Impact of DNA ligase 1 and IIIα interactions with APE1 and polß on the efficiency of base excision repair pathway at the downstream steps.

Base excision repair (BER) requires a tight coordination between the repair enzymes through protein-protein interactions and involves gap filling by DNA polymerase (pol) ß and subsequent nick sealing by DNA ligase (LIG) 1 or LIGIIIα at the downstream steps. Apurinic/apyrimidinic-endonuclease 1 (APE1), by its exonuclease activity, proofreads 3' mismatches incorporated by polß during BER. We previously reported that the interruptions in the functional interplay between polß and the BER ligases result in faulty repair events. Yet, how the protein interactions of LIG1 and LIGIIIα could affect the repair pathway coordination during nick sealing at the final steps remains unknown. Here, we demonstrate that LIGIIIα interacts more tightly with polß and APE1 than LIG1, and the N-terminal noncatalytic region of LIG1 as well as the catalytic core and BRCT domain of LIGIIIα mediate interactions with both proteins. Our results demonstrated less efficient nick sealing of polß nucleotide insertion products in the absence of LIGIIIα zinc-finger domain and LIG1 N-terminal region. Furthermore, we showed a coordination between APE1 and LIG1/LIGIIIα during the removal of 3' mismatches from the nick repair intermediate on which both BER ligases can seal noncanonical ends or gap repair intermediate leading to products of single deletion mutagenesis. Overall results demonstrate the importance of functional coordination from gap filling by polß coupled to nick sealing by LIG1/LIGIIIα in the presence of proofreading by APE1, which is mainly governed by protein-protein interactions and protein-DNA intermediate communications, to maintain repair efficiency at the downstream steps of the BER pathway.

Impact of DNA ligase inhibition on the nick sealing of polß nucleotide insertion products at the downstream steps of base excision repair pathway.

DNA ligase (LIG) I and IIIα finalize base excision repair (BER) by sealing a nick product after nucleotide insertion by DNA polymerase (pol) ß at the downstream steps. We previously demonstrated that a functional interplay between polß and BER ligases is critical for efficient repair, and polß mismatch or oxidized nucleotide insertions confound final ligation step. Yet, how targeting downstream enzymes with small molecule inhibitors could affect this coordination remains unknown. Here, we report that DNA ligase inhibitors, L67 and L82-G17, slightly enhance hypersensitivity to oxidative stress-inducing agent, KBrO3, in polß+/+ cells more than polß-/- null cells. We showed less efficient ligation after polß nucleotide insertions in the presence of the DNA ligase inhibitors. Furthermore, the mutations at the ligase inhibitor binding sites (G448, R451, A455) of LIG1 significantly affect nick DNA binding affinity and nick sealing efficiency. Finally, our results demonstrated that the BER ligases seal a gap repair intermediate by the effect of polß inhibitor that diminishes gap filling activity. Overall, our results contribute to understand how the BER inhibitors against downstream enzymes, polß, LIG1, and LIGIIIα, could impact the efficiency of gap filling and subsequent nick sealing at the final steps leading to the formation of deleterious repair intermediates.

Structural and biochemical characterization of LIG1 during mutagenic nick sealing of oxidatively damaged ends at the final step of DNA repair.

DNA ligase 1 (LIG1) joins broken strand-breaks in the phosphodiester backbone to finalize DNA repair pathways. We previously reported that LIG1 fails on nick repair intermediate with 3'-oxidative damage incorporated by DNA polymerase (pol) ß at the downstream steps of base excision repair (BER) pathway. Here, we determined X-ray structures of LIG1/nick DNA complexes containing 3'-8oxodG and 3'-8oxorG opposite either a templating Cytosine or Adenine and demonstrated that the ligase active site engages with mutagenic repair intermediates during steps 2 and 3 of the ligation reaction referring to the formation of DNA-AMP intermediate and a final phosphodiester bond, respectively. Furthermore, we showed the mutagenic nick sealing of DNA substrates with 3'-8oxodG:A and 3'-8oxorG:A by LIG1 wild-type, immunodeficiency disease-associated variants, and DNA ligase 3α (LIG3α) in vitro . Finally, we observed that LIG1 and LIG3α seal resulting nick after an incorporation of 8oxorGTP:A by polß and AP-Endonuclease 1 (APE1) can clean oxidatively damaged ends at the final steps. Overall, our findings uncover a mechanistic insight into how LIG1 discriminates DNA or DNA/RNA junctions including oxidative damage and a functional coordination between the downstream enzymes, polß, APE1, and BER ligases, to process mutagenic repair intermediates to maintain repair efficiency.

Uncovering nick DNA binding by LIG1 at the single-molecule level.

DNA ligases repair the strand breaks are made continually and naturally throughout the genome, if left unrepaired and allowed to persist, they can lead to genome instability in the forms of lethal double-strand (ds) breaks, deletions, and duplications. DNA ligase 1 (LIG1) joins Okazaki fragments during the replication machinery and seals nicks at the end of most DNA repair pathways. Yet, how LIG1 recognizes its target substrate is entirely missing. Here, we uncover the dynamics of nick DNA binding by LIG1 at the single-molecule level. Our findings reveal that LIG1 binds to dsDNA both specifically and non-specifically and exhibits diffusive behavior to form a stable complex at the nick. Furthermore, by comparing with the LIG1 C-terminal protein, we demonstrate that the N-terminal non-catalytic region promotes binding enriched at nick sites and facilitates an efficient nick search process by promoting 1D diffusion along the DNA. Our findings provide a novel single-molecule insight into the nick binding by LIG1, which is critical to repair broken phosphodiester bonds in the DNA backbone to maintain genome integrity.

Molecular editing of NSC-666719 enabling discovery of benzodithiazinedioxide-guanidines as anticancer agents.

DNA polymerase ß (Polß) is crucial for the base excision repair (BER) pathway of DNA damage repair and is an attractive target for suppressing tumorigenesis as well as chemotherapeutic intervention of cancer. In this study, a unique strategy of scaffold-hopping-based molecular editing of a bioactive agent NSC-666719 was investigated, which led to the development of new molecular motifs with Polß inhibitory activity. NSC compound and its analogs (two series) were prepared, focusing on pharmacophore-based molecular diversity. Most compounds showed higher activities than the parent NSC-666719 and exhibited effects on apoptosis. The inhibitory activity of Polß was evaluated in both in vitro reconstituted and in vivo intact cell systems. Compound 10e demonstrated significant Polß interaction and inhibition characteristics, including direct, non-covalent, reversible, and comparable binding affinity. The investigated approach is useful, and the discovered novel analogs have a high potential for developing as anticancer therapeutics.

Structures of LIG1 provide a mechanistic basis for understanding a lack of sugar discrimination against a ribonucleotide at the 3'-end of nick DNA.

Human DNA ligase 1 (LIG1) is the main replicative ligase that seals Okazaki fragments during nuclear replication and finalizes DNA repair pathways by joining DNA ends of the broken strand breaks in the three steps of the ligation reaction. LIG1 can tolerate the RNA strand upstream of the nick, yet an atomic insight into the sugar discrimination mechanism by LIG1 against a ribonucleotide at the 3'-terminus of nick DNA is unknown. Here, we determined X-ray structures of LIG1/3'-RNA-DNA hybrids and captured the ligase during pre- and post-step 3 the ligation reaction. Furthermore, the overlays of 3'-rA:T and 3'-rG:C step 3 structures with step 2 structures of canonical 3'-dA:T and 3'-dG:C uncover a network of LIG1/DNA interactions through Asp570 and Arg871 side chains with 2'-OH of the ribose at nick showing a final phosphodiester bond formation and the other ligase active site residues surrounding the AMP site. Finally, we demonstrated that LIG1 can ligate the nick DNA substrates with pre-inserted 3'-ribonucleotides as efficiently as Watson-Crick base-paired ends in vitro. Together, our findings uncover a novel atomic insight into a lack of sugar discrimination by LIG1 and the impact of improper sugar on the nick sealing of ribonucleotides at the last step of DNA replication and repair.

Unfilled gaps by polß lead to aberrant ligation by LIG1 at the downstream steps of base excision repair pathway.

Base excision repair (BER) involves the tightly coordinated function of DNA polymerase ß (polß) and DNA ligase I (LIG1) at the downstream steps. Our previous studies emphasize that defective substrate-product channeling, from gap filling by polß to nick sealing by LIG1, can lead to interruptions in repair pathway coordination. Yet, the molecular determinants that dictate accurate BER remains largely unknown. Here, we demonstrate that a lack of gap filling by polß leads to faulty repair events and the formation of deleterious DNA intermediates. We dissect how ribonucleotide challenge and cancer-associated mutations could adversely impact the ability of polß to efficiently fill the one nucleotide gap repair intermediate which subsequently results in gap ligation by LIG1, leading to the formation of single-nucleotide deletion products. Moreover, we demonstrate that LIG1 is not capable of discriminating against nick DNA containing a 3'-ribonucleotide, regardless of base-pairing potential or damage. Finally, AP-Endonuclease 1 (APE1) shows distinct substrate specificity for the exonuclease removal of 3'-mismatched bases and ribonucleotides from nick repair intermediate. Overall, our results reveal that unfilled gaps result in impaired coordination between polß and LIG1, defining a possible type of mutagenic event at the downstream steps where APE1 could provide a proofreading role to maintain BER efficiency.

Impact of polß/XRCC1 Interaction Variants on the Efficiency of Nick Sealing by DNA Ligase IIIα in the Base Excision Repair Pathway.

Base excision repair (BER) requires a coordination from gap filling by DNA polymerase (pol) ß to subsequent nick sealing by DNA ligase (LIG) IIIα at downstream steps of the repair pathway. X-ray cross-complementing protein 1 (XRCC1), a non-enzymatic scaffolding protein, forms repair complexes with polß and LIGIIIα. Yet, the impact of the polß mutations that affect XRCC1 interaction and protein stability on the repair pathway coordination during nick sealing by LIGIIIα remains unknown. Our results show that the polß colon cancer-associated variant T304 exhibits a reduced interaction with XRCC1 and the mutations in the interaction interface of V303 loop (L301R/V303R/V306R) and at the lysine residues (K206A/K244A) that prevent ubiquitin-mediated degradation of the protein exhibit a diminished repair protein complex formation with XRCC1. Furthermore, we demonstrate no significant effect on gap and nick DNA binding affinity of wild-type polß by these mutations. Finally, our results reveal that XRCC1 leads to an efficient channeling of nick repair products after nucleotide incorporation by polß variants to LIGIIIα, which is compromised by the L301R/V303R/V306R and K206A/K244A mutations. Overall, our findings provide insight into how the mutations in the polß/XRCC1 interface and the regions affecting protein stability could dictate accurate BER pathway coordination at the downstream steps involving nick sealing by LIGIIIα.

Structures of LIG1 active site mutants reveal the importance of DNA end rigidity for mismatch discrimination.

ATP-dependent DNA ligases catalyze phosphodiester bond formation in the conserved three-step chemical reaction of nick sealing. Human DNA ligase I (LIG1) finalizes almost all DNA repair pathways following DNA polymerase-mediated nucleotide insertion. We previously reported that LIG1 discriminates mismatches depending on the architecture of the 3'-terminus at a nick, however the contribution of conserved active site residues to faithful ligation remains unknown. Here, we comprehensively dissect the nick DNA substrate specificity of LIG1 active site mutants carrying Ala(A) and Leu(L) substitutions at Phe(F)635 and Phe(F)F872 residues and show completely abolished ligation of nick DNA substrates with all 12 non-canonical mismatches. LIG1EE/AA structures of F635A and F872A mutants in complex with nick DNA containing A:C and G:T mismatches demonstrate the importance of DNA end rigidity, as well as uncover a shift in a flexible loop near 5'-end of the nick, which causes an increased barrier to adenylate transfer from LIG1 to the 5'-end of the nick. Furthermore, LIG1EE/AA/8oxoG:A structures of both mutants demonstrated that F635 and F872 play critical roles during steps 1 or 2 of the ligation reaction depending on the position of the active site residue near the DNA ends. Overall, our study contributes towards a better understanding of the substrate discrimination mechanism of LIG1 against mutagenic repair intermediates with mismatched or damaged ends and reveals the importance of conserved ligase active site residues to maintain ligation fidelity.

Structures of LIG1 active site mutants reveal the importance of DNA end rigidity for mismatch discrimination.

ATP-dependent DNA ligases catalyze phosphodiester bond formation in the conserved three-step chemical reaction of nick sealing. Human DNA ligase I (LIG1) finalizes almost all DNA repair pathways following DNA polymerase-mediated nucleotide insertion. We previously reported that LIG1 discriminates mismatches depending on the architecture of the 3'-terminus at a nick, however the contribution of conserved active site residues to faithful ligation remains unknown. Here, we comprehensively dissect the nick DNA substrate specificity of LIG1 active site mutants carrying Ala(A) and Leu(L) substitutions at Phe(F)635 and Phe(F)F872 residues and show completely abolished ligation of nick DNA substrates with all 12 non-canonical mismatches. LIG1 EE/AA structures of F635A and F872A mutants in complex with nick DNA containing A:C and G:T mismatches demonstrate the importance of DNA end rigidity, as well as uncover a shift in a flexible loop near 5'-end of the nick, which causes an increased barrier to adenylate transfer from LIG1 to the 5'-end of the nick. Furthermore, LIG1 EE/AA /8oxoG:A structures of both mutants demonstrated that F635 and F872 play critical roles during steps 1 or 2 of the ligation reaction depending on the position of the active site residue near the DNA ends. Overall, our study contributes towards a better understanding of the substrate discrimination mechanism of LIG1 against mutagenic repair intermediates with mismatched or damaged ends and reveals the importance of conserved ligase active site residues to maintain ligation fidelity.

Structures of LIG1 that engage with mutagenic mismatches inserted by polß in base excision repair.

DNA ligase I (LIG1) catalyzes the ligation of the nick repair intermediate after gap filling by DNA polymerase (pol) ß during downstream steps of the base excision repair (BER) pathway. However, how LIG1 discriminates against the mutagenic 3'-mismatches incorporated by polß at atomic resolution remains undefined. Here, we determine the X-ray structures of LIG1/nick DNA complexes with G:T and A:C mismatches and uncover the ligase strategies that favor or deter the ligation of base substitution errors. Our structures reveal that the LIG1 active site can accommodate a G:T mismatch in the wobble conformation, where an adenylate (AMP) is transferred to the 5'-phosphate of a nick (DNA-AMP), while it stays in the LIG1-AMP intermediate during the initial step of the ligation reaction in the presence of an A:C mismatch at the 3'-strand. Moreover, we show mutagenic ligation and aberrant nick sealing of dG:T and dA:C mismatches, respectively. Finally, we demonstrate that AP-endonuclease 1 (APE1), as a compensatory proofreading enzyme, removes the mismatched bases and interacts with LIG1 at the final BER steps. Our overall findings provide the features of accurate versus mutagenic outcomes coordinated by a multiprotein complex including polß, LIG1, and APE1 to maintain efficient repair.

The scaffold protein XRCC1 stabilizes the formation of polß/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) ß 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 polß/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 polß 8-oxodGTP insertion is enhanced in the presence of XRCC1. Our results demonstrated a stabilizing effect of XRCC1 on the formation of polß/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 polß 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 polß/XRCC1 complex formation, polß 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.

DNA ligase I fidelity mediates the mutagenic ligation of pol ß oxidized and mismatch nucleotide insertion products in base excision repair.

DNA ligase I (LIG1) completes the base excision repair (BER) pathway at the last nick-sealing step after DNA polymerase (pol) ß gap-filling DNA synthesis. However, the mechanism by which LIG1 fidelity mediates the faithful substrate-product channeling and ligation of repair intermediates at the final steps of the BER pathway remains unclear. We previously reported that pol ß 8-oxo-2'-deoxyribonucleoside 5'-triphosphate insertion confounds LIG1, leading to the formation of ligation failure products with a 5'-adenylate block. Here, using reconstituted BER assays in vitro, we report the mutagenic ligation of pol ß 8-oxo-2'-deoxyribonucleoside 5'-triphosphate insertion products and an inefficient ligation of pol ß Watson-Crick-like dG:T mismatch insertion by the LIG1 mutant with a perturbed fidelity (E346A/E592A). Moreover, our results reveal that the substrate discrimination of LIG1 for the nicked repair intermediates with preinserted 3'-8-oxodG or mismatches is governed by mutations at both E346 and E592 residues. Finally, we found that aprataxin and flap endonuclease 1, as compensatory DNA-end processing enzymes, can remove the 5'-adenylate block from the abortive ligation products harboring 3'-8-oxodG or the 12 possible noncanonical base pairs. These findings contribute to the understanding of the role of LIG1 as an important determinant in faithful BER and how a multiprotein complex (LIG1, pol ß, aprataxin, and flap endonuclease 1) can coordinate to prevent the formation of mutagenic repair intermediates with damaged or mismatched ends at the downstream steps of the BER pathway.

DNA ligase I variants fail in the ligation of mutagenic repair intermediates with mismatches and oxidative DNA damage.

DNA ligase I (LIG1) joins DNA strand breaks during DNA replication and repair transactions and contributes to genome integrity. The mutations (P529L, E566K, R641L and R771W) in LIG1 gene are described in patients with LIG1-deficiency syndrome that exhibit immunodeficiency. LIG1 senses 3'-DNA ends with a mismatch or oxidative DNA base inserted by a repair DNA polymerase. However, the ligation efficiency of the LIG1 variants for DNA polymerase-promoted mutagenesis products with 3'-DNA mismatches or 8-oxo-2'-deoxyguanosine (8-oxodG) remains undefined. Here, we report that R641L and R771W fail in the ligation of nicked DNA with 3'-8-oxodG, leading to an accumulation of 5'-AMP-DNA intermediates in vitro. Moreover, we found that the presence of all possible 12 non-canonical base pairs variously impacts the ligation efficiency by P529L and R771W depending on the architecture at the DNA end, whereas E566K exhibits no activity against all substrates tested. Our results contribute to the understanding of the substrate specificity and mismatch discrimination of LIG1 for mutagenic repair intermediates and the effect of non-synonymous mutations on ligase fidelity.

Pol ß gap filling, DNA ligation and substrate-product channeling during base excision repair opposite oxidized 5-methylcytosine modifications.

DNA methylation on cytosine in CpG islands generates 5-methylcytosine (5mC), and further modification of 5mC can result in the oxidized variants 5-hydroxymethyl (5hmC), 5-formyl (5fC), and 5-carboxy (5caC). Base excision repair (BER) is crucial for both genome maintenance and active DNA demethylation of modified cytosine products and involves substrate-product channeling from nucleotide insertion by DNA polymerase (pol) ß to the subsequent ligation step. Here, we report that, in contrast to the pol ß mismatch insertion products (dCTP, dATP, and dTTP), the nicked products after pol ß dGTP insertion can be ligated by DNA ligase I or DNA ligase III/XRCC1 complex when a 5mC oxidation modification is present opposite in the template position in vitro. A Pol ß K280A mutation, which perturbates the stabilization of these base modifications within the active site, hinders the BER ligases. Moreover, the nicked repair intermediates that mimic pol ß mismatch insertion products, i.e., with 3'-preinserted dGMP or dTMP opposite templating 5hmC, 5fC or 5caC, can be efficiently ligated, whereas preinserted 3'-dAMP or dCMP mismatches result in failed ligation reactions. These findings herein contribute to our understanding of the insertion tendencies of pol ß opposite different cytosine base forms, the ligation properties of DNA ligase I and DNA ligase III/XRCC1 complex in the context of gapped and nicked damage-containing repair intermediates, and the efficiency and fidelity of substrate channeling during the final steps of BER in situations involving oxidative 5mC base modifications in the template strand.

The ligation of pol ß mismatch insertion products governs the formation of promutagenic base excision DNA repair intermediates.

DNA ligase I and DNA ligase III/XRCC1 complex catalyze the ultimate ligation step following DNA polymerase (pol) ß nucleotide insertion during base excision repair (BER). Pol ß Asn279 and Arg283 are the critical active site residues for the differentiation of an incoming nucleotide and a template base and the N-terminal domain of DNA ligase I mediates its interaction with pol ß. Here, we show inefficient ligation of pol ß insertion products with mismatched or damaged nucleotides, with the exception of a Watson-Crick-like dGTP insertion opposite T, using BER DNA ligases in vitro. Moreover, pol ß N279A and R283A mutants deter the ligation of the promutagenic repair intermediates and the presence of N-terminal domain of DNA ligase I in a coupled reaction governs the channeling of the pol ß insertion products. Our results demonstrate that the BER DNA ligases are compromised by subtle changes in all 12 possible noncanonical base pairs at the 3'-end of the nicked repair intermediate. These findings contribute to understanding of how the identity of the mismatch affects the substrate channeling of the repair pathway and the mechanism underlying the coordination between pol ß and DNA ligase at the final ligation step to maintain the BER efficiency.

Pol µ ribonucleotide insertion opposite 8-oxodG facilitates the ligation of premutagenic DNA repair intermediate.

DNA polymerase (pol) µ primarily inserts ribonucleotides into a single-nucleotide gapped DNA intermediate, and the ligation step plays a critical role in the joining of noncomplementary DNA ends during nonhomologous end joining (NHEJ) for the repair of double-strand breaks (DSBs) caused by reactive oxygen species. Here, we report that the pol µ insertion products of ribonucleotides (rATP or rCTP), instead of deoxyribonucleotides, opposite 8-oxo-2'-deoxyguanosine (8-oxodG) are efficiently ligated and the presence of Mn2+ stimulates this coupled reaction in vitro. Moreover, our results point to a role of pol µ in mediating ligation during the mutagenic bypass of 8-oxodG, while 3'-preinserted noncanonical base pairs (3'-rA or 3'-rC) on NHEJ repair intermediates compromise the end joining by DNA ligase I or the DNA ligase IV/XRCC4 complex.

Interplay between DNA Polymerases and DNA Ligases: Influence on Substrate Channeling and the Fidelity of DNA Ligation.

DNA ligases are a highly conserved group of nucleic acid enzymes that play an essential role in DNA repair, replication, and recombination. This review focuses on functional interaction between DNA polymerases and DNA ligases in the repair of single- and double-strand DNA breaks, and discusses the notion that the substrate channeling during DNA polymerase-mediated nucleotide insertion coupled to DNA ligation could be a mechanism to minimize the release of potentially mutagenic repair intermediates. Evidence suggesting that DNA ligases are essential for cell viability includes the fact that defects or insufficiency in DNA ligase are casually linked to genome instability. In the future, it may be possible to develop small molecule inhibitors of mammalian DNA ligases and/or their functional protein partners that potentiate the effects of chemotherapeutic compounds and improve cancer treatment outcomes.

Pol µ dGTP mismatch insertion opposite T coupled with ligation reveals promutagenic DNA repair intermediate.

Incorporation of mismatched nucleotides during DNA replication or repair leads to transition or transversion mutations and is considered as a predominant source of base substitution mutagenesis in cancer cells. Watson-Crick like dG:dT base pairing is considered to be an important source of genome instability. Here we show that DNA polymerase (pol) µ insertion of 7,8-dihydro-8'-oxo-dGTP (8-oxodGTP) or deoxyguanosine triphosphate (dGTP) into a model double-strand break DNA repair substrate with template base T results in efficient ligation by DNA ligase. These results indicate that pol µ-mediated dGTP mismatch insertion opposite template base T coupled with ligation could be a feature of mutation prone nonhomologous end joining during double-strand break repair.

XRCC1 phosphorylation affects aprataxin recruitment and DNA deadenylation activity.

Aprataxin (APTX) is a DNA-adenylate hydrolase that removes 5'-AMP blocking groups from abortive ligation repair intermediates. XRCC1, a multi-domain protein without catalytic activity, interacts with a number of known repair proteins including APTX, modulating and coordinating the various steps of DNA repair. CK2-phosphorylation of XRCC1 is thought to be crucial for its interaction with the FHA domain of APTX. In light of conflicting reports, the importance of XRCC1 phosphorylation and APTX function is not clear. In this study, a phosphorylation mutant of XRCC1 designed to eliminate APTX binding was stably expressed in Xrcc1-/- cells. Analysis of APTX-GFP accumulation at micro-irradiation damage confirmed that phosphorylated XRCC1 is required for APTX recruitment. APTX-mediated DNA deadenylation activity (i.e., 5'-AMP removal) was measured in extracts of cells expressing wild-type XRCC1 or the XRCC1 phosphorylation mutant, and compared with activity in APTX-deficient and APTX-complemented human cells. APTX activity was lower in extracts from Xrcc1-/- and XRCC1 phosphorylation mutant cells compared to the robust activity in extract from wild-type XRCC1 expressing cells. Taken together, results verify that interaction with phosphorylated XRCC1 is a requirement for significant APTX recruitment to cellular DNA damage and enzymatic activity in cell extracts.