDNA) encoding electron transport chain proteins. Reactive oxygen species formed as byproducts from oxidative phosphorylation in these organelles can cause oxidative deamination of cytosine and lead to uracil in mtDNA. Upon mtDNA replication, these lesions, if unrepaired, can lead to mutations. Until recently, it was thought that there was no DNA repair in mitochondria, but lately there is evidence that some lesions are efficiently repaired in these organelles. In the study of nuclear DNA repair, the in vitro repair measurements in cell extracts have provided major insights into the mechanisms. The use of whole-cell extract based DNA repair methods has revealed that mammalian nuclear base excision repair (BER) diverges into two pathways: the single-nucleotide replacement and long patch repair mechanisms. Similar in vitro methods have not been available for the study of mitochondrial BER. We have established an in vitro DNA repair system supported by rat liver mitochondrial protein extract and DNA substrates containing a single uracil opposite to a guanine. Using this approach, we examined the repair pathways and the identity of the DNA polymerase involved in mitochondrial BER (mtBER). Employing restriction analysis of in vitro repaired DNA to map the repair patch size, we demonstrate that only one nucleotide is incorporated during the repair process. Thus, in contrast to BER in the nucleus, mtBER of uracil in DNA is solely accomplished by single-nucleotide replacement.     

Role of DNA polymerase beta in the excision step of long patch mammalian base excision repair.

The two base excision repair (BER) subpathways in mammalian cells are characterized by the number of nucleotides synthesized into the excision patch. They are the "single-nucleotide" BER pathway and the "long patch" (several nucleotides incorporated) BER pathway. Both of these subpathways involve excision of a damaged base and/or nearby nucleotides and DNA synthesis to fill the excision gap. Whereas DNA polymerase beta (pol beta) is known to participate in the single-nucleotide BER pathway, the identity of polymerases involved in long patch BER has remained unclear. By analyzing products of long patch excision generated during BER of a uracil-containing DNA substrate in mammalian cell extracts we find that long patch excision depends on pol beta. We show that the excision of the characteristic 5'-deoxyribose phosphate containing oligonucleotide (dRP-oligo) is deficient in extracts from pol beta null cells and is rescued by addition of purified pol beta. Also, pol beta-neutralizing antibody inhibits release of the dRP-oligo in wild-type cell extracts, and the addition of pol beta after inhibition with antibody completely restores the excision reaction. The results indicate that pol beta plays an essential role in long patch BER by conducting strand displacement synthesis and controlling the size of the excised flap.     

Repair of dihydrouracil supported by base excision repair in mNTH1 knock-out cell extracts

In mammalian cells, thymine glycols and other oxidized pyrimidines such as 5,6-dihydrouracil are removed from DNA by the NTH1 protein, a bifunctional DNA-N-glycosylase. However, mNTH1 knock-out mice in common with other DNA glycosylase-deficient mice do not show any severe abnormalities associated with accumulation of DNA damage and mutations. In the present study we used an in vitro repair system to investigate the mechanism for the removal of 5,6-dihydrouracil from DNA by mNTH1-deficient cell-free extracts derived from testes of mNTH1 knock-out mice. We found that these extracts are able to support the removal of 5,6-dihydrouracil from DNA at about 20% of the efficiency of normal extracts. Furthermore, we also found that single-nucleotide patch base excision repair is the major pathway for removal of 5,6-dihydrouracil in mNTH1-deficient cell extracts, suggesting the involvement of other DNA glycosylase(s) in the removal of oxidized pyrimidines.     

Protection against methylation-induced cytotoxicity by DNA polymerase beta-dependent long patch base excision repair

Using a plasmid-based uracil-containing DNA substrate, we found that the long patch base excision repair (BER) activity of a wild-type mouse fibroblast extract was partially inhibited by an antibody to DNA polymerase beta (beta-pol). This suggests that beta-pol participates in long patch BER, in addition to single-nucleotide BER. In single-nucleotide BER, the deoxyribose phosphate (dRP) in the abasic site is removed by the lyase activity of beta-pol. Methoxyamine (MX) can react with the aldehyde of an abasic site, making it refractory to the beta-elimination step of the dRP lyase mechanism, thus blocking single-nucleotide BER. MX exposure sensitizes wild-type, but not beta-pol null mouse embryonic fibroblasts, to the cytotoxic effects of methyl methanesulfonate (MMS) and methylnitrosourea. Expression of beta-pol in the null cells restores the ability of MX to modulate sensitivity to MMS. The beta-pol null cells are known to be hypersensitive to MMS and methylnitrosourea, and in the presence of MX (i.e. under conditions where single-nucleotide BER is blocked) the null cells are still considerably more sensitive than wild-type. The data are consistent with a role of beta-pol in long patch BER, which helps protect cells against methylation damage-induced cytotoxicity.     

FEN1 stimulation of DNA polymerase beta mediates an excision step in mammalian long patch base excision repair

In mammalian cells, single-base lesions, such as uracil and abasic sites, appear to be repaired by at least two base excision repair (BER) subpathways: "single-nucleotide BER" requiring DNA synthesis of just one nucleotide and "long patch BER" requiring multi-nucleotide DNA synthesis. In single-nucleotide BER, DNA polymerase beta (beta-pol) accounts for both gap filling DNA synthesis and removal of the 5'-deoxyribose phosphate (dRP) of the abasic site, whereas the involvement of various DNA polymerases in long patch BER is less well understood. Recently, we found that beta-pol plays a role in mammalian cell extract-mediated long patch BER, in that formation of a key excision product, 5'-dRP-trinucleotide (5'-dRP-N(3)), is dependent upon beta-pol (Dianov, G. L., Prasad, R., Wilson, S. H., and Bohr, V.A. (1999) J. Biol. Chem. 274, 13741-13743). The structure-specific endonuclease flap endonuclease 1 (FEN1) has also been suggested to be involved in long patch BER excision. Here, we demonstrate by immunodepletion experiments that 5'-dRP-N(3) excision in long patch BER of uracil-DNA in a human lymphoid cell extract is, indeed, dependent upon FEN1. Next, we reconstituted the excision step of long patch BER using purified human proteins and an oligonucleotide substrate with 5'-dRP at the margin of a one-nucleotide gap. Formation of the excision product 5'-dRP-N(3) was dependent upon both strand displacement DNA synthesis by beta-pol and FEN1 excision. FEN1 stimulated strand displacement DNA synthesis of beta-pol. FEN1 acting either alone, or without DNA synthesis by beta-pol, produced a two-nucleotide excision product, 5'-dRP-N(1), but not 5'-dRP-N(3). These results demonstrate that human FEN1 and beta-pol can cooperate in long patch BER excision and specify the predominant excision product seen with a cell extract.     

Mismatch and base excision repair proficiency in murine embryonic stem cells

Accumulation of mutations in embryonic stem (ES) cells would be detrimental to an embryo derived from these cells, and would adversely affect multiple organ systems and tissue types. ES cells have evolved multiple mechanisms to preserve genomic integrity that extend beyond those found in differentiated cell types. The present study queried whether mismatch repair (MMR) and base-excision repair (BER) may play a role in the maintenance of murine ES cell genomes. The MMR proteins Msh2 and Msh6 are highly elevated in mouse ES cells compared with mouse embryo fibroblasts (MEFs), as are Pms2 and Mlh1, albeit to a lesser extent. Cells transfected with an MMR reporter plasmid showed that MMR repair capacity is low in MEFs, but highly active in wildtype ES cells. As expected, an ES cell line defective in MMR was several-fold less effective in repair level than wildtype ES cells. Like proteins that participate in MMR, the level of proteins involved in BER was elevated in ES cells compared with MEFs. When BER activity was examined biochemically using a uracil-containing oligonucleotide template, repair activity was higher in ES cells compared with MEFs. The data are consistent with the suggestion that ES cells have multiple mechanisms, including highly active MMR and BER that preserve genetic integrity and minimize the accumulation of mutations.     

Mitochondrial DNA, base excision repair and neurodegeneration

Neurodegeneration is a growing public health concern because of the rapid increase in median and maximum life expectancy in the developed world. Mitochondrial dysfunction seems to play a critical role in neurodegeneration, likely owing to the high energy demand of the central nervous system and its sole reliance on oxidative metabolism for energy production. Loss of mitochondrial function has been clearly demonstrated in several neuropathologies, most notably those associated with age, like Alzheimer's, Parkinson's and Huntington's diseases. Among the common features observed in such conditions is the accumulation of oxidative DNA damage, in particular in the mitochondrial DNA, suggesting that mitochondrial DNA instability may play a causative role in the development of these diseases. In this review we examine the evidence for the accumulation of oxidative DNA damage in mitochondria, and its relationship with loss of mitochondrial function and cell death in neural tissues. Oxidative DNA damage is repaired mainly by the base excision repair pathway. Thus, we review the molecular events and enzymes involved in base excision repair in mitochondria, and explore the possible role of alterations in mitochondrial base excision repair activities in premature aging and age-associated neurodegenerative diseases.     

DNA polymerase beta promotes recruitment of DNA ligase III alpha-XRCC1 to sites of base excision repair

Base excision repair is a major pathway for the removal of simple lesions in DNA including base damage and base loss (abasic site). Base excision repair requires the coordinated action of several repair and ancillary proteins, the impairment of which can lead to genetic instability. Using a protein-DNA cross-linking assay during repair in human whole cell extracts, we monitored proteins involved in the initial steps of repair of a substrate containing a site-specific abasic site to address the molecular events following incision of the abasic site by AP endonuclease. We find that after dissociation of AP endonuclease from the incised abasic site, both DNA polymerase beta (Pol beta) and the DNA ligase IIIalpha-XRCC1 heterodimer efficiently bind/cross-link to the substrate DNA. We also find that the cross-linking efficacy of the DNA ligase IIIalpha-XRCC1 heterodimer was decreased about 2-fold in the Pol beta-deficient cell extract but was rescued by addition of purified wild type but not a mutant Pol beta protein that does not interact with the DNA ligase IIIalpha-XRCC1 heterodimer. We further demonstrate that Pol beta and the DNA ligase IIIalpha-XRCC1 heterodimer are present at equimolar concentrations in whole cell extracts and that Pol beta has a 7-fold higher affinity to the incised abasic site containing substrate than DNA ligase IIIalpha. Using gel filtration of whole cell extracts prepared at physiological salt conditions (0.15 M NaCl), we find no evidence for a stable preexisting complex of DNA Pol beta with the DNA ligase IIIalpha-XRCC1 heterodimer. Taken together, these data suggest that following incision by AP endonuclease, DNA Pol beta recognizes and binds to the incised abasic site and promotes recruitment of the DNA ligase IIIalpha-XRCC1 heterodimer through its interaction with XRCC1.     

DNA polymerase beta is the major dRP lyase involved in repair of oxidative base lesions in DNA by mammalian cell extracts.

The repair of oxidative base lesions in DNA is a coordinated chain of reactions that includes removal of the damaged base, incision of the phosphodiester backbone at the abasic sugar residue, incorporation of an undamaged nucleotide and sealing of the DNA strand break. Although removal of a damaged base in mammalian cells is initiated primarily by a damage-specific DNA glycosylase, several lyases and DNA polymerases may contribute to the later stages of repair. DNA polymerase beta (Pol beta) was implicated recently as the major polymerase involved in repair of oxidative base lesions; however, the identity of the lyase participating in the repair of oxidative lesions is unclear. We studied the mechanism by which mammalian cell extracts process DNA substrates containing a single 8-oxoguanine or 5,6-dihydrouracil at a defined position. We find that, when repair synthesis proceeds through a Pol beta-dependent single nucleotide replacement mechanism, the 5'-deoxyribosephosphate lyase activity of Pol beta is essential for repair of both lesions.