Repair of radiation-induced DNA double-strand breaks is dependent upon radiation quality and the structural complexity of double-strand breaks

Mammalian cells primarily repair DSBs by nonhomologous end joining (NHEJ). To assess the ability of human cells to mediate end joining of complex DSBs such as those produced by chemicals, oxidative events, or high- and low-LET radiation, we employed an in vitro double-strand break repair assay using plasmid DNA linearized by these various agents. We found that human HeLa cell extracts support end joining of complex DSBs and form multimeric plasmid products from substrates produced by the radiomimetic drug bleomycin, 60Co gamma rays, and the effects of 125I decay in DNA. End joining was found to be dependent on the type of DSB-damaging agent, and it decreased as the cytotoxicity of the DSB-inducing agent increased. In addition to the inhibitory effects of DSB end-group structures on repair, NHEJ was found to be strongly inhibited by lesions proximal to DSB ends. The initial repair rate for complex non-ligatable bleomycin-induced DSBs was sixfold less than that of similarly configured (blunt-ended) but less complex (ligatable) restriction enzyme-induced DSBs. Repair of DSBs produced by gamma rays was 15-fold less efficient than repair of restriction enzyme-induced DSBs. Repair of the DSBs produced by 125I was near the lower limit of detection in our assay and was at least twofold lower than that of gamma-ray-induced DSBs. In addition, DSB ends produced by 125I were shown to be blocked by 3'-nucleotide fragments: the removal of these by E. coli endonuclease IV permitted ligation.     

Nonhomologous end joining of complex DNA double-strand breaks with proximal thymine glycol and interplay with base excision repair

DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol at the first (Tg1), second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3′ terminus, was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break. However, Tg as the terminal or penultimate base was a major barrier to end joining (>10-fold reduction in ligated products) and an absolute barrier when present at both ends. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised from Tg1, Tg2 and Tg3 largely if not exclusively after DSB ligation. However, Tg was rapidly excised from the Tg5 substrate, resulting in a reduced level of DSB ligation, as well as slow concomitant resection of the opposite strand. Ligase reactions containing only purified Ku, XRCC4, ligase IV and XLF showed that ligation of Tg3 and Tg5 was efficient and only partially XLF-dependent, whereas ligation of Tg1 and Tg2 was inefficient and only detectable in the presence of XLF. Overall, the results suggest that promoting ligation of DSBs with proximal base damage may be an important function of XLF, but that Tg can still be a major impediment to repair, being relatively resistant to both trimming and ligation. Moreover, it appears that base excision repair of Tg can sometimes interfere with repair of DSBs that would otherwise be readily rejoined.     

Overhang polarity of chromosomal double-strand breaks impacts kinetics and fidelity of yeast non-homologous end joining

Non-homologous end joining (NHEJ) is the main repair pathway for DNA double-strand breaks (DSBs) in cells with limited 5′ resection. To better understand how overhang polarity of chromosomal DSBs affects NHEJ, we made site-specific 5′-overhanging DSBs (5′ DSBs) in yeast using an optimized zinc finger nuclease at an efficiency that approached HO-induced 3′ DSB formation. When controlled for the extent of DSB formation, repair monitoring suggested that chromosomal 5′ DSBs were rejoined more efficiently than 3′ DSBs, consistent with a robust recruitment of NHEJ proteins to 5′ DSBs. Ligation-mediated qPCR revealed that Mre11-Rad50-Xrs2 rapidly modified 5′ DSBs and facilitated protection of 3′ DSBs, likely through recognition of overhang polarity by the Mre11 nuclease. Next-generation sequencing revealed that NHEJ at 5′ DSBs had a higher mutation frequency, and validated the differential requirement of Pol4 polymerase at 3′ and 5′ DSBs. The end processing enzyme Tdp1 did not impact joining fidelity at chromosomal 5′ DSBs as in previous plasmid studies, although Tdp1 was recruited to only 5′ DSBs in a Ku-independent manner. These results suggest distinct DSB handling based on overhang polarity that impacts NHEJ kinetics and fidelity through differential recruitment and action of DSB modifying enzymes.     

Requirement for XLF/Cernunnos in alignment-based gap filling by DNA polymerases λ and μ for nonhomologous end joining in human whole-cell extracts

XLF/Cernunnos is a core protein of the nonhomologous end-joining pathway of DNA double-strand break repair. To better define the role of Cernunnos in end joining, whole-cell extracts were prepared from Cernunnos-deficient human cells. These extracts effected little joining of DNA ends with cohesive 5′ or 3′ overhangs, and no joining at all of partially complementary 3′ overhangs that required gap filling prior to ligation. Assays in which gap-filled but unligated intermediates were trapped using dideoxynucleotides revealed that there was no gap filling on aligned DSB ends in the Cernunnos-deficient extracts. Recombinant Cernunnos protein restored gap filling and end joining of partially complementary overhangs, and stimulated joining of cohesive ends more than twentyfold. XLF-dependent gap filling was nearly eliminated by immunodepletion of DNA polymerase λ, but was restored by addition of either polymerase λ or polymerase μ. Thus, Cernunnos is essential for gap filling by either polymerase during nonhomologous end joining, suggesting that it plays a major role in aligning the two DNA ends in the repair complex.     

ATM regulates Mre11-dependent DNA end-degradation and microhomology-mediated end joining

The human disorder ataxia telangiectasia (AT), which is characterized by genetic instability and neurodegeneration, results from mutation of the ataxia telangiectasia mutated (ATM) kinase. The loss of ATM leads to cell cycle checkpoint deficiencies and other DNA damage signaling defects that do not fully explain all pathologies associated with A-T including neuronal loss. In addressing this enigma, we find here that ATM suppresses DNA double-strand break (DSB) repair by microhomology-mediated end joining (MMEJ). We show that ATM repression of DNA end-degradation is dependent on its kinase activities and that Mre11 is the major nuclease behind increased DNA end-degradation and MMEJ repair in A-T. Assessment of MMEJ by an in vivo reporter assay system reveals decreased levels of MMEJ repair in Mre11-knockdown cells and in cells treated with Mre11-nuclease inhibitor mirin. Structure-based modeling of Mre11 dimer engaging DNA ends suggests the 5′ ends of a bridged DSB are juxtaposed such that DNA unwinding and 3′–5′ exonuclease activities may collaborate to facilitate simultaneous pairing of extended 5′ termini and exonucleolytic degradation of the 3′ ends in MMEJ. Together our results provide an integrated understanding of ATM and Mre11 in MMEJ: ATM has a critical regulatory function in controlling DNA end-stability and error-prone DSB repair and Mre11 nuclease plays a major role in initiating MMEJ in mammalian cells. These functions of ATM and Mre11 could be particularly important in neuronal cells, which are post-mitotic and therefore depend on mechanisms other than homologous recombination between sister chromatids to repair DSBs.Key words: ATM, Mre11, MRN complex, DNA degradation, double-strand break repair, microhomology-mediated end joining, PI-3-kinase-like kinases     

Repair of a minimal DNA double-strand break by NHEJ requires DNA-PKcs and is controlled by the ATM/ATR checkpoint

Mammalian cells primarily rejoin DNA double-strand breaks (DSBs) by the non-homologous end-joining (NHEJ) pathway. The joining of the broken DNA ends appears directly without template and accuracy is ensured by the NHEJ factors that are under ATM/ATR regulated checkpoint control. In the current study we report the engineering of a mono-specific DNA damaging agent. This was used to study the molecular requirements for the repair of the least complex DSB in vivo. Single-chain PvuII restriction enzymes fused to protein delivery sequences transduce cells efficiently and induce blunt end DSBs in vivo. We demonstrate that beside XRCC4/LigaseIV and KU, the DNA-PK catalytic subunit (DNA-PKcs) is also essential for the joining of this low complex DSB in vivo. The appearance of blunt end 3′-hydroxyl and 5′-phosphate DNA DSBs induces a significantly higher frequency of anaphase bridges in cells that do not contain functional DNA-PKcs, suggesting an absolute requirement for DNA-PKcs in the control of chromosomal stability during end joining. Moreover, these minimal blunt end DSBs are sufficient to induce a p53 and ATM/ATR checkpoint function.     

Deficiency in 3'-phosphoglycolate processing in human cells with a hereditary mutation in tyrosyl-DNA phosphodiesterase (TDP1)

Tyrosyl-DNA phosphodiesterase (TDP1) is a DNA repair enzyme that removes peptide fragments linked through tyrosine to the 3′ end of DNA, and can also remove 3′-phosphoglycolates (PGs) formed by free radical-mediated DNA cleavage. To assess whether TDP1 is primarily responsible for PG removal during in vitro end joining of DNA double-strand breaks (DSBs), whole-cell extracts were prepared from lymphoblastoid cells derived either from spinocerebellar ataxia with axonal neuropathy (SCAN1) patients, who have an inactivating mutation in the active site of TDP1, or from closely matched normal controls. Whereas extracts from normal cells catalyzed conversion of 3′-PG termini, both on single-strand oligomers and on 3′ overhangs of DSBs, to 3′-phosphate termini, extracts of SCAN1 cells did not process either substrate. Addition of recombinant TDP1 to SCAN1 extracts restored 3′-PG removal, allowing subsequent gap filling on the aligned DSB ends. Two of three SCAN1 lines examined were slightly more radiosensitive than normal cells, but only for fractionated radiation in plateau phase. The results suggest that the TDP1 mutation in SCAN1 abolishes the 3′-PG processing activity of the enzyme, and that there are no other enzymes in cell extracts capable of processing protruding 3′-PG termini. However, the lack of severe radiosensitivity suggests that there must be alternative, TDP1-independent pathways for repair of 3′-PG DSBs.     

Proofreading Activity of DNA Polymerase Pol2 Mediates 3′-End Processing during Nonhomologous End Joining in Yeast

Genotoxic agents that cause double-strand breaks (DSBs) often generate damage at the break termini. Processing enzymes, including nucleases and polymerases, must remove damaged bases and/or add new bases before completion of repair. Artemis is a nuclease involved in mammalian nonhomologous end joining (NHEJ), but in Saccharomyces cerevisiae the nucleases and polymerases involved in NHEJ pathways are poorly understood. Only Pol4 has been shown to fill the gap that may form by imprecise pairing of overhanging 3′ DNA ends. We previously developed a chromosomal DSB assay in yeast to study factors involved in NHEJ. Here, we use this system to examine DNA polymerases required for NHEJ in yeast. We demonstrate that Pol2 is another major DNA polymerase involved in imprecise end joining. Pol1 modulates both imprecise end joining and more complex chromosomal rearrangements, and Pol3 is primarily involved in NHEJ-mediated chromosomal rearrangements. While Pol4 is the major polymerase to fill the gap that may form by imprecise pairing of overhanging 3′ DNA ends, Pol2 is important for the recession of 3′ flaps that can form during imprecise pairing. Indeed, a mutation in the 3′-5′ exonuclease domain of Pol2 dramatically reduces the frequency of end joins formed with initial 3′ flaps. Thus, Pol2 performs a key 3′ end-processing step in NHEJ.     

Biochemical evidence for Ku-independent backup pathways of NHEJ

Cells of higher eukaryotes process within minutes double strand breaks (DSBs) in their genome using a non-homologous end joining (NHEJ) apparatus that engages DNA-PKcs, Ku, DNA ligase IV, XRCC4 and other as of yet unidentified factors. Although chemical inhibition, or mutation, in any of these factors delays processing, cells ultimately remove the majority of DNA DSBs using an alternative pathway operating with an order of magnitude slower kinetics. This alternative pathway is active in mutants deficient in genes of the RAD52 epistasis group and frequently joins incorrect ends. We proposed, therefore, that it reflects an alternative form of NHEJ that operates as a backup (B-NHEJ) to the DNA-PK-dependent (D-NHEJ) pathway, rather than homology directed repair of DSBs. The present study investigates the role of Ku in the coordination of these pathways using as a model end joining of restriction endonuclease linearized plasmid DNA in whole cell extracts. Efficient, error-free, end joining observed in such in vitro reactions is strongly inhibited by anti-Ku antibodies. The inhibition requires DNA-PKcs, despite the fact that Ku efficiently binds DNA ends in the presence of antibodies, or in the absence of DNA-PKcs. Strong inhibition of DNA end joining is also mediated by wortmannin, an inhibitor of DNA-PKcs, in the presence but not in the absence of Ku, and this inhibition can be rescued by pre-incubating the reaction with double stranded oligonucleotides. The results are compatible with a role of Ku in directing end joining to a DNA-PK dependent pathway, mediated by efficient end binding and productive interactions with DNA-PKcs. On the other hand, efficient end joining is observed in extracts of cells lacking DNA-PKcs, as well as in Ku-depleted extracts in line with the operation of alternative pathways. Extracts depleted of Ku and DNA-PKcs rejoin blunt ends, as well as homologous ends with 3′ or 5′ protruding single strands with similar efficiency, but addition of Ku suppresses joining of blunt ends and homologous ends with 3′ overhangs. We propose that the affinity of Ku for DNA ends, particularly when cooperating with DNA-PKcs, suppresses B-NHEJ by quickly and efficiently binding DNA ends and directing them to D-NHEJ for rapid joining. A chromatin-based model of DNA DSB rejoining accommodating biochemical and genetic results is presented and deviations between in vitro and in vivo results discussed.     

Tracking the processing of damaged DNA double-strand break ends by ligation-mediated PCR: increased persistence of 3′-phosphoglycolate termini in SCAN1 cells

To track the processing of damaged DNA double-strand break (DSB) ends in vivo, a method was devised for quantitative measurement of 3′-phosphoglycolate (PG) termini on DSBs induced by the non-protein chromophore of neocarzinostatin (NCS-C) in the human Alu repeat. Following exposure of cells to NCS-C, DNA was isolated, and labile lesions were chemically stabilized. All 3′-phosphate and 3′-hydroxyl ends were enzymatically capped with dideoxy termini, whereas 3′-PG ends were rendered ligatable, linked to an anchor, and quantified by real-time Taqman polymerase chain reaction. Using this assay and variations thereof, 3′-PG and 3′-phosphate termini on 1-base 3′ overhangs of NCS-C-induced DSBs were readily detected in DNA from the treated lymphoblastoid cells, and both were largely eliminated from cellular DNA within 1 h. However, the 3′-PG termini were processed more slowly than 3′-phosphate termini, and were more persistent in tyrosyl-DNA phosphodiesterase 1-mutant SCAN1 than in normal cells, suggesting a significant role for tyrosyl-DNA phosphodiesterase 1 in removing 3′-PG blocking groups for DSB repair. DSBs with 3′-hydroxyl termini, which are not directly induced by NCS-C, were formed rapidly in cells, and largely eliminated by further processing within 1 h, both in Alu repeats and in heterochromatic α-satellite DNA. Moreover, absence of DNA-PK in M059J cells appeared to accelerate resolution of 3′-PG ends.     

End Resection at Double-Strand Breaks: Mechanism and Regulation

RecA/Rad51 catalyzed pairing of homologous DNA strands, initiated by polymerization of the recombinase on single-stranded DNA (ssDNA), is a universal feature of homologous recombination (HR). Generation of ssDNA from a double-strand break (DSB) requires nucleolytic degradation of the 5′-terminated strands to generate 3′-ssDNA tails, a process referred to as 5′–3′ end resection. The RecBCD helicase–nuclease complex is the main end-processing machine in Gram-negative bacteria. Mre11-Rad50 and Mre11-Rad50-Xrs2/Nbs1 can play a direct role in end resection in archaea and eukaryota, respectively, by removing end-blocking lesions and act indirectly by recruiting the helicases and nucleases responsible for extensive resection. In eukaryotic cells, the initiation of end resection has emerged as a critical regulatory step to differentiate between homology-dependent and end-joining repair of DSBs.DSBs can arise accidentally during normal cell metabolism or after exposure of cells to DNA-damaging agents, and also serve as intermediates in a number of programmed recombination events in eukaryotic cells (Mehta and Haber 2014). The repair of DSBs is critical for maintenance of genome integrity, and misrepair, or failure to repair, is associated with chromosome rearrangements, chromosome loss, or even cell death. Both prokaryotic and eukaryotic cells have evolved elaborate mechanisms for the recognition and repair of DSBs. The two predominant repair mechanisms are HR and non-homologous end joining (NHEJ). HR relies on the presence of an intact homologous duplex to template repair of the broken strands, whereas NHEJ repairs DSBs by direct ligation of the DNA ends. For DSBs to be repaired by HR, the ends must first be degraded to generate long 3′-ssDNA tails, a process referred to as 5′–3′ end resection. The 3′-ssDNA tails are then bound by a member of the RecA/Rad51 family of proteins to initiate homologous pairing and serve as primers for DNA synthesis following strand invasion. Strand invasion intermediates are further processed by helicases and/or nucleases (Bizard and Hickson 2014; Wyatt and West 2014), and ultimately by gap-filling DNA synthesis and ligation, to generate mature recombinant products. The DNA end-resection step of HR is conserved in all domains of life, but the mechanisms used for generating ssDNA are distinct. Here, we review the basic machinery for DNA end resection in bacteria, archaea, and eukaryota and the regulation of end resection in eukaryotic cells.     

Ligation-assisted homologous recombination enables precise genome editing by deploying both MMEJ and HDR

CRISPR/Cas12a is a single effector nuclease that, like CRISPR/Cas9, has been harnessed for genome editing based on its ability to generate targeted DNA double strand breaks (DSBs). Unlike the blunt-ended DSB generated by Cas9, Cas12a generates sticky-ended DSB that could potentially aid precise genome editing, but this unique feature has thus far been underutilized. In the current study, we found that a short double-stranded DNA (dsDNA) repair template containing a sticky end that matched one of the Cas12a-generated DSB ends and a homologous arm sharing homology with the genomic region adjacent to the other end of the DSB enabled precise repair of the DSB and introduced a desired nucleotide substitution. We termed this strategy ‘Ligation-Assisted Homologous Recombination’ (LAHR). Compared to the single-stranded oligo deoxyribonucleotide (ssODN)-mediated homology directed repair (HDR), LAHR yields relatively high editing efficiency as demonstrated for both a reporter gene and endogenous genes. We found that both HDR and microhomology-mediated end joining (MMEJ) mechanisms are involved in the LAHR process. Our LAHR genome editing strategy, extends the repertoire of genome editing technologies and provides a broader understanding of the type and role of DNA repair mechanisms involved in genome editing.     

Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaks

Genome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays (‘PathSig-dPCR’) for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.     

DNA end resection: Many nucleases make light work

Double-strand breaks (DSBs) are deleterious DNA lesions and if left unrepaired result in severe genomic instability. Cells use two main pathways to repair DSBs: homologous recombination (HR) or non-homologous end joining (NHEJ) depending on the phase of the cell cycle and the nature of the DSB ends. A key step where pathway choice is exerted is in the ‘licensing’ of 5′–3′ resection of the ends to produce recombinogenic 3′ single-stranded tails. These tails are substrate for binding by Rad51 to initiate pairing and strand invasion with homologous duplex DNA. Moreover, the single-stranded DNA generated after end processing is important to activate the DNA damage response. The mechanism of end processing is the focus of this review and we will describe recent findings that shed light on this important initiating step for HR. The conserved MRX/MRN complex appears to be a major regulator of DNA end processing. Sae2/CtIP functions with the MRX complex, either to activate the Mre11 nuclease or via the intrinsic endonuclease, in an initial step to trim the DSB ends. In a second step, redundant systems remove long tracts of DNA to reveal extensive 3′ single-stranded tails. One system is dependent on the helicase Sgs1 and the nuclease Dna2, and the other on the 5′–3′ exonuclease Exo1.     

Distinct mechanisms of nonhomologous end joining in the repair of site-directed chromosomal breaks with noncomplementary and complementary ends

DNA double-strand breaks (DSBs) are considered the most important type of DNA damage inflicted by ionizing radiation. The molecular mechanisms of DSB repair by nonhomologous end joining (NHEJ) have not been well studied in live mammalian cells, due in part to the lack of suitable chromosomal repair assays. We previously introduced a novel plasmid-based assay to monitor NHEJ of site-directed chromosomal I-SceI breaks. In the current study, we expanded the analysis of chromosomal NHEJ products in murine fibroblasts to focus on the error-prone rejoining of DSBs with noncomplementary ends, which may serve as a model for radiation damage repair. We found that noncomplementary ends were efficiently repaired using microhomologies of 1-2 nucleotides (nt) present in the single-stranded overhangs, thereby keeping repair-associated end degradation to a minimum (2-3 nt). Microhomology-mediated end joining was disrupted by Wortmannin, a known inhibitor of DNA-PKcs. However, Wortmannin did not significantly impair the proficiency of end joining. In contrast to noncomplementary ends, the rejoining of cohesive ends showed only a minor dependence on microhomologies but produced fivefold larger deletions than the repair of noncomplementary ends. Together, these data suggest the presence of several distinct NHEJ mechanisms in live cells, which are characterized by the degree of sequence deletion and microhomology use. Our NHEJ assay should prove a useful system to further elucidate the genetic determinants and molecular mechanisms of site-directed DSBs in living cells.     

Single-stranded DNA oligomers stimulate error-prone alternative repair of DNA double-strand breaks through hijacking Ku protein

In humans, DNA double-strand breaks (DSBs) are repaired by two mutually-exclusive mechanisms, homologous recombination or end-joining. Among end-joining mechanisms, the main process is classical non-homologous end-joining (C-NHEJ) which relies on Ku binding to DNA ends and DNA Ligase IV (Lig4)-mediated ligation. Mostly under Ku- or Lig4-defective conditions, an alternative end-joining process (A-EJ) can operate and exhibits a trend toward microhomology usage at the break junction. Homologous recombination relies on an initial MRN-dependent nucleolytic degradation of one strand at DNA ends. This process, named DNA resection generates 3′ single-stranded tails necessary for homologous pairing with the sister chromatid. While it is believed from the current literature that the balance between joining and recombination processes at DSBs ends is mainly dependent on the initiation of resection, it has also been shown that MRN activity can generate short single-stranded DNA oligonucleotides (ssO) that may also be implicated in repair regulation. Here, we evaluate the effect of ssO on end-joining at DSB sites both in vitro and in cells. We report that under both conditions, ssO inhibit C-NHEJ through binding to Ku and favor repair by the Lig4-independent microhomology-mediated A-EJ process.     

Gap Filling Activities of Pseudomonas DNA Ligase D (LigD) Polymerase and Functional Interactions of LigD with the DNA End-binding Ku Protein

Many bacterial pathogens, including Pseudomonas aeruginosa, have a nonhomologous end joining (NHEJ) system of DNA double strand break (DSB) repair driven by Ku and DNA ligase D (LigD). LigD is a multifunctional enzyme composed of a ligase domain fused to an autonomous polymerase module (POL) that adds ribonucleotides or deoxyribonucleotides to DSB ends and primer-templates. LigD POL and the eukaryal NHEJ polymerase λ are thought to bridge broken DNA ends via contacts with a duplex DNA segment downstream of the primer terminus, a scenario analogous to gap repair. Here, we characterized the gap repair activity of Pseudomonas LigD POL, which is more efficient than simple templated primer extension and relies on a 5′-phosphate group on the distal gap strand end to confer apparent processivity in filling gaps of 3 or 4 nucleotides. Mutations of the His-553, Arg-556, and Lys-566 side chains implicated in DNA 5′-phosphate binding eliminate the preferential filling of 5′-phosphate gaps. Mutating Phe-603, which is imputed to stack on the nucleobase of the template strand that includes the 1st bp of the downstream gap duplex segment, selectively affects incorporation of the final gap-closing nucleotide. We find that Pseudomonas Ku stimulates POL-catalyzed ribonucleotide addition to a plasmid DSB end and promotes plasmid end joining by full-length Pseudomonas LigD. A series of incremental truncations from the C terminus of the 293-amino acid Ku polypeptide identifies Ku-(1–229) as sufficient for homodimerization and LigD stimulation. The slightly longer Ku-(1–253) homodimer forms stable complexes at both ends of linear plasmid DNA that protect the DSBs from digestion by 5′- and 3′-exonucleases.     

Analysis of DNA-dependent protein kinase-mediated DNA end joining by two-photon fluorescence cross-correlation spectroscopy

Nonhomologous end joining (NHEJ) is the primary mechanism by which mammalian cells repair DNA double-strand breaks (DSBs). Proteins known to play a role in NHEJ include the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the Ku 70/Ku 80 heterodimer (Ku), XRCC4, and DNA ligase IV. One of the main roles of the DNA-PKcs-Ku complex is to bring the ends of the DSB together in a process termed synapsis, prior to end joining. Synapsis results in the autophosphorylation of DNA-PKcs, which is required to make the DNA ends available for ligation. Here, we describe a novel assay using two-photon fluorescence cross-correlation spectroscopy that allows for the analysis of DNA synapsis and end joining in solution using purified proteins. We demonstrate that although autophosphorylation-defective DNA-PKcs does not support DNA ligase-mediated DNA end joining, like wild-type (WT) DNA-PKcs, it is capable of Ku-dependent DNA synapsis in solution. Moreover, we show that, in the presence of Ku, both WT DNA-PKcs and autophosphorylation-defective DNA-PKcs promote the formation of multiple, large multi-DNA complexes in solution, suggesting that, rather than align two opposing DNA ends, multiple DNA-PK molecules may serve to bring multiple DNA ends into the NHEJ complex.     

Involvement of polynucleotide kinase in a poly(ADP-ribose) polymerase-1-dependent DNA double-strand breaks rejoining pathway

Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genomic integrity. In mammalian cells, DSBs are preferentially repaired by the non-homologous end-joining pathway relying on DNA-PK activity, but other mechanisms may promote end-joining. We previously described a DSB repair pathway that requires synapsis of DNA ends by poly(ADP-ribose) polymerase-1 (PARP-1) and ligation by the XRCC1/DNA ligase III complex (XL). Here, the repair of non-ligatable DNA ends by this pathway was examined in human cell extracts. The phosphorylation of the 5'-terminal end was shown to represent a limiting step for the repair process. Polynucleotide kinase (hPNK) was identified as the 5'-DNA kinase associated with the PARP-1-dependent end-joining pathway because (i) hPNK was co-recruited to DNA ends together with PARP-1 and XL, (ii) ligation of 5'-OH terminal breaks was compromised in hPNK-depleted extracts and restored upon addition of recombinant hPNK, and (iii) recombinant hPNK was necessary for end-joining of 5'-OH terminal breaks reconstituted with the PARP-1/XL complex. Also, using an assay enabling us to follow the ligation kinetics of each strand of a DSB, we established that the two strands at the junction can be processed and joined independently, so that one strand can be ligated without a ligatable nick on the other strand at the DSB site. Taken together these results reveal functional parallels between the PARP-1 and DNA-PK-dependent end-joining processes.