Modulation of the 5'-deoxyribose-5-phosphate lyase and DNA synthesis activities of mammalian DNA polymerase beta by apurinic/apyrimidinic endonuclease 1

The Ape1 protein initiates the repair of apurinic/apyrimidinic sites during mammalian base excision repair (BER) of DNA. Ape1 catalyzes hydrolysis of the 5'-phosphodiester bond of abasic DNA to create nicks flanked by 3'-hydroxyl and 5'-deoxyribose 5-phosphate (dRP) termini. DNA polymerase (pol) beta catalyzes both DNA synthesis at the 3'-hydroxyl terminus and excision of the 5'-dRP moiety prior to completion of BER by DNA ligase. During BER, Ape1 recruits pol beta to the incised apurinic/apyrimidinic site and stimulates 5'-dRP excision by pol beta. The activities of these two enzymes are thus coordinated during BER. To examine further the coordination of BER, we investigated the ability of Ape1 to modulate the deoxynucleotidyltransferase and 5'-dRP lyase activities of pol beta. We report here that Ape1 stimulates 5'-dRP excision by a mechanism independent of its apurinic/apyrimidinic endonuclease activity. We also demonstrate a second mechanism, independent of Ape1, in which conditions that support DNA synthesis by pol beta also enhance 5'-dRP excision. Ape1 modulates the gap-filling activity of pol beta by specifically inhibiting synthesis on an incised abasic substrate but not on single-nucleotide gapped DNA. In contrast to the wild-type Ape1 protein, a catalytically impaired mutant form of Ape1 did not affect DNA synthesis by pol beta. However, this mutant protein retained the ability to stimulate 5'-dRP excision by pol beta. Simultaneous monitoring of 5'-dRP excision and DNA synthesis by pol beta demonstrated that the 5'-dRP lyase activity lags behind the polymerase activity despite the coordination of these two steps by Ape1 during BER.     

Trypanosoma cruzi contains a single detectable uracil-DNA glycosylase and repairs uracil exclusively via short patch base excision repair

Enzymes involved in genomic maintenance of human parasites are attractive targets for parasite-specific drugs. The parasitic protozoan Trypanosoma cruzi contains at least two enzymes involved in the protection against potentially mutagenic uracil, a deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and a uracil-DNA glycosylase belonging to the highly conserved UNG-family. Uracil-DNA glycosylase activities excise uracil from DNA and initiate a multistep base-excision repair (BER) pathway to restore the correct nucleotide sequence. Here we report the biochemical characterisation of T.cruzi UNG (TcUNG) and its contribution to the total uracil repair activity in T.cruzi. TcUNG is shown to be the major uracil-DNA glycosylase in T.cruzi. The purified recombinant TcUNG exhibits substrate preference for removal of uracil in the order ssU>U:G>U:A, and has no associated thymine-DNA glycosylase activity. T.cruzi apparently repairs U:G DNA substrate exclusively via short-patch BER, but the DNA polymerase involved surprisingly displays a vertebrate POLdelta-like pattern of inhibition. Back-up UDG activities such as SMUG, TDG and MBD4 were not found, underlying the importance of the TcUNG enzyme in protection against uracil in DNA and as a potential target for drug therapy.     

The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome: DOMINANT NEGATIVE FUNCTION OF THE CTD*

The human DNA glycosylase NEIL1 was recently demonstrated to initiate prereplicative base excision repair (BER) of oxidized bases in the replicating genome, thus preventing mutagenic replication. A significant fraction of NEIL1 in cells is present in large cellular complexes containing DNA replication and other repair proteins, as shown by gel filtration. However, how the interaction of NEIL1 affects its recruitment to the replication site for prereplicative repair was not investigated. Here, we show that NEIL1 binarily interacts with the proliferating cell nuclear antigen clamp loader replication factor C, DNA polymerase δ, and DNA ligase I in the absence of DNA via its non-conserved C-terminal domain (CTD); replication factor C interaction results in ∼8-fold stimulation of NEIL1 activity. Disruption of NEIL1 interactions within the BERosome complex, as observed for a NEIL1 deletion mutant (N311) lacking the CTD, not only inhibits complete BER in vitro but also prevents its chromatin association and reduced recruitment at replication foci in S phase cells. This suggests that the interaction of NEIL1 with replication and other BER proteins is required for efficient repair of the replicating genome. Consistently, the CTD polypeptide acts as a dominant negative inhibitor during in vitro repair, and its ectopic expression sensitizes human cells to reactive oxygen species. We conclude that multiple interactions among BER proteins lead to large complexes, which are critical for efficient BER in mammalian cells, and the CTD interaction could be targeted for enhancing drug/radiation sensitivity of tumor cells.     

1H NMR studies of DNA recognition by the glucocorticoid receptor: complex of the DNA binding domain with a half-site response element.

The complex of the rat glucocorticoid receptor (GR) DNA binding domain (DBD) and half-site sequence of the consensus glucocorticoid response element (GRE) has been studied by two-dimensional 1H NMR spectroscopy. The DNA fragment is a 10 base-pair oligonucleotide, 5'd(GCTGTTCTGC)3'.5'd-(GCAGAACAGC)3', containing the stronger binding GRE half-site hexamer, with GC base pairs at each end. The 93-residue GR-DBD contains an 86-residue segment corresponding to residues 440-525 of the rat GR. Eleven NOE cross peaks between the protein and DNA have been identified, and changes in the chemical shift of the DNA protons upon complex formation have been analyzed. Using these protein-DNA contact points, it can be concluded that (i) the "recognition helix" formed by residues C460-E469 lies in the major groove of the DNA; (ii) the GR-DBD is oriented on the GRE half-site such that residues A477-D481, forming the so-called D-loop, are available for protein-protein interaction in the GR-DBD dimer on the intact consensus GRE; and (iii) the 5-methyl of the second thymine in the half-site and valine 462 interact, confirming indirect evidence [Truss et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7180-7184; Mader et al. (1989) Nature 338, 271-274] that both play an important role in GR-DBD DNA binding. These findings are consistent with the model proposed by H?rd et al. [(1990) Science 249, 157-160] and the X-ray crystallographic complex structure determined by Luisi et al. [(1991) Nature 352, 497-505].     

Post-replicative base excision repair in replication foci.

Base excision repair (BER) is initiated by a DNA glycosylase and is completed by alternative routes, one of which requires proliferating cell nuclear antigen (PCNA) and other proteins also involved in DNA replication. We report that the major nuclear uracil-DNA glycosylase (UNG2) increases in S phase, during which it co-localizes with incorporated BrdUrd in replication foci. Uracil is rapidly removed from replicatively incorporated dUMP residues in isolated nuclei. Neutralizing antibodies to UNG2 inhibit this removal, indicating that UNG2 is the major uracil-DNA glycosylase responsible. PCNA and replication protein A (RPA) co-localize with UNG2 in replication foci, and a direct molecular interaction of UNG2 with PCNA (one binding site) and RPA (two binding sites) was demonstrated using two-hybrid assays, a peptide SPOT assay and enzyme-linked immunosorbent assays. These results demonstrate rapid post-replicative removal of incorporated uracil by UNG2 and indicate the formation of a BER complex that contains UNG2, RPA and PCNA close to the replication fork.     

Uracil-DNA glycosylase of Thermoplasma acidophilum directs long-patch base excision repair, which is promoted by deoxynucleoside triphosphates and ATP/ADP, into short-patch repair

Hydrolytic deamination of cytosine to uracil in DNA is increased in organisms adapted to high temperatures. Hitherto, the uracil base excision repair (BER) pathway has only been described in two archaeons, the crenarchaeon Pyrobaculum aerophilum and the euryarchaeon Archaeoglobus fulgidus, which are hyperthermophiles and use single-nucleotide replacement. In the former the apurinic/apyrimidinic (AP) site intermediate is removed by the sequential action of a 5'-acting AP endonuclease and a 5'-deoxyribose phosphate lyase, whereas in the latter the AP site is primarily removed by a 3'-acting AP lyase, followed by a 3'-phosphodiesterase. We describe here uracil BER by a cell extract of the thermoacidophilic euryarchaeon Thermoplasma acidophilum, which prefers a similar short-patch repair mode as A. fulgidus. Importantly, T. acidophilumcell extract also efficiently executes ATP/ADP-stimulated long-patch BER in the presence of deoxynucleoside triphosphates, with a repair track of ～15 nucleotides. Supplementation of recombinant uracil-DNA glycosylase (rTaUDG; ORF Ta0477) increased the formation of short-patch at the expense of long-patch repair intermediates, and additional supplementation of recombinant DNA ligase (rTalig; Ta1148) greatly enhanced repair product formation. TaUDG seems to recruit AP-incising and -excising functions to prepare for rapid single-nucleotide insertion and ligation, thus excluding slower and energy-costly long-patch BER.     

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.     

Down-regulation of DNA polymerase beta accompanies somatic hypermutation in human BL2 cell lines

Somatic hypermutation (SHM) is a fundamental process in immunoglobulin gene maturation that results in increased affinity of antibodies toward antigens. In one hypothesis explaining SHM in human B cells, the process is initiated by enzymatic deamination of cytosine to uracil in the immunoglobulin gene V-region and this in turn triggers mutation-prone forms of uracil-DNA base excision repair (BER). Yet, an uncertainty with this model is that BER of uracil-DNA in mammalian cells is generally error-free, wherein DNA polymerase beta (pol beta) conducts gap-filling synthesis by insertion of bases according to Watson-Crick rules. To evaluate this inconsistency, we examined pol beta expression in various SHM proficient human BL2 cell line subclones. We report that expression of pol beta in SHM proficient cell lines was strongly down-regulated. In contrast, in other BL2 subclones, we found that SHM was deficient and that pol beta expression was much higher than in the SHM proficient subclones. We also found that overexpression of recombinant human pol beta in a SHM proficient subclone abrogated its capacity for SHM. These results suggest that down-regulation of the normal BER gap-filling DNA polymerase, pol beta, accompanies induced SHM in BL2 cells. This is consistent with the hypothesis that normal error-free BER must be silenced to make way for an error-prone BER process that may be required during somatic hypermutation.     

DNA intermediates at the Escherichia coli replication fork. II. Studies using dut and ung mutants in vitro.

The incorporation of uracil into and excision from DNA were studied in vitro using lysates on cellophane discs made from Escherichia coli strains with defects in the enzymes dUTPase (dut) and uracil-DNA glycosylase (ung).Results with dut ung lysates indicate that dUTP is competitively incorporated with dTTP at the replication fork. Such incorporation is not due to DNA polymerase I. There is a mild discrimination (2.5-fold) against incorporation of dUTP versus dTTP. These data, together with in vivo uracil incorporation data (Tye et al., 1978) permit a rough estimate of the pool of dUTP in vivo (~0.5% of the dTTP pool).These in vitro data indicate that uracil-DNA glycosylase is the initial step in at least 90% of uracil excision events. However, in a strain defective in uracil-DNA glycosylase (ung-1), uracil-containing DNA is still more subject to single-strand scission than non-uracil-containing DNA, albeit at a rate at least tenfold less than in an ung⁺ strain.A number of qualitative statements may also be made about different steps in uracil incorporation and subsequent excision and repair events. When high levels of dUTP are added in vitro, a dut ung⁺ strain has a higher steady-state level of uracil in newly synthesized DNA than does an isogenic dut⁺ ung strain. Thus the dUTPase in these lysates has a higher capacity to be overloaded than does the excision system (i.e. uracil DNA glycosylase). However, the DNA sealing system (presumably DNA polymerase I and DNA ligase) apparently can handle all single-strand interruptions being introduced by uracil excision at the maximal rate, at least so that DNA synthesis can continue.     

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   

A mollicute (mycoplasma) DNA repair enzyme: purification and characterization of uracil-DNA glycosylase.

The DNA repair enzyme uracil-DNA glycosylase from Mycoplasma lactucae (831-C4) was purified 1,657-fold by using affinity chromatography and chromatofocusing techniques. The only substrate for the enzyme was DNA that contained uracil residues, and the Km of the enzyme was 1.05 +/- 0.12 microM for dUMP containing DNA. The product of the reaction was uracil, and it acted as a noncompetitive inhibitor of the uracil-DNA glycosylase with a Ki of 5.2 mM. The activity of the enzyme was insensitive to Mg2+, Mn2+, Zn2+, Ca2+, and Co2+ over the concentration range tested, and the activity was not inhibited by EDTA. The enzyme activity exhibited a biphasic response to monovalent cations and to polyamines. The enzyme had a pI of 6.4 and existed as a nonspherical monomeric protein with a molecular weight of 28,500 +/- 1,200. The uracil-DNA glycosylase from M. lactucae was inhibited by the uracil-DNA glycosylase inhibitor from bacteriophage PBS-2, but the amount of inhibitor required for 50% inhibition of the mycoplasmal enzyme was 2.2 and 8 times greater than that required to cause 50% inhibition of the uracil-DNA glycosylases from Escherichia coli and Bacillus subtilis, respectively. Previous studies have reported that some mollicutes lack uracil-DNA glycosylase activity, and the results of this study demonstrate that the uracil-DNA glycosylase from M. lactucae has a higher Km for uracil-containing DNA than those of the glycosylases of other procaryotic organisms. Thus, the low G + C content of the DNA from some mollicutes and the A.T-biased mutation pressure observed in these organisms may be related to their decreased capacity to remove uracil residues from DNA.     

Biochemical Reconstitution of the Mimiviral Base Excision Repair Pathway

Viruses are believed to be the obligate intracellular parasites that only carry genes essential for infecting and hijacking the host cell machinery. However, a recently discovered group of viruses belonging to the phylum nucleocytovirocota, also known as the nucleo-cytoplasmic large DNA viruses (NCLDVs), possess a number of genes that code for proteins predicted to be involved in metabolism, and DNA replication, and repair. In the present study, first, using proteomics of viral particles, we show that several proteins required for the completion of the DNA base excision repair (BER) pathway are packaged within the virions of Mimivirus as well as related viruses while they are absent from the virions of Marseillevirus and Kurlavirus that are NCLDVs with smaller genomes. We have thoroughly characterized three putative base excision repair enzymes from Mimivirus, a prototype NCLDV and successfully reconstituted the BER pathway using the purified recombinant proteins. The mimiviral uracil-DNA glycosylase (mvUDG) excises uracil from both ssDNA and dsDNA, a novel finding contrary to earlier studies. The putative AP-endonuclease (mvAPE) specifically cleaves at the abasic site created by the glycosylase while also exhibiting the 3′-5′ exonuclease activity. The Mimivirus polymerase X protein (mvPolX) can bind to gapped DNA substrates and perform single nucleotide gap-filling followed by downstream strand displacement. Furthermore, we show that when reconstituted in vitro, mvUDG, mvAPE, and mvPolX function cohesively to repair a uracil-containing DNA predominantly by long patch BER and together, may participate in the BER pathway during the early phase of Mimivirus life-cycle.     

The scaffold protein XRCC1 stabilizes the formation of polβ/gap DNA and ligase IIIα/nick DNA complexes in base excision repair

The base excision repair (BER) pathway involves gap filling by DNA polymerase (pol) β and subsequent nick sealing by ligase IIIα. X-ray cross-complementing protein 1 (XRCC1), a nonenzymatic scaffold protein, assembles multiprotein complexes, although the mechanism by which XRCC1 orchestrates the final steps of coordinated BER remains incompletely defined. Here, using a combination of biochemical and biophysical approaches, we revealed that the polβ/XRCC1 complex increases the processivity of BER reactions after correct nucleotide insertion into gaps in DNA and enhances the handoff of nicked repair products to the final ligation step. Moreover, the mutagenic ligation of nicked repair intermediate following polβ 8-oxodGTP insertion is enhanced in the presence of XRCC1. Our results demonstrated a stabilizing effect of XRCC1 on the formation of polβ/dNTP/gap DNA and ligase IIIα/ATP/nick DNA catalytic ternary complexes. Real-time monitoring of protein–protein interactions and DNA-binding kinetics showed stronger binding of XRCC1 to polβ than to ligase IIIα or aprataxin, and higher affinity for nick DNA with undamaged or damaged ends than for one nucleotide gap repair intermediate. Finally, we demonstrated slight differences in stable polβ/XRCC1 complex formation, polβ and ligase IIIα protein interaction kinetics, and handoff process as a result of cancer-associated (P161L, R194W, R280H, R399Q, Y576S) and cerebellar ataxia-related (K431N) XRCC1 variants. Overall, our findings provide novel insights into the coordinating role of XRCC1 and the effect of its disease-associated variants on substrate-product channeling in multiprotein/DNA complexes for efficient BER.     

The rate of base excision repair of uracil is controlled by the initiating glycosylase

Uracil in DNA is repaired by base excision repair (BER) initiated by a DNA glycosylase, followed by strand incision, trimming of ends, gap filling and ligation. Uracil in DNA comes in two distinct forms; U:A pairs, typically resulting from replication errors, and mutagenic U:G mismatches, arising from cytosine deamination. To identify proteins critical to the rate of repair of these lesions, we quantified overall repair of U:A pairs, U:G mismatches and repair intermediates (abasic sites and nicked abasic sites) in vitro. For this purpose we used circular DNA substrates and nuclear extracts of eight human cell lines with wide variation in the content of BER proteins. We identified the initiating uracil-DNA glycosylase UNG2 as the major overall rate-limiting factor. UNG2 is apparently the sole glycosylase initiating BER of U:A pairs and generally initiated repair of almost 90% of the U:G mismatches. Surprisingly, TDG contributed at least as much as single-strand selective monofunctional uracil-DNA glycosylase 1 (SMUG1) to BER of U:G mismatches. Furthermore, in a cell line that expressed unusually high amounts of TDG, this glycosylase contributed to initiation of as much as approximately 30% of U:G repair. Repair of U:G mismatches was generally faster than that of U:A pairs, which agrees with the known substrate preference of UNG-type glycosylases. Unexpectedly, repair of abasic sites opposite G was also generally faster than when opposite A, and this could not be explained by the properties of the purified APE1 protein. It may rather reflect differences in substrate recognition or repair by different complex(es). Lig III is apparently a minor rate-regulator for U:G repair. APE1, Pol beta, Pol delta, PCNA, XRCC1 and Lig I did not seem to be rate-limiting for overall repair of any of the substrates. These results identify damaged base removal as the major rate-limiting step in BER of uracil in human cells.     

Arabidopsis Uracil DNA Glycosylase (UNG) Is Required for Base Excision Repair of Uracil and Increases Plant Sensitivity to 5-Fluorouracil

Uracil in DNA arises by misincorporation of dUMP during replication and by hydrolytic deamination of cytosine. This common lesion is actively removed through a base excision repair (BER) pathway initiated by a uracil DNA glycosylase (UDG) activity that excises the damage as a free base. UDGs are classified into different families differentially distributed across eubacteria, archaea, yeast, and animals, but remain to be unambiguously identified in plants. We report here the molecular characterization of AtUNG (Arabidopsis thaliana uracil DNA glycosylase), a plant member of the Family-1 of UDGs typified by Escherichia coli Ung. AtUNG exhibits the narrow substrate specificity and single-stranded DNA preference that are characteristic of Ung homologues. Cell extracts from atung^−/− mutants are devoid of UDG activity, and lack the capacity to initiate BER on uracil residues. AtUNG-deficient plants do not display any apparent phenotype, but show increased resistance to 5-fluorouracil (5-FU), a cytostatic drug that favors dUMP misincorporation into DNA. The resistance of atung^−/− mutants to 5-FU is accompanied by the accumulation of uracil residues in DNA. These results suggest that AtUNG excises uracil in vivo but generates toxic AP sites when processing abundant U:A pairs in dTTP-depleted cells. Altogether, our findings point to AtUNG as the major UDG activity in Arabidopsis.     

Reprint of “Oxidant and environmental toxicant-induced effects compromise DNA ligation during base excision DNA repair”

DNA lesions arise from many endogenous and environmental agents, and such lesions can promote deleterious events leading to genomic instability and cell death. Base excision repair (BER) is the main DNA repair pathway responsible for repairing single strand breaks, base lesions and abasic sites in mammalian cells. During BER, DNA substrates and repair intermediates are channeled from one step to the next in a sequential fashion so that release of toxic repair intermediates is minimized. This includes handoff of the product of gap-filling DNA synthesis to the DNA ligation step. The conformational differences in DNA polymerase β (pol β) associated with incorrect or oxidized nucleotide (8-oxodGMP) insertion could impact channeling of the repair intermediate to the final step of BER, i.e., DNA ligation by DNA ligase I or the DNA Ligase III/XRCC1 complex. Thus, modified DNA ligase substrates produced by faulty pol β gap-filling could impair coordination between pol β and DNA ligase. Ligation failure is associated with 5′-AMP addition to the repair intermediate and accumulation of strand breaks that could be more toxic than the initial DNA lesions. Here, we provide an overview of the consequences of ligation failure in the last step of BER. We also discuss DNA-end processing mechanisms that could play roles in reversal of impaired BER.     

DNA base excision repair in human malaria parasites is predominantly by a long-patch pathway

Mammalian cells repair apurinic/apyrimidinic (AP) sites in DNA by two distinct pathways: a polymerase beta (pol beta)-dependent, short- (one nucleotide) patch base excision repair (BER) pathway, which is the major route, and a PCNA-dependent, long- (several nucleotide) patch BER pathway. The ability of a cell-free lysate prepared from asexual Plasmodium falciparum malaria parasites to remove uracil and repair AP sites in a variety of DNA substrates was investigated. We found that the lysate contained uracil DNA glycosylase, AP endonuclease, DNA polymerase, flap endonuclease, and DNA ligase activities. This cell-free lysate effectively repaired a regular or synthetic AP site on a covalently closed circular (ccc) duplex plasmid molecule or a long (382 bp), linear duplex DNA fragment, or a regular or reduced AP site in short (28 bp), duplex oligonucleotides. Repair of the AP sites in the various DNA substrates involved a long-patch BER pathway. This biology is different from mammalian cells, yeast, Xenopus, and Escherichia coli, which predominantly repair AP sites by a one-nucleotide patch BER pathway. The apparent absence of a short-patch BER pathway in P. falciparum may provide opportunities to develop antimalarial chemotherapeutic strategies for selectively damaging the parasites in vivo and will allow the characterization of the long-patch BER pathway without having to knock-out or inactivate a short-patch BER pathway, which is necessary in mammalian cells.     

Repair of U/G and U/A in DNA by UNG2-associated repair complexes takes place predominantly by short-patch repair both in proliferating and growth-arrested cells

Nuclear uracil-DNA glycosylase UNG2 has an established role in repair of U/A pairs resulting from misincorporation of dUMP during replication. In antigen-stimulated B-lymphocytes UNG2 removes uracil from U/G mispairs as part of somatic hypermutation and class switch recombination processes. Using antibodies specific for the N-terminal non-catalytic domain of UNG2, we isolated UNG2-associated repair complexes (UNG2-ARC) that carry out short-patch and long-patch base excision repair (BER). These complexes contain proteins required for both types of BER, including UNG2, APE1, POLbeta, POLdelta, XRCC1, PCNA and DNA ligase, the latter detected as activity. Short-patch repair was the predominant mechanism both in extracts and UNG2-ARC from proliferating and less BER-proficient growth-arrested cells. Repair of U/G mispairs and U/A pairs was completely inhibited by neutralizing UNG-antibodies, but whereas added recombinant SMUG1 could partially restore repair of U/G mispairs, it was unable to restore repair of U/A pairs in UNG2-ARC. Neutralizing antibodies to APE1 and POLbeta, and depletion of XRCC1 strongly reduced short-patch BER, and a fraction of long-patch repair was POLbeta dependent. In conclusion, UNG2 is present in preassembled complexes proficient in BER. Furthermore, UNG2 is the major enzyme initiating BER of deaminated cytosine (U/G), and possibly the sole enzyme initiating BER of misincorporated uracil (U/A).     

AP endonuclease-independent DNA base excision repair in human cells

The paradigm for repair of oxidized base lesions in genomes via the base excision repair (BER) pathway is based on studies in Escherichia coli, in which AP endonuclease (APE) removes all 3' blocking groups (including 3' phosphate) generated by DNA glycosylase/AP lyases after base excision. The recently discovered mammalian DNA glycosylase/AP lyases, NEIL1 and NEIL2, unlike the previously characterized OGG1 and NTH1, generate DNA strand breaks with 3' phosphate termini. Here we show that in mammalian cells, removal of the 3' phosphate is dependent on polynucleotide kinase (PNK), and not APE. NEIL1 stably interacts with other BER proteins, DNA polymerase beta (pol beta) and DNA ligase IIIalpha. The complex of NEIL1, pol beta, and DNA ligase IIIalpha together with PNK suggests coordination of NEIL1-initiated repair. That NEIL1/PNK could also repair the products of other DNA glycosylases suggests a broad role for this APE-independent BER pathway in mammals.