Long-patch Base Excision Repair Pathway Research Articles

Recognition of a Clickable Abasic Site Analog by DNA Polymerases and DNA Repair Enzymes.

Azide-alkyne cycloaddition ("click chemistry") has found wide use in the analysis of molecular interactions in living cells. 5-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (EAP) is a recently developed apurinic/apyrimidinic (AP) site analog functionalized with an ethynyl moiety, which can be introduced into cells in DNA constructs to perform labeling or cross-linking in situ. However, as a non-natural nucleoside, EAP could be subject to removal by DNA repair and misreading by DNA polymerases. Here, we investigate the interaction of this clickable AP site analog with DNA polymerases and base excision repair enzymes. Similarly to the natural AP site, EAP was non-instructive and followed the "A-rule", directing residual but easily detectable incorporation of dAMP by E. coli DNA polymerase I Klenow fragment, bacteriophage RB69 DNA polymerase and human DNA polymerase β. On the contrary, EAP was blocking for DNA polymerases κ and λ. EAP was an excellent substrate for the major human AP endonuclease APEX1 and E. coli AP exonucleases Xth and Nfo but was resistant to the AP lyase activity of DNA glycosylases. Overall, our data indicate that EAP, once within a cell, would represent a replication block and would be removed through an AP endonuclease-initiated long-patch base excision repair pathway.

Participation of RecJ in the base excision repair pathway of Deinococcus radiodurans.

RecJ reportedly participates in the base excision repair (BER) pathway, but structural and functional data are scarce. Herein, the Deinococcus radiodurans RecJ (drRecJ) deletion strain exhibited extreme sensitivity to hydrogen peroxide and methyl-methanesulphonate, as well as a high spontaneous mutation rate and an accumulation of unrepaired abasic sites in vivo, indicating the involvement of drRecJ in the BER pathway. The binding affinity and nuclease activity preference of drRecJ toward DNA substrates containing a 5′-P-dSpacer group, a 5′-deoxyribose-phosphate (dRP) mimic, were established. A 1.9 Å structure of drRecJ in complex with 5′-P-dSpacer-modified single-stranded DNA (ssDNA) revealed a 5′-monophosphate binding pocket and occupancy of 5′-dRP in the drRecJ nuclease core. The mechanism for RecJ 5′-dRP catalysis was explored using structural and biochemical data, and the results implied that drRecJ is not a canonical 5′-dRP lyase. Furthermore, in vitro reconstitution assays indicated that drRecJ tends to participate in the long-patch BER pathway rather than the short-patch BER pathway.

Abstract LB-265: 5’ gap-mediated long patch base excision repair

Abstract The base excision repair (BER) pathway is one of the most frequently used DNA repair mechanisms in the cell and modulates many human pathophysiological conditions related to DNA damage. Through live cell and in vitro reconstitution experiments, we have discovered a novel step in a sub-pathway of the conventional long-patch BER that involves formation of a 9 nt gap 5’ to the lesion, which is mediated by RECQ1 DNA helicase and ERCC1-XPF endonuclease in cooperation with PARP1 and RPA. The novel step is employed during repair of a variety of DNA lesions, including oxidative and alkylation damage. Moreover, RECQ1 regulates the autoribosylation status of PARP1 and the choice between long-patch and single-nucleotide BER, which regulates cellular toxicity to DNA damage. Taken together, our results propose a revised model of the long-patch BER pathway and characterize a crucial regulation point for pathway choice in BER. Citation Format: Jordan Woodrick, Sharon Camacho, Swetha Parvathaneni, Sudha Sharma, Rabindra Roy. 5’ gap-mediated long patch base excision repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-265. doi:10.1158/1538-7445.AM2017-LB-265

5-Fluorouracil mediated anti-cancer activity in colon cancer cells is through the induction of Adenomatous Polyposis Coli: Implication of the long-patch base excision repair pathway.

Colorectal cancer (CRC) patients with APC mutations do not benefit from 5-FU therapy. It was reported that APC physically interacts with POLβ and FEN1, thus blocking LP-BER via APC's DNA repair inhibitory (DRI) domain in vitro. The aim of this study was to elucidate how APC status affects BER and the response of CRC to 5-FU. HCT-116, HT-29, and LOVO cells varying in APC status were treated with 5-FU to evaluate expression, repair, and survival responses. HCT-116 expresses wild-type APC; HT-29 expresses an APC mutant that contains DRI domain; LOVO expresses an APC mutant lacking DRI domain. 5-FU increased the expression of APC and decreased the expression of FEN1 in HCT-116 and HT-29 cells, which were sensitized to 5-FU when compared to LOVO cells. Knockdown of APC in HCT-116 rendered cells resistant to 5-FU, and FEN1 levels remained unchanged. Re-expression of full-length APC in LOVO cells caused sensitivity to 5-FU, and decreased expression of FEN1. These knockdown and addback studies confirmed that the DRI domain is necessary for the APC-mediated reduction in LP-BER and 5-FU. Modelling studies showed that 5-FU can interact with the DRI domain of APC via hydrogen bonding and hydrophobic interactions. 5-FU resistance in CRC occurs with mutations in APC that disrupt or eliminate the DRI domain's interaction with LP-BER. Understanding the type of APC mutation should better predict 5-FU resistance in CRC than simply characterizing APC status as wild-type or mutant.

The mechanism of human tyrosyl-DNA phosphodiesterase 1 in the cleavage of AP site and its synthetic analogs

The mechanism of hydrolysis of the apurinic/apyrimidinic (AP) site and its synthetic analogs by using tyrosyl-DNA phosphodiesterase 1 (Tdp1) was analyzed. Tdp1 catalyzes the cleavage of AP site and the synthetic analog of the AP site, 3-hydroxy-2(hydroxymethyl)-tetrahydrofuran (THF), in DNA by hydrolysis of the phosphodiester bond between the substituent and 5' adjacent phosphate. The product of Tdp1 cleavage in the case of the AP site is unstable and is hydrolyzed with the formation of 3'- and 5'-margin phosphates. The following repair demands the ordered action of polynucleotide kinase phosphorylase, with XRCC1, DNA polymerase β, and DNA ligase. In the case of THF, Tdp1 generates break with the 5'-THF and the 3'-phosphate termini. Tdp1 is also able to effectively cleave non-nucleotide insertions in DNA, decanediol and diethyleneglycol moieties by the same mechanism as in the case of THF cleavage. The efficiency of Tdp1 catalyzed hydrolysis of AP-site analog correlates with the DNA helix distortion induced by the substituent. The following repair of 5'-THF and other AP-site analogs can be processed by the long-patch base excision repair pathway.

Clustered DNA Lesions Containing 5-Formyluracil and AP Site: Repair via the BER System

Lesions in the DNA arise under ionizing irradiation conditions or various chemical oxidants as a single damage or as part of a multiply damaged site within 1–2 helical turns (clustered lesion). Here, we explored the repair opportunity of the apurinic/apyrimidinic site (AP site) composed of the clustered lesion with 5-formyluracil (5-foU) by the base excision repair (BER) proteins. We found, that if the AP site is shifted relative to the 5-foU of the opposite strand, it could be repaired primarily via the short-patch BER pathway. In this case, the cleavage efficiency of the AP site-containing DNA strand catalyzed by human apurinic/apyrimidinic endonuclease 1 (hAPE1) decreased under AP site excursion to the 3'-side relative to the lesion in the other DNA strand. DNA synthesis catalyzed by DNA polymerase lambda was more accurate in comparison to the one catalyzed by DNA polymerase beta. If the AP site was located exactly opposite 5-foU it was expected to switch the repair to the long-patch BER pathway. In this situation, human processivity factor hPCNA stimulates the process.

Mutations in DNA2 Link Progressive Myopathy to Mitochondrial DNA Instability

Syndromes associated with multiple mtDNA deletions are due to different molecular defects that can result in a wide spectrum of predominantly adult-onset clinical presentations, ranging from progressive external ophthalmoplegia (PEO) to multisystemic disorders of variable severity. The autosomal-dominant form of PEO is genetically heterogeneous. Recently, causative mutations have been reported in several nuclear genes that encode proteins of the mtDNA replisome machinery (POLG, POLG2, and C10orf2) or that are involved in pathways for the synthesis of deoxyribonuclotides (ANT1 and RRM2B). Despite these findings, putative mutations remain unknown in half of the subjects with PEO. We report the identification, by exome sequencing, of mutations in DNA2 in adult-onset individuals with a form of mitochondrial myopathy featuring instability of muscle mtDNA. DNA2 encodes a helicase/nuclease family member that is most likely involved in mtDNA replication, as well as in the long-patch base-excision repair (LP-BER) pathway. Invitro biochemical analysis of purified mutant proteins revealed a severe impairment of nuclease, helicase, and ATPase activities. These results implicate human DNA2 and the LP-BER pathway in the pathogenesis of adult-onset disorders of mtDNA maintenance.

5,6-Dihydrothymine Impairs the Base Excision Repair Pathway of a Closely Opposed AP Site or Single-Strand Break

Ionizing radiation can induce clustered DNA damage (two or more lesions formed within one to two helical turns of DNA by passage of a single ionization track). Using oligonucleotide constructs containing clustered DNA lesions at defined positions, evidence is presented demonstrating that a persistent 5,6-dihydrothymine (DHT) lesion reduces the efficiency of rejoining, in mammalian nuclear extracts, of an opposing AP site or SSB when within 5 bp. The efficiency of repair of the SSB is reduced when DHT is present on the opposing strand in both the 3' and 5' orientation; however, the efficiency of the repair of the AP site is reduced only when DHT is present 3' to the AP site. DNA polymerase beta and ligation are particularly impaired by DHT. It was also shown that in the presence of DHT there is a greater dependence on the long-patch base excision repair pathway than when DHT is absent. In addition, immunodepletion of XRCC1 from the nuclear extracts slows down the initial rate of repair of the AP site in both the presence and absence of DHT, but immunodepletion of XRCC1 has no influence on the repair of an SSB.

Poly(ADP-ribose) polymerase 1 regulates activity of DNA polymerase β in long patch base excision repair

Poly(ADP-ribose)polymerase 1 (PARP1), functioning as DNA nick-sensor, interacts with base excision repair (BER) DNA intermediates containing single-strand breaks. When bound to DNA breaks, PARP1 catalyzes synthesis of poly(ADP-ribose) covalently attached to itself and some nuclear proteins. Autopoly(ADP-ribosyl)ation of PARP1 facilitates its dissociation from DNA breaks and is considered as a factor regulating DNA repair. In the study, using system reconstituted from purified BER proteins, bovine testis nuclear extract and model BER DNA intermediates, we examined the influence of PARP1 and its autopoly(ADP-ribosyl)ation on DNA polymerase β (Pol β)-mediated long patch (LP) BER DNA synthesis that is accomplished through a cooperation between Pol β and apurinic/apyrimidinic endonuclease1 (APE1) or flap endonuclease 1 (FEN1) and gap-filling activity of Pol β. PARP1 upon interaction with nicked LP BER DNA intermediated, formed after gap-filling, was shown to suppress the subsequent steps in LP pathway. PARP1 interferes with APE1-dependent stimulation of DNA synthesis by Pol β via strand-displacement mechanism. PARP1 also represses Pol β/FEN1-mediated LP BER DNA synthesis via a “gap translation” mechanism inhibiting FEN1 activity on the nicked DNA intermediate. Poly(ADP-ribosyl)ation of PARP1 abolishes its inhibitory influence on LP BER DNA synthesis catalyzed by Pol β both via APE1-mediated strand-displacement and FEN1-mediated “gap translation” mechanism. Thus PARP1 may act as a negative regulator of Pol β activity in LP BER pathway and poly(ADP-ribosyl)ation of PARP1 seems to play a critical role in enablement of Pol β-mediated DNA synthesis in this process. In contrast, interaction of PARP1 with one nucleotide gapped DNA mimicking the intermediate of short patch (SP) BER slightly inhibits the gap-filling activity of Pol β and the overall efficiency of SP BER is practically unaffected by PARP1. Thus, PARP1 differentially influences DNA synthesis in SP- and LP BER pathways.

Long Patch Base Excision Repair Proceeds via Coordinated Stimulation of the Multienzyme DNA Repair Complex

Base excision repair, a major repair pathway in mammalian cells, is responsible for correcting DNA base damage and maintaining genomic integrity. Recent reports show that the Rad9-Rad1-Hus1 complex (9-1-1) stimulates enzymes proposed to perform a long patch-base excision repair sub-pathway (LP-BER), including DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), DNA polymerase beta (pol beta), flap endonuclease 1 (FEN1), and DNA ligase I (LigI). However, 9-1-1 was found to produce minimal stimulation of FEN1 and LigI in the context of a complete reconstitution of LP-BER. We show here that pol beta is a robust stimulator of FEN1 and a moderate stimulator of LigI. Apparently, there is a maximum possible stimulation of these two proteins such that after responding to pol beta or another protein in the repair complex, only a small additional response to 9-1-1 is allowed. The 9-1-1 sliding clamp structure must serve primarily to coordinate enzyme actions rather than enhancing rate. Significantly, stimulation by the polymerase involves interaction of primer terminus-bound pol beta with FEN1 and LigI. This observation provides compelling evidence that the proposed LP-BER pathway is actually employed in cells. Moreover, this pathway has been proposed to function by sequential enzyme actions in a "hit and run" mechanism. Our results imply that this mechanism is still carried out, but in the context of a multienzyme complex that remains structurally intact during the repair process.

DNA repair in mammalian cells: Base excision repair: the long and short of it.

Base excision repair (BER) is the primary DNA repair pathway that corrects base lesions that arise due to oxidative, alkylation, deamination, and depurinatiation/depyrimidination damage. BER facilitates the repair of damaged DNA via two general pathways - short-patch and long-patch. The shortpatch BER pathway leads to a repair tract of a single nucleotide. Alternatively, the long-patch BER pathway produces a repair tract of at least two nucleotides. The BER pathway is initiated by one of many DNA glycosylases, which recognize and catalyze the removal of damaged bases. The completion of the BER pathway is accomplished by the coordinated action of at least three additional enzymes. These downstream enzymes carry out strand incision, gap-filling and ligation. The high degree of BER conservation between E. coli and mammals has lead to advances in our understanding of mammalian BER. This review will provide a general overview of the mammalian BER pathway. (Part of a Multi-author Review).

DNA2 Resolves Expanding Flap in Mitochondrial Base Excision Repair

In a recent issue of Molecular Cell, Zheng et al. (2008) demonstrated that human DNA2, originally identified in yeast as a nuclear DNA replication and repair factor, functions exclusively in mammalian mitochondria in the recently discovered long-patch base excision repair pathway.

Adenomatous polyposis coli-mediated hypersensitivity of mouse embryonic fibroblast cell lines to methylmethane sulfonate treatment: implication of base excision repair pathways

The role of adenomatous polyposis coli (APC) has been implicated in various cellular functions including cell migration, cell-cell adhesion, cell cycle control, chromosomal segregation and apoptosis. Recently, we discovered a novel role of APC in DNA base excision repair (BER) and showed that APC interacts with DNA polymerase beta (Pol-beta) and flap endonuclease 1 and interferes long-patch base excision repair (LP-BER) by blocking strand displacement synthesis. Many times, the chemotherapeutic drugs induce DNA alkylation damage, which is primarily repaired by the BER pathway. Thus, the efficacy of such drugs can be increased by APC resulting in the blockage of LP-BER. In the present study, we tested this hypothesis by using isogenic wild-type and Pol-beta-knockout mouse embryonic fibroblast (MEF) cell lines in which the Apc gene was knocked down by the small interfering RNA technique and treated with methylmethane sulfonate (MMS). The MEF-Apc(WT)/Polbeta-/- cells were hypersensitive to MMS treatment compared with the MEF-Apc(WT)/Polbeta+/+ cells. However, once the Apc gene was knocked down, these cells became more resistant to MMS treatment, suggesting that the MMS-induced hypersensitivity was associated with Apc. We then determined whether the hypersensitivity of MEF-Apc(WT)/Polbeta-/- and MEF-Apc(WT)/Polbeta+/+ cell lines were due to decreased Pol-beta-independent and Pol-beta-dependent LP-BER pathways, respectively. The results of in vivo and in vitro LP-BER assays supported our findings. Furthermore, Apc-mediated hypersensitivity to MMS treatment was correlated with increased apoptosis of MEF-Apc(WT)/Polbeta-/- and MEF-Apc(WT)/Polbeta+/+ as compared with MEF-Apc(KD)/Polbeta-/- and MEF-Apc(KD)/Polbeta+/+ cells. These results suggest that an increased level of Apc can increase the efficacy of DNA-alkylating drugs used as a curative therapy.

Mechanism of Adenomatous Polyposis Coli (APC)-Mediated Blockage of Long-Patch Base Excision Repair

Recently, we found an interaction between adenomatous polyposis coli (APC) and DNA polymerase beta (pol-beta) and showed that APC blocks strand-displacement synthesis of long-patch base excision repair (LP-BER) (Narayan, S., Jaiswal, A. S., and Balusu, R. (2005) J. Biol. Chem. 280, 6942-6949); however, the mechanism is not clear. Using an in vivo LP-BER assay system, we now show that the LP-BER is higher in APC-/- cells than in APC+/+ cells. In addition to pol-beta, the pull-down experiments showed that the full-length APC also interacted with flap endonuclease 1 (Fen-1). To further characterize the interaction of APC with pol-beta and Fen-1, we performed a domain-mapping of APC and found that both pol-beta and Fen-1 interact with a 138-amino acids peptide from the APC at the DRI-domain. Our functional assays showed that APC blocks pol-beta-mediated 1-nucleotide (1-nt) as well as strand-displacement synthesis of reduced abasic, nicked-, or 1-nt gapped-DNA substrates. Further studies demonstrated that APC blocks 5'-flap endonuclease as well as the 5'-3' exonuclease activity of Fen-1 resulting in the blockage of LP-BER. From these results, we concluded that APC can have three different effects on the LP-BER pathway. First, APC can block pol-beta-mediated 1-nt incorporation and strand-displacement synthesis. Second, APC can block LP-BER by blocking the coordinated formation and removal of the strand-displaced flap. Third, APC can block LP-BER by blocking hit-and-run synthesis. These studies will have important implications for APC in DNA damage-induced carcinogenesis and chemoprevention.

Monte Carlo Simulation of Base and Nucleotide Excision Repair of Clustered DNA Damage Sites. I. Model Properties and Predicted Trends

DNA is constantly damaged through endogenous processes and by exogenous agents, such as ionizing radiation. Base excision repair (BER) and nucleotide excision repair (NER) help maintain the stability of the genome by removing many different types of DNA damage. We present a Monte Carlo excision repair (MCER) model that simulates key steps in the short-patch and long-patch BER pathways and the NER pathway. The repair of both single and clustered damages, except double-strand breaks (DSBs), is simulated in the MCER model. Output from the model includes estimates of the probability that a cluster is repaired correctly, the fraction of the clusters converted into DSBs through the action of excision repair enzymes, the fraction of the clusters repaired with mutations, and the expected number of repair cycles needed to completely remove a clustered damage site. The quantitative implications of alternative hypotheses regarding the postulated repair mechanisms are investigated through a series of parameter sensitivity studies. These sensitivity studies are also used to help define the putative repair characteristics of clustered damage sites other than DSBs.

Comparison of functional properties of mammalian DNA polymerase λ and DNA polymerase β in reactions of DNA synthesis related to DNA repair

DNA polymerase λ (Pol λ) is a novel enzyme of the family X of DNA polymerases. Pol λ has some properties in common with DNA polymerase β (Pol β). The substrate properties of Pol λ were compared to Pol β using DNAs mimicking short-patch (SP) and long-patch (LP) base excision repair (BER) intermediates as well as recessed template primers. In the present work, the influence of several BER proteins such as flap-endonuclease-1 (FEN1), PCNA, and apurinic/apyrimidinic endonuclease-1 (APE1) on the activity of Pol λ was investigated. Pol λ is unable to catalyze strand displacement synthesis using nicked DNA, although this enzyme efficiently incorporates a dNMP into a one-nucleotide gap. FEN1 and PCNA stimulate the strand displacement activity of Pol λ. FEN1 processes nicked DNA, thus removing a barrier to Pol λ DNA synthesis. It results in a one-nucleotide gapped DNA molecule that is a favorite substrate of Pol λ. Photocrosslinking and functional assay show that Pol λ is less efficient than Pol β in binding to nicked DNA. APE1 has no influence on the strand displacement activity of Pol λ though it stimulates strand displacement synthesis catalyzed with Pol β. It is suggested that Pol λ plays a role in the SP BER rather than contributes to the LP BER pathway.

Exchangeability of mammalian DNA ligases between base excision repair pathways.

In mammalian cells, DNA ligase IIIalpha and DNA ligase I participate in the short- and long-patch base excision repair pathways, respectively. Using an in vitro repair assay employing DNA ligase-depleted cell extracts and DNA substrates containing a single lesion repaired either through short-patch (regular abasic site) or long-patch (reduced abasic site) base excision repair pathways, we addressed the question whether DNA ligases are specific to each pathway or if they are exchangeable. We find that immunodepletion of DNA ligase I did not affect the short-patch repair pathway but blocked long-patch repair, suggesting that DNA ligase IIIalpha is not able to substitute DNA ligase I during long-patch repair. In contrast, immunodepletion of DNA ligase IIIalpha did not significantly affect either pathway. Moreover, repair of normal abasic sites in wild-type and X-ray cross-complementing gene 1 (XRCC1)-DNA ligase IIIalpha-immunodepleted cell extracts involved similar proportions of short- and long-patch repair events. This suggests that DNA ligase I was able to efficiently substitute the XRCC1-DNA ligase IIIalpha complex during short-patch repair.

The Werner Syndrome Protein Stimulates DNA Polymerase β Strand Displacement Synthesis via Its Helicase Activity

Werner syndrome is a hereditary premature aging disorder characterized by genomic instability. Genetic analysis and protein interaction studies indicate that the defective gene product (WRN) may play an important role in DNA replication, recombination, and repair. DNA polymerase beta (pol beta) is a central participant in both short and long-patch base excision repair (BER) pathways, which function to process most spontaneous, alkylated, and oxidative DNA damage. We report here a physical interaction between WRN and pol beta, and using purified proteins reconstitute of a portion of the long-patch BER pathway to examine a potential role for WRN in this repair response. We demonstrate that WRN stimulates pol beta strand displacement DNA synthesis and that this stimulation is dependent on the helicase activity of WRN. In addition, a truncated WRN protein, containing primarily the helicase domain, retains helicase activity and is sufficient to mediate the stimulation of pol beta. The WRN helicase also unwinds a BER substrate, providing evidence that WRN plays a role in unwinding DNA repair intermediates. Based on these findings, we propose a novel mechanism by which WRN may mediate pol beta-directed long-patch BER.

Repair of Clustered DNA Lesions: SEQUENCE-SPECIFIC INHIBITION OF LONG-PATCH BASE EXCISION REPAIR BY 8-OXOGUANINE

Ionizing radiation induces clustered DNA damage where two or more lesions are located proximal to each other on the same or opposite DNA strands. It has been suggested that individual lesions within a cluster are removed sequentially and that the presence of a vicinal lesion(s) may affect the rate and fidelity of DNA repair. In this study, we addressed the question of how 8-oxoguanine located opposite to normal or reduced abasic sites would affect the repair of these sites by the base excision repair system. We have found that an 8-oxoguanine located opposite to an abasic site does not affect either the efficiency or fidelity of repair synthesis by DNA polymerase beta. In contrast, an 8-oxoguanine located one nucleotide 3'-downstream of the abasic site significantly reduces both strand displacement synthesis supported by DNA polymerase beta or delta and cleavage by flap endonuclease of the generated flap, thus inhibiting the long-patch base excision repair pathway.

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.