Biochemical evidence for Ku-independent backup pathways of NHEJ - PubMed (original) (raw)

Biochemical evidence for Ku-independent backup pathways of NHEJ

Huichen Wang et al. Nucleic Acids Res. 2003.

Expression of concern in

Abstract

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.

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Figures

Figure 1

Figure 1

Levels and activity of Ku, as well as DNA end joining in WCEs of HeLa and M059-J cells. (A) Western blot showing the levels of Ku70, Ku80, DNA ligase IV, XRCC4 and DNA-PKcs in extracts of HeLa and M059-J cells. Fifty micrograms of extract were loaded per lane. Note the comparable abundance of Ku, DNA ligase IV and XRCC4 in the extracts of the two cell lines, as well as the absence of DNA-PKcs in extracts from M059-J cells. (B) EMSA with extracts of HeLa and M059-J cells. 0.2 ng of a 32 bp, 32P-labeled, double stranded DNA probe was incubated with different amounts of extract at 25°C for 30 min and loaded on a 6% polyacrylamide gel. The lower band indicates unbound probe, whereas the higher bands indicate DNA–protein complexes. Note the comparable DNA binding activity present in HeLa and M059-J cell extracts. (C) DNA end joining in extracts of HeLa and M059-J cells. Reactions were assembled with the indicated amount of extract and 0.25 µg plasmid substrate and incubated at 25°C for 1 h. Products were analyzed by electrophoresis in a 0.7% agarose gel run at 45 V (2 V/cm) for 5 h. Gels were stained in SYBR Gold and scanned in a FluorImager (Molecular Dynamics). Quantification was carried out using the ImageQuant software (Molecular Dynamics). The results were used to calculate the percent of plasmid migrating as a dimer and other higher order forms and is given at the bottom of the gel (% end joining). The migration distances of the input plasmid substrate (linear), as well as of dimers and other higher order forms (multimers) are indicated. Note the high level of DNA end-joining activity in the extracts of both cell lines.

Figure 2

Figure 2

Serum of polymyositis-scleroderma overlap syndrome patients inhibits in vitro DNA end joining. (A) Twenty micrograms of extract of either HeLa or M059-J cells were incubated in 16 µl of DNA end-joining reaction-mixture (without DNA and ATP) in which 2 µl of serum from OY patient were added, after appropriate dilution in phosphate buffered saline, to achieve the final levels in µl serum per reaction indicated on top of the gel, and were incubated at 25°C for 10 min. After this pre-incubation period, DNA and ATP were added in half of the reaction-mixture to assay for DNA end joining, whereas the remaining reaction-mixture was processed for EMSA (results not shown). Note the inhibition of DNA end joining with increasing serum concentration in reactions assembled with extracts of HeLa cells and the reduced inhibition in reactions assembled with extracts of M059-J cells. (B) Similar to (A), but for reactions assembled in the presence of identical amounts of NHS from a healthy individual. (C) Quantitative evaluation of the DNA end-joining results shown in (A) and (B) using ImageQuant. Results are shown normalized to the percent rejoining measured for each set of reactions when incubated in the absence of serum. Actual values for percent end joining are shown at the bottom of the gels in (A) and (B). Note the inhibition in DNA end joining with increasing amount of OY serum in the reaction, and the difference in the effect between M059-J and HeLa cells, particularly when considering the effect of NHS.

Figure 3

Figure 3

DNA end joining in extracts of HeLa and M059-J cells after depletion of Ku. (A) Two hundred microgram extracts of HeLa or M059-J cells (∼20 µl) were incubated with a 1:1 mixture of Sepharose A and G beads (20 µl) that had been pre-incubated overnight with 5 µl of OY serum, or NHS. The extract-beads mixture was incubated at 4°C for 1 h and beads were removed by centrifugation. The procedure was repeated a second time and extracts were analyzed by western blotting for the levels of Ku70. Similar results were obtained when Ku80 was assayed (not shown). Note the practically complete depletion of Ku in extracts treated with OY serum and the unchanged levels of the protein in extracts treated with NHS compared to those observed in untreated controls. The beads-fraction contains large amounts of Ku protein after treatment with OY serum, but no protein after treatment with NHS. (B) EMSA analysis under the conditions outlined in Figure 1 for extracts of HeLa and M059-J cells treated as described in (A). Note the absence of detectable DNA binding in extracts treated with OY serum. (C) DNA-PK activity in HeLa WCE and Ku-depleted HeLa WCE (HeLa WCE/–Ku). 1, 2.5, 5, 10 µg of cell extract was mixed in 20 µl DNA-PK reaction buffer with and without calf thymus DNA. Reactions were incubated at 30°C for 30 min. The DNA-PK activity was calculated as described in Materials and Methods. (D) DNA end joining in reactions assembled with untreated control extracts (20 µg), or equal amounts of extracts treated with either OY serum, or NHS. End-joining activity is indicated at the bottom of the gel as % end joining. Note the efficient end joining in Ku-depleted cell extracts of either HeLa or M059-J cells. (E) Twenty micrograms of extract of either HeLa WCE, Ku-depleted HeLa WCE, or Ku-depleted HeLa WCE supplemented with 50 ng purified recombinant Ku protein were incubated in 18 µl of DNA end-joining reaction-mixture (without DNA and ATP) with 0.1 µl of OY serum, or 0.1 µl of NHS at 25°C for 10 min. After this pre-incubation period, DNA and ATP were added to start the reaction. Note that OY serum does not inhibit the DNA end joining in Ku-depleted HeLa WCE, and that the inhibition can be rescued by recombinant Ku. (F) DNA end joining in reactions assembled with extracts of HeLa or M059-J cells (10 µg) and increasing amounts of purified Ku. Percent end joining is indicated at the bottom of the gel. Note the inhibitory effect of Ku that is less pronounced in reactions assembled with extracts of M059-J cells.

Figure 4

Figure 4

The role of Ku in the fidelity of DNA end joining, as well as in the joining of different types of DNA ends. (A) Reactions were assembled under standard conditions using normal or Ku-depleted extracts (20 µg) of either HeLa or M059-J cells and DNA end joining was allowed to take place at 25°C for 1 h (lanes marked –Sal I). After gel electrophoresis, products were purified and the fidelity of end joining assayed by re-digestion with SalI. The lanes marked +Sal I show the digested products. Percent- undigested plasmid was calculated by ImageQuant and the values are shown at the bottom of the gel. Note that products of the end joining reaction remain sensitive to SalI under all conditions examined (92% digestibility) suggesting end joining without sequence modification around the end. (B) Types of ends generated and recognition sequences of BamHI, SalI, PstI and SmaI. (C) DNA end joining in HeLa cell extracts under standard conditions using as substrate pSP65 plasmid, linearized with the indicated restriction endonucleases. Percent end joining is shown at the bottom of the gel. Note the reduced end joining with substrate prepared by digestion with BamHI, PstI and SmaI. (D) As in (C) but with extracts of M059-J cells. Note the increase in DNA end joining, as compared to HeLa cells, with substrate prepared by digestion with PstI. (E) As in (D) but with Ku-depleted M059-J cell extracts (using OY serum). Note that the end-bias of the joining reaction is significantly reduced when compared to HeLa cells.

Figure 5

Figure 5

Inhibition of DNA end joining by wortmannin requires Ku and can be reversed by short dsDNA oligonucleotides. (A) DNA end joining in reactions assembled with 20 µg extract of HeLa or M059-J cells in the presence or absence of 1 µM wortmannin. Percent end joining is given at the bottom of the gel. Reaction mixtures containing the cell extract, but no substrate DNA or ATP were incubated with wortmannin or DMSO (the solvent of wortmannin) at 25°C for 10 min. End joining was initiated by adding DNA and ATP and was allowed to take place at 25°C for 1h. Note the strong inhibition of DNA end joining by wortmannin in extracts of HeLa cells, and the lack of inhibition in extracts of M059-J cells. (B) Reactions were assembled with Ku-depleted extracts of M059-J cells (20 µg) in the presence or absence of wortmannin as outlined in (A). Two hundred nanograms of purified DNA-PKcs, or 100 ng of purified Ku were added to the indicated reactions starting from and including the pre-incubation period. Percent end joining is given at the bottom of the gel. Note that wortmannin induced inhibition requires Ku. See Figure 3D for results of reactions assembled with Ku, with or without wortmannin. (C) Sequence of the double stranded and single stranded oligonucleotides (32mers) used in the DNA end joining reactions shown in (D) and (E). (D) DNA end joining in reactions assembled under standard conditions with extracts (20 µg) of HeLa cells in the presence or absence of 1 µM wortmannin and the indicated amounts of the dsDNA shown in (C). Reaction mixtures containing extract but no ATP or substrate DNA were incubated with wortmannin or DMSO at 25°C for 10 min. Subsequently the indicated amount of dsDNA was added and the reaction was incubated at 25°C for an additional 10 min. Finally, substrate DNA (0.25 µg per reaction, ∼1.1 pmol) and ATP were added to initiate end joining. Percent end joining is given at the bottom of the gel. Note the lack of inhibition in the presence of dsDNA alone, and the reversion of the wortmannin effect by more than 0.5 pmol dsDNA. (E) As in (D) but with reactions supplemented with the indicated amounts of single-stranded DNA. Note the lack of effect on DNA end joining under these conditions, in the presence or absence of wortmannin.

Figure 6

Figure 6

A hypothetical model for the operation of D-NHEJ and B-NHEJ in chromatin-packed DNA. D-NHEJ: (A) neighboring chromatin loops of two chromosomes (Chr 1 and Chr 2) with attachments to the nuclear matrix are shown. In the absence of DNA DSBs, DNA-PKcs is drawn attached to the nuclear matrix and Ku (5–10-fold excess) distributed throughout the nucleus. (B) Exposure to IR induces a DSB in Chr 2 that is recognized by Ku. This recognition step and the availability of DNA-PKcs allow the cell to direct the rejoining of the DSB to the D-NHEJ pathway. (C) Within minutes after induction, interactions between Ku and DNA-PKcs, on the one hand, and chromatin remodeling, on the other hand, facilitate synapsing of the ends and joining by DNA ligase IV/XRCC4, a process also facilitated by as of yet uncharacterized factors. Optimal interaction of factors and processes involved in this step require the chromatin context to ensure the exceptional speed characterizing this pathway of end joining. (D) Although the rejoining process does not ensure restoration of the original sequence in the vicinity of the DSB, the extremely rapid processing favors synapsing of correct ends and suppresses thus the formation of exchange-type chromosome aberrations. Therefore, when chromosome integrity is the endpoint, D-NHEJ can be considered an error-free process. B-NHEJ: In the absence of Ku (intermediate results are expected in case of Ku haploinsufficiency), DSBs are induced by IR with similar efficiency, but are not directed to D-NHEJ despite the presence of DNA-PKcs. Under these conditions, DSBs are recognized and processed by as yet uncharacterized components of B-NHEJ that can also operate outside the chromatin context. Although B-NHEJ is capable of processing the great majority of IR-induced DNA DSBs, it does so with kinetics an order of magnitude slower than D-NHEJ, presumably because of inefficient synapsing of DNA ends. As a result the DNA ends remain open for long periods of time and can be partly degraded and/or interact with DNA DSBs in neighboring loops induced randomly, or through the replication of damaged DNA at a later time point, to form exchanges. Exchanges can also be formed through the interaction between two IR-induced breaks that are brought into proximity after irradiation by changes in chromatin conformation. (D) An exchange between loops from different chromosomes can lead to a reciprocal exchange (shown), or to the formation of a dicentrics chromosome and an acentric fragment (not shown).

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