Interplay of two major repair pathways in the processing of complex double-strand DNA breaks

Radiation-induced complex double-strand breaks (DSBs) characterised by base lesions, abasic sites or single-strand breaks in close proximity to the break termini, are believed to be a major cause of the biological effects of ionising radiation exposure. It has been hypothesised that complex DSBs pose problems for the repair machinery of the cell. Using a biochemical approach, we have investigated the challenge to two major repair processes: base excision repair and ligation of DSB ends. Double-stranded oligonucleotides were synthesised with 8-oxo-7,8-dihydroguanine (8-oxoG) at defined positions relative to readily ligatable 3'-hydroxy or 5'-phosphate termini. The break termini interfere with removal of 8-oxoG during base excision repair as elucidated from the severely reduced efficiency of 8-oxoG removal by OGG1 with AP endonuclease-1 when in close proximity to break termini. NEIL-1, however, can partially restore processing of complex DSBs in an AP endonuclease-1 independent manner. The influence of 8-oxoG on ligation shows delayed rejoining if 8-oxoG is positioned two to three bases from the 3'-hydroxy or six bases from the 5'-phosphate termini. When two 8-oxoG lesions are positioned across the break junction ligation is severely retarded. This reduced efficiency of repair indicates that complex DSBs are likely to persist longer than simple DSBs in cells, and as a consequence are more significant in contributing to the biological effects of ionising radiation.     

Double-strand DNA break formation mediated by flap endonuclease-1

Double-strand DNA breaks are the most lethal type of DNA damage induced by ionizing radiations. Previously, we reported that double-strand DNA breaks can be enzymatically produced from two DNA damages located on opposite DNA strands 18 or 30 base pairs apart in a cell-free double-strand DNA break formation assay (Vispé, S., and Satoh, M. S. (2000) J. Biol. Chem. 275, 27386-27392). In the assay that we developed, these two DNA damages are converted into single-strand interruptions by enzymes involved in base excision repair. We showed that these single-strand interruptions are converted into double-strand DNA breaks; however, it was not due to spontaneous denaturation of DNA. Thus, we proposed a model in which DNA polymerase delta/epsilon, by producing repair patches at single-strand interruptions, collide, resulting in double-strand DNA break formation. We tested the model and investigated whether other enzymes/factors are involved in double-strand DNA break formation. Here we report that, instead of DNA polymerase delta/epsilon, flap endonuclease-1 (FEN-1), an enzyme involved in base excision repair, is responsible for the formation of double-strand DNA break in the assay. Furthermore, by transfecting a flap endonuclease-1 expression construct into cells, thus altering their flap endonuclease-1 content, we found an increased number of double-strand DNA breaks after gamma-ray irradiation of these cells. These results suggest that flap endonuclease-1 acts as a double-strand DNA break formation factor. Because FEN-1 is an essential enzyme that plays its roles in DNA repair and DNA replication, DSBs may be produced in cells as by-products of the activity of FEN-1.     

CK2 phosphorylation-dependent interaction between aprataxin and MDC1 in the DNA damage response

Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1, resolves abortive DNA ligation intermediates during DNA repair. Here, we demonstrate that aprataxin localizes at sites of DNA damage induced by high LET radiation and binds to mediator of DNA-damage checkpoint protein 1 (MDC1/NFBD1) through a phosphorylation-dependent interaction. This interaction is mediated via the aprataxin FHA domain and multiple casein kinase 2 di-phosphorylated S-D-T-D motifs in MDC1. X-ray structural and mutagenic analysis of aprataxin FHA domain, combined with modelling of the pSDpTD peptide interaction suggest an unusual FHA binding mechanism mediated by a cluster of basic residues at and around the canonical pT-docking site. Mutation of aprataxin FHA Arg29 prevented its interaction with MDC1 and recruitment to sites of DNA damage. These results indicate that aprataxin is involved not only in single strand break repair but also in the processing of a subset of double strand breaks presumably through its interaction with MDC1.     

APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks

Aprataxin and polynucleotide kinase (PNK) are DNA end processing factors that are recruited into the DNA single- and double-strand break repair machinery through phosphorylation-specific interactions with XRCC1 and XRCC4, respectively. These interactions are mediated through a divergent class of forkhead-associated (FHA) domain that binds to peptide sequences in XRCC1 and XRCC4 that are phosphorylated by casein kinase 2 (CK2). Here, we identify the product of the uncharacterized open reading frame C2orf13 as a novel member of this FHA domain family of proteins and we denote this protein APLF (aprataxin- and PNK-like factor). We show that APLF interacts with XRCC1 in vivo and in vitro in a manner that is stimulated by CK2. Yeast two-hybrid analyses suggest that APLF also interacts with the double-strand break repair proteins XRCC4 and XRCC5 (Ku86). We also show that endogenous and yellow fluorescent protein-tagged APLF accumulates at sites of H(2)O(2) or UVA laser-induced chromosomal DNA damage and that this is achieved through at least two mechanisms: one that requires the FHA domain-mediated interaction with XRCC1 and a second that is independent of XRCC1 but requires a novel type of zinc finger motif located at the C terminus of APLF. Finally, we demonstrate that APLF is phosphorylated in a DNA damage- and ATM-dependent manner and that the depletion of APLF from noncycling human SH-SY5Y neuroblastoma cells reduces rates of chromosomal DNA strand break repair following ionizing radiation. These data identify APLF as a novel component of the cellular response to DNA strand breaks in human cells.     

The structural basis for substrate recognition by mammalian polynucleotide kinase 3' phosphatase

Mammalian polynucleotide kinase 3' phosphatase (PNK) plays a key role in the repair of DNA damage, functioning as part of both the nonhomologous end-joining (NHEJ) and base excision repair (BER) pathways. Through its two catalytic activities, PNK ensures that DNA termini are compatible with extension and ligation by either removing 3'-phosphates from, or by phosphorylating 5'-hydroxyl groups on, the ribose sugar of the DNA backbone. We have now determined crystal structures of murine PNK with DNA molecules bound to both of its active sites. The structure of ssDNA engaged with the 3'-phosphatase domain suggests a mechanism of substrate interaction that assists DNA end seeking. The structure of dsDNA bound to the 5'-kinase domain reveals a mechanism of DNA bending that facilitates recognition of DNA ends in the context of single-strand and double-strand breaks and suggests a close functional cooperation in substrate recognition between the kinase and phosphatase active sites.     

The ataxia-oculomotor apraxia 1 gene product has a role distinct from ATM and interacts with the DNA strand break repair proteins XRCC1 and XRCC4

Ataxia-oculomotor apraxia 1 (AOA1) is an autosomal recessive neurodegenerative disease that is reminiscent of ataxia-telangiectasia (A-T). AOA1 is caused by mutations in the gene encoding aprataxin, a protein whose physiological function is currently unknown. We report here that, in contrast to A-T, AOA1 cell lines exhibit neither radioresistant DNA synthesis nor a reduced ability to phosphorylate downstream targets of ATM following DNA damage, suggesting that AOA1 lacks the cell cycle checkpoint defects that are characteristic of A-T. In addition, AOA1 primary fibroblasts exhibit only mild sensitivity to ionising radiation, hydrogen peroxide, and methyl methanesulphonate (MMS). Strikingly, however, aprataxin physically interacts in vitro and in vivo with the DNA strand break repair proteins XRCC1 and XRCC4. Aprataxin possesses a divergent forkhead associated (FHA) domain that closely resembles the FHA domain present in polynucleotide kinase, and appears to mediate the interactions with CK2-phosphorylated XRCC1 and XRCC4 through this domain. Aprataxin is therefore physically associated with both the DNA single-strand and double-strand break repair machinery, raising the possibility that AOA1 is a novel DNA damage response-defective disease.     

Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV

Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA ligation, involving the Xrcc4-DNA ligase IV complex. DNA end processing is often a prerequisite for ligation, but the coordination of these events is poorly understood. We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5'-OH and 3'-phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4. Analysis of the PNK FHA-Xrcc4 interaction revealed that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains. Disruption of the Xrcc4-PNK interaction in vivo is associated with increased radiosensitivity and slower repair kinetics of DSBs, in conjunction with a diminished efficiency of DNA end joining in vitro. Therefore, these results suggest a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation.     

Effects of deletion and site-directed mutations on ligation steps of NAD+-dependent DNA ligase: a biochemical analysis of BRCA1 C-terminal domain

DNA strand joining entails three consecutive steps: enzyme adenylation to form AMP-ligase, substrate adenylation to form AMP-DNA, and nick closure. In this study, we investigate the effects on ligation steps by deletion and site-directed mutagenesis of the BRCA1 C-terminal (BRCT) domain using NAD(+)-dependent DNA ligase from Thermus species AK16D. Deletion of the BRCT domain resulted in substantial loss of ligation activity, but the mutant was still able to form an AMP-ligase intermediate, suggesting that the defects caused by deletion of the entire BRCT domain occur primarily at steps after enzyme adenylation. The lack of AMP-DNA accumulation by the domain deletion mutant as compared to the wild-type ligase indicates that the BRCT domain plays a role in the substrate adenylation step. Gel mobility shift analysis suggests that the BRCT domain and helix-hairpin-helix subdomain play a role in DNA binding. Similar to the BRCT domain deletion mutant, the G617I mutant showed a low ligation activity and lack of accumulation of AMP-DNA intermediate. However, the G617I mutant was only weakly adenylated, suggesting that a point mutation in the BRCT domain could also affect the enzyme adenylation step. The significant reduction of ligation activity by G634I appears to be attributable to a defect at the substrate adenylation step. The greater ligation of mismatched substrates by G638I is accountable by accelerated conversion of the AMP-DNA intermediate to a ligation product at the final nick closure step. The mutational effects of the BRCT domain on ligation steps in relation to protein-DNA and potential protein-protein interactions are discussed.     

PNKP在DNA损伤修复中的作用

多聚核苷酸激酶/磷酸酶(polynucleotide kinase/phosphatase,PNKP)是一种DNA末端修复酶,同时具有激酶和磷酸酶活性,在DNA单链断裂修复途径、碱基切除修复途径以及DNA双链断裂修复中的非同源末端连接途径中发挥着至关重要的作用。近年来,由于一种与PNKP相关的常染色体隐性遗传病——MCSZ综合征的发现,使得人们对PNKP的关注度进一步增加。笔者从与PNKP相互作用的X射线交叉互补修复基因1(X-ray repair cross-complementing group 1,XRCC1)、X射线交叉互补修复基因4(X-ray repair cross-complementing group 4,XRCC4)和毛细血管扩张性共济失调突变基因(ataxia-telangiectasia mutated,ATM)入手,对PNKP在DNA损伤修复中的作用进行概述。     

Down-regulation of DNA repair synthesis at DNA single-strand interruptions in poly(ADP-ribose) polymerase-1 deficient murine cell extracts

The functional involvement of poly(ADP-ribose) polymerase-1 (PARP-1) in the repair of DNA single- and double-strand breaks, DNA base damage, and related repair substrate intermediates remains unclear. Using an in vitro DNA repair assay and cell extracts derived from PARP-1 deficient or wild-type murine embryonic fibroblasts, we investigated the DNA synthesis and ligation steps associated with the rejoining of DNA single-strand interruptions containing 3'-OH, and either 5'-OH or 5'-P termini. Complete repair leading to DNA rejoining was similar between PARP-1 deficient cells and wild-type controls and poly(ADP-ribose) synthesis was, as expected, greatly reduced in PARP-1 deficient cell extracts. The incorporation of [32P]dCMP into repaired DNA at the site of a lesion was reduced two-three-fold in PARP-1 deficient cell extracts, demonstrating a decrease in repair patch size. Addition of purified PARP-1 to levels approximating those present in wild-type extracts did not stimulate DNA repair synthesis. We conclude that PARP-1 is not required for the efficient processing and rejoining of single-strand interruptions with defined 3'-OH and 5'-OH or 5'-P termini. Decreased DNA repair synthesis observed in PARP-1 deficient cell extracts is associated with reduced cellular expression of several factors required for long-patch base excision repair (BER), including FEN-1 and DNA ligase I.     

Molecular mechanism of DNA deadenylation by the neurological disease protein aprataxin

The human neurological disease known as ataxia with oculomotor apraxia 1 is caused by mutations in the APTX gene that encodes Aprataxin (APTX) protein. APTX is a member of the histidine triad superfamily of nucleotide hydrolases and transferases but is distinct from other family members in that it acts upon DNA. The target of APTX is 5'-adenylates at DNA nicks or breaks that result from abortive DNA ligation reactions. In this work, we show that APTX acts as a nick sensor, which provides a mechanism to assess the adenylation status of unsealed nicks. When an adenylated nick is encountered by APTX, base pairing at the 5' terminus of the nick is disrupted as the adenylate is accepted into the active site of the enzyme. Adenylate removal occurs by a two-step process that proceeds through a transient AMP-APTX covalent intermediate. These results pinpoint APTX as the first protein to adopt canonical histidine triad-type reaction chemistry for the repair of DNA.     

The molecular architecture of the mammalian DNA repair enzyme, polynucleotide kinase

Mammalian polynucleotide kinase (PNK) is a key component of both the base excision repair (BER) and nonhomologous end-joining (NHEJ) DNA repair pathways. PNK acts as a 5'-kinase/3'-phosphatase to create 5'-phosphate/3'-hydroxyl termini, which are a necessary prerequisite for ligation during repair. PNK is recruited to repair complexes through interactions between its N-terminal FHA domain and phosphorylated components of either pathway. Here, we describe the crystal structure of intact mammalian PNK and a structure of the PNK FHA bound to a cognate phosphopeptide. The kinase domain has a broad substrate binding pocket, which preferentially recognizes double-stranded substrates with recessed 5' termini. In contrast, the phosphatase domain efficiently dephosphorylates single-stranded 3'-phospho termini as well as double-stranded substrates. The FHA domain is linked to the kinase/phosphatase catalytic domain by a flexible tether, and it exhibits a mode of target selection based on electrostatic complementarity between the binding surface and the phosphothreonine peptide.     

End modification of a linear DNA duplex enhances NER-mediated excision of an internal Pt(II)-lesion

The study of DNA repair has been facilitated by the development of extract-based in vitro assay systems and the use of synthetic DNA duplexes that contain site-specific lesions as repair substrates. Unfortunately, exposed DNA termini can be a liability when working in crude cell extracts because they are targets for DNA end-modifying enzymes and binding sites for proteins that recognize DNA termini. In particular, the double-strand break repair protein Ku is an abundant DNA end-binding protein that has been shown to interfere with nucleotide excision repair (NER) in vitro. To facilitate the investigation of NER in whole-cell extracts, we explored ways of modifying the exposed ends of synthetic repair substrates to prevent Ku binding and improve in vitro NER efficiency. Replacement of six contiguous phosphodiester linkages at the 3'-ends of the duplex repair substrate with nuclease-resistant nonionic methylphosphonate linkages resulted in a 280-fold decrease in binding affinity between Ku and the modified duplex. These results are consistent with the published crystal structure of a Ku/DNA complex [Walker et al. (2001) Nature 412, 607-614] and show that the 3'-terminal phosphodiester linkages of linear DNA duplexes are important determinants in DNA end-binding by Ku. Using HeLa whole-cell extracts and a 149-base pair DNA duplex repair substrate, we tested the effects of modification of exposed DNA termini on NER-mediated in vitro excision of a 1,3-GTG-Pt(II) intrastrand cross-link. Methylphosphonate modification at the 3'-ends of the repair substrate resulted in a 1.6-fold increase in excision. Derivatization of the 5'-ends of the duplex with biotin and subsequent conjugation with streptavidin to block Ku binding resulted in a 2.3-fold increase excision. By combining these modifications, we were able to effectively reduce Ku-derived interference of NER excision in vitro and observed a 4.4-fold increase in platinum lesion excision. These modifications are easy to incorporate into synthetic oligonucleotides and may find general utility whenever synthetic linear duplex DNAs are used as substrates to investigate DNA repair in whole-cell extracts.     

Securing genome stability by orchestrating DNA repair: removal of radiation-induced clustered lesions in DNA

In addition to double- and single-strand DNA breaks and isolated base modifications, ionizing radiation induces clustered DNA damage, which contains two or more lesions closely spaced within about two helical turns on opposite DNA strands. Post-irradiation repair of single-base lesions is routinely performed by base excision repair and a DNA strand break is involved as an intermediate. Simultaneous processing of lesions on opposite DNA strands may generate double-strand DNA breaks and enhance nonhomologous end joining, which frequently results in the formation of deletions. Recent studies support the possibility that the mechanism of base excision repair contributes to genome stability by diminishing the formation of double-strand DNA breaks during processing of clustered lesions.     

Specificity of the dRP/AP lyase of Ku promotes nonhomologous end joining (NHEJ) fidelity at damaged ends

Nonhomologous end joining (NHEJ) is essential for efficient repair of chromosome breaks. However, the NHEJ ligation step is often obstructed by break-associated nucleotide damage, including base loss (abasic site or 5'-dRP/AP sites). Ku, a 5'-dRP/AP lyase, can excise such damage at ends in preparation for the ligation step. We show here that this activity is greatest if the abasic site is within a short 5' overhang, when this activity is necessary and sufficient to prepare such termini for ligation. In contrast, Ku is less active near 3' strand termini, where excision would leave a ligation-blocking α,β-unsaturated aldehyde. The Ku AP lyase activity is also strongly suppressed by as little as two paired bases 5' of the abasic site. Importantly, in vitro end joining experiments show that abasic sites significantly embedded in double-stranded DNA do not block the NHEJ ligation step. Suppression of the excision activity of Ku in this context therefore is not essential for ligation and further helps NHEJ retain terminal sequence in junctions. We show that the DNA between the 5' terminus and the abasic site can also be retained in junctions formed by cellular NHEJ, indicating that these sites are at least partly resistant to other abasic site-cleaving activities as well. High levels of the 5'-dRP/AP lyase activity of Ku are thus restricted to substrates where excision of an abasic site is required for ligation, a degree of specificity that promotes more accurate joining.     

Defective DNA Ligation during Short-Patch Single-Strand Break Repair in Ataxia Oculomotor Apraxia 1

Ataxia oculomotor apraxia 1 (AOA1) results from mutations in aprataxin, a component of DNA strand break repair that removes AMP from 5′ termini. Despite this, global rates of chromosomal strand break repair are normal in a variety of AOA1 and other aprataxin-defective cells. Here we show that short-patch single-strand break repair (SSBR) in AOA1 cell extracts bypasses the point of aprataxin action at oxidative breaks and stalls at the final step of DNA ligation, resulting in the accumulation of adenylated DNA nicks. Strikingly, this defect results from insufficient levels of nonadenylated DNA ligase, and short-patch SSBR can be restored in AOA1 extracts, independently of aprataxin, by the addition of recombinant DNA ligase. Since adenylated nicks are substrates for long-patch SSBR, we reasoned that this pathway might in part explain the apparent absence of a chromosomal SSBR defect in aprataxin-defective cells. Indeed, whereas chemical inhibition of long-patch repair did not affect SSBR rates in wild-type mouse neural astrocytes, it uncovered a significant defect in Aptx^−/− neural astrocytes. These data demonstrate that aprataxin participates in chromosomal SSBR in vivo and suggest that short-patch SSBR arrests in AOA1 because of insufficient nonadenylated DNA ligase.Oxidative stress is an etiological factor in many neurological diseases, including Alzheimer''s disease, Parkinson''s disease, and Huntington''s disease. One type of macromolecule damaged by reactive oxygen species is DNA, and oxidative damage to DNA has been suggested to be a significant factor in these and other neurological conditions (2). In particular, a number of rare hereditary neurodegenerative disorders have provided direct support for the notion that unrepaired DNA damage causes neural dysfunction. Not least of these are the recessive spinocerebellar ataxias, a number of which are associated with mutations in DNA damage response proteins (17). The archetypal DNA damage-associated spinocerebellar ataxia is ataxia-telangiectasia (A-T), in which mutations in ATM protein result in defects in the detection and signaling of DNA double-strand breaks (DSBs) (3). A-T-like disorder is a related disease that exhibits neurological features similar to those of A-T, resulting from mutation of Mre11, a component of the MRN complex that operates in conjunction with ATM during DSB detection and signaling (28).Two additional spinocerebellar ataxias are spinocerebellar ataxia with axonal neuropathy 1 (SCAN1) and ataxia oculomotor apraxia 1 (AOA1), in which the TDP1 and aprataxin proteins are mutated, respectively (9, 19, 27). Both TDP1 and aprataxin are components of the DNA strand break repair machinery (recently reviewed in references 6 and 24). Whereas SCAN1 is currently limited to nine individuals from a single family, AOA1 is one of the commonest recessive spinocerebellar ataxias. Aprataxin is a member of the histidine triad superfamily of nucleotide hydrolases/transferases and has been reported to remove phosphate and phosphoglycolate moieties from the 3′ termini of DNA strand breaks (26). Aprataxin can also remove AMP from a variety of ligands in vitro, including adenosine polyphosphates, AMP-lysine, AMP-NH₂ (adenine monophosphoramidate), and adenylated DNA in which AMP is covalently attached to the 5′ terminus of a DNA single-strand break (SSB) or DSB (1, 16, 23, 25). To date, aprataxin activity is greatest on AMP-DNA, suggesting that this may be the physiological substrate of this enzyme.In vitro, DNA strand breaks with 5′-AMP termini can arise from premature DNA ligase activity. DNA ligases adenylate 5′ termini at DNA breaks to enable nucleophilic attack of the resulting pyrophosphate bonds by 3′-hydroxyl termini, thereby resealing the breaks. However, DNA adenylation by DNA ligases can occur prematurely, before a 3′-hydroxyl terminus is available. Aprataxin reverses these premature DNA adenylation events, in vitro at least, effectively “resetting” the DNA ligation reaction to the beginning (1). Whether or not 5′-AMP arises in DNA in vivo or is a physiological substrate of aprataxin, however, is unknown. Moreover, attempts to measure DNA strand break repair rates in vivo are conflicting and have failed to identify a consistent defect in DNA SSB repair (SSBR) or DSB repair (DSBR) in AOA1 cells (14, 15, 20). It is thus not clear whether or not defects in DNA strand break repair can account for this neurodegenerative disease.Here we have resolved the discrepancy between the requirements for aprataxin in vitro and in vivo by identifying the stage at which SSBR reactions fail in vitro and by carefully analyzing chromosomal SSBR rates in vivo. We show that short-patch SSBR reactions are defective in AOA1 cell extracts at the final step of DNA ligation, resulting in the accumulation of adenylated DNA nicks, and that this defect can be rescued in AOA1 extracts independently of aprataxin by addition of recombinant DNA ligase. We also find that treatment with aphidicolin, an inhibitor of DNA polymerase δ (Pol δ) and Pol ɛ, unveils a measurable defect in chromosomal SSBR in Aptx^−/− primary neural astrocytes, suggesting that the adenylated nicks that arise from the short-patch repair defect can be channeled into long-patch repair in vivo. These data demonstrate that aprataxin participates in chromosomal SSBR and suggest that this process arrests in AOA1, at oxidative SSBs, due to insufficient levels of nonadenylated DNA ligase.     

APE1-dependent repair of DNA single-strand breaks containing 3'-end 8-oxoguanine

DNA single-strand breaks containing 3′-8-oxoguanine (3′-8-oxoG) ends can arise as a consequence of ionizing radiation and as a result of DNA polymerase infidelity by misincorporation of 8-oxodGMP. In this study we examined the mechanism of repair of 3′-8-oxoG within a single-strand break using purified base excision repair enzymes and human whole cell extracts. We find that 3′-8-oxoG inhibits ligation by DNA ligase IIIα or DNA ligase I, inhibits extension by DNA polymerase β and that the lesion is resistant to excision by DNA glycosylases involved in the repair of oxidative lesions in human cells. However, we find that purified human AP-endonuclease 1 (APE1) is able to remove 3′-8-oxoG lesions. By fractionation of human whole cell extracts and immunoprecipitation of fractions containing 3′-8-oxoG excision activity, we further demonstrate that APE1 is the major activity involved in the repair of 3′-8-oxoG lesions in human cells and finally we reconstituted repair of the 3′-8-oxoG-containing oligonucleotide duplex with purified human enzymes including APE1, DNA polymerase β and DNA ligase IIIα.     

Genetic interactions between HNT3/Aprataxin and RAD27/FEN1 suggest parallel pathways for 5′ end processing during base excision repair

Mutations in Aprataxin cause the neurodegenerative syndrome ataxia oculomotor apraxia type 1. Aprataxin catalyzes removal of adenosine monophosphate (AMP) from the 5′ end of a DNA strand, which results from an aborted attempt to ligate a strand break containing a damaged end. To gain insight into which DNA lesions are substrates for Aprataxin action in vivo, we deleted the Saccharomyces cerevisiae HNT3 gene, which encodes the Aprataxin homolog, in combination with known DNA repair genes. While hnt3Δ single mutants were not sensitive to DNA damaging agents, loss of HNT3 caused synergistic sensitivity to H₂O₂ in backgrounds that accumulate strand breaks with blocked termini, including apn1Δ apn2Δ tpp1Δ and ntg1Δ ntg2Δ ogg1Δ. Loss of HNT3 in rad27Δ cells, which are deficient in long-patch base excision repair (LP-BER), resulted in synergistic sensitivity to H₂O₂ and MMS, indicating that Hnt3 and LP-BER provide parallel pathways for processing 5′ AMPs. Loss of HNT3 also increased the sister chromatid exchange frequency. Surprisingly, HNT3 deletion partially rescued H₂O₂ sensitivity in recombination-deficient rad51Δ and rad52Δ cells, suggesting that Hnt3 promotes formation of a repair intermediate that is resolved by recombination.     

The DNA-dependent protein kinase interacts with DNA to form a protein-DNA complex that is disrupted by phosphorylation

DNA double-strand breaks are a serious threat to genome stability and cell viability. One of the major pathways for the repair of DNA double-strand breaks in human cells is nonhomologous end-joining. Biochemical and genetic studies have shown that the DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV, and Artemis are essential components of the nonhomologous end-joining pathway. DNA-PK is composed of a large catalytic subunit, DNA-PKcs, and a heterodimer of Ku70 and Ku80 subunits. Current models predict that the Ku heterodimer binds to ends of double-stranded DNA, then recruits DNA-PKcs to form the active protein kinase complex. XRCC4 and DNA ligase IV are subsequently required for ligation of the DNA ends. Magnesium-ATP and the protein kinase activity of DNA-PKcs are essential for DNA double-strand break repair. However, little is known about the physiological targets of DNA-PK. We have previously shown that DNA-PKcs and Ku undergo autophosphorylation, and that this correlates with loss of protein kinase activity. Here we show, using electron spectroscopic imaging, that DNA-PKcs and Ku interact with multiple DNA molecules to form large protein-DNA complexes that converge at the base of multiple DNA loops. The number of large protein complexes and the amount of DNA associated with them were dramatically reduced under conditions that promote phosphorylation of DNA-PK. Moreover, treatment of autophosphorylated DNA-PK with the protein phosphatase 1 catalytic subunit restored complex formation. We propose that autophosphorylation of DNA-PK plays an important regulatory role in DNA double-strand break repair by regulating the assembly and disassembly of the DNA-PK-DNA complex.     

Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells

In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excision (BER) and single strand break repair (SSBR). Here, using conditional knockout strategies for LIG3 and concomitant inactivation of the LIG1 and LIG4 genes, we show that in DT40 cells LigIII efficiently supports semi-conservative DNA replication. Our observations demonstrate a high functional versatility for the evolutionary new LigIII in DNA replication and mitochondrial metabolism, and suggest the presence of an alternative pathway for Okazaki fragment ligation.