Cyclin A overexpression induces chromosomal double-strand breaks in mammalian cells

Cyclin A is a major regulator in vertebrate cell cycle, associated with cyclin-dependent kinase (Cdk), and involved in S-phase progression and entry into mitosis. It has been known that cyclin A overexpression not only causes premature S-phase entry but also induces prolongation of S phase. Here we show that ectopic expression of cyclin A leads to extensive γ?H2AX focus formation, which is indicative of DNA double-strand breaks. Likewise, cyclin E, but not cyclin B1 and cyclin D1, also induced the γ?H2AX focus formation, suggesting that these DNA lesions may be induced via aberrant DNA replication process. Moreover, the γ?H2AX focus formation was suppressed by co-expressing p21Cip1/Waf1 or dominant-negative Cdk2 mutant, suggesting that aberrant cyclin A-Cdk2 activation induces the chromosomal double-strand breaks.     

Repair of double-strand breaks by homologous recombination in mismatch repair-defective mammalian cells

Chromosomal double-strand breaks (DSBs) stimulate homologous recombination by several orders of magnitude in mammalian cells, including murine embryonic stem (ES) cells, but the efficiency of recombination decreases as the heterology between the repair substrates increases (B. Elliott, C. Richardson, J. Winderbaum, J. A. Nickoloff, and M. Jasin, Mol. Cell. Biol. 18:93-101, 1998). We have now examined homologous recombination in mismatch repair (MMR)-defective ES cells to investigate both the frequency of recombination and the outcome of events. Using cells with a targeted mutation in the msh2 gene, we found that the barrier to recombination between diverged substrates is relaxed for both gene targeting and intrachromosomal recombination. Thus, substrates with 1.5% divergence are 10-fold more likely to undergo DSB-promoted recombination in Msh2(-/-) cells than in wild-type cells. Although mutant cells can repair DSBs efficiently, examination of gene conversion tracts in recombinants demonstrates that they cannot efficiently correct mismatched heteroduplex DNA (hDNA) that is formed adjacent to the DSB. As a result, >20-fold more of the recombinants derived from mutant cells have uncorrected tracts compared with recombinants from wild-type cells. The results indicate that gene conversion repair of DSBs in mammalian cells frequently involves mismatch correction of hDNA rather than double-strand gap formation. In cells with MMR defects, therefore, aberrant recombinational repair may be an additional mechanism that contributes to genomic instability and possibly tumorigenesis.     

Processing of clustered DNA damage generates additional double-strand breaks in mammalian cells post-irradiation

Clustered DNA damage sites, in which two or more lesions are formed within a few helical turns of the DNA after passage of a single radiation track, are signatures of DNA modifications induced by ionizing radiation in mammalian cells. Mutant hamster cells (xrs-5), deficient in non-homologous end joining (NHEJ), were irradiated at 37 degrees C to determine whether any additional double-strand breaks (DSBs) are formed during processing of gamma-radiation-induced DNA clustered damage sites. A class of non-DSB clustered DNA damage, corresponding to approximately 30% of the initial yield of DSBs, is converted into DSBs reflecting an artefact of preparation of genomic DNA for pulsed field gel electrophoresis. These clusters are removed within 4 min in both NHEJ-deficient and wild-type CHO cells. In xrs-5 cells, a proportion of non-DSB clustered DNA damage, representing approximately 10% of the total yield of non-DSB clustered DNA damage sites, are also converted into DSBs within approximately 30 min post-gamma but not post-alpha irradiation through cellular processing at 37 degrees C. That the majority of radiation-induced non-DSB clustered DNA damage sites are resistant to conversion into DSBs may be biologically significant at environmental levels of radiation exposure, as a non-DSB clustered damage site rather than a DSB, which only constitutes a minor proportion, is more likely to be induced in irradiated cells.     

Effect of double-strand breaks on homologous recombination in mammalian cells and extracts.

We examined the effect of double-strand breaks on homologous recombination between two plasmids in human cells and in nuclear extracts prepared from human and rodent cells. Two pSV2neo plasmids containing nonreverting, nonoverlapping deletions were cotransfected into cells or incubated with cell extracts. Generation of intact neo genes was monitored by the ability of the DNA to confer G418r to cells or Neor to bacteria. We show that double-strand breaks at the sites of the deletions enhanced recombination frequency, whereas breaks outside the neo gene had no effect. Examination of the plasmids obtained from experiments involving the cell extracts revealed that gene conversion events play an important role in the generation of plasmids containing intact neo genes. Studies with plasmids carrying multiple polymorphic genetic markers revealed that markers located within 1,000 base pairs could be readily coconverted. The frequency of coconversion decreased with increasing distance between the markers. The plasmids we constructed along with the in vitro system should permit a detailed analysis of homologous recombinational events mediated by mammalian enzymes.     

Dysfunctional mammalian telomeres join with DNA double-strand breaks

In addition to joining broken DNA strands, several non-homologous end-joining (NHEJ) proteins have a second seemingly antithetical role in constructing functional telomeres, the nucleoprotein structures at the termini of linear eukaryotic chromosomes that prevent joining between natural chromosome ends. Although NHEJ deficiency impairs double-strand break (DSB) repair, it also promotes inappropriate chromosomal end fusions that are observed microscopically as dicentric chromosomes with telomeric DNA sequence at points of joining. Here, we test the proposition that unprotected telomeres can fuse not only to other dysfunctional telomeres, but also to ends created by DSBs. Severe combined immunodeficiency (scid) is caused by a mutation in the catalytic subunit of DNA-dependent protein kinase (DNA-PK), an enzyme required for both efficient DSB repair and telomeric end-capping. Cells derived from wild-type, Trp53-/-, scid, and Trp53-/-/scid mice were exposed to gamma radiation to induce DSBs, and chromosomal aberrations were analyzed using a novel cytogenetic technique that can detect joining of a telomere to a DSB end. Telomere-DSB fusions were observed in both cell lines having the scid mutation, but not in wild-type nor Trp53-/- cells. Over a range of 25-340 cGy, half of the visible exchange-type chromosomal aberrations in Trp53-/-/scid cells involved telomere-DSB fusions. Our results demonstrate that unprotected telomeres are not only sensed as, but also acted upon, by the DNA repair machinery as if they were DSB ends. By opening a new pathway for misrepair, telomere-DSB fusion decreases the overall fidelity of DSB repair. The high frequency of these events in scid cells indicates telomere dysfunction makes a strong, and previously unsuspected, contribution to the characteristic radiation sensitivity associated with DNA-PK deficiency.     

Capture of DNA sequences at double-strand breaks in mammalian chromosomes

To study double-strand break (DSB)-induced mutations in mammalian chromosomes, we transfected thymidine kinase (tk)-deficient mouse fibroblasts with a DNA substrate containing a recognition site for yeast endonuclease I-SceI embedded within a functional tk gene. To introduce a genomic DSB, cells were electroporated with a plasmid expressing endonuclease I-SceI, and clones that had lost tk function were selected. Among 253 clones analyzed, 78% displayed small deletions or insertions of several nucleotides at the DSB site. Surprisingly, approximately 8% of recovered mutations involved the capture of one or more DNA fragments. Among 21 clones that had captured DNA, 10 harbored a specific segment of the I-SceI expression plasmid mapping between two replication origins on the plasmid. Four clones had captured a long terminal repeat sequence from an intracisternal A particle (an endogenous retrovirus-like sequence) and one had captured what appears to be a cDNA copy of a moderately repetitive B2 sequence. Additional clones displayed segments of the tk gene and/or microsatellite sequences copied into the DSB. This first systematic study of DNA capture at DSBs in a mammalian genome suggests that DSB repair may play a considerable role in the evolution of eukaryotic genomes.     

Evidence that extrachromosomal double-strand break repair can be coupled to the repair of chromosomal double-strand breaks in mammalian cells

Transfected linear DNA molecules are substrates for double-strand break (DSB) repair in mammalian cells. The DSB repair process can involve recombination between the transfected DNA molecules, between the transfected molecules and chromosomal DNA, or both. In order to determine whether these different types of repair events are linked, we devised assays enabling us to follow the fate of linear extrachromosomal DNA molecules involved in both interplasmid and chromosome-plasmid recombination, in the presence or absence of a pre-defined chromosomal DSB. Plasmid-based vectors were designed that could either recombine via interplasmid recombination or chromosome-plasmid recombination to produce a functional beta-galactosidase (betagal) fusion gene. By measuring the frequency of betagal+ cells at 36 h post-transfection versus the frequency of betagal+ clones after 14 days, we found that the number of cells containing extrachromosomal recombinant DNA molecules at 36 h (i.e., betagal+), either through interplasmid or chromosome-plasmid recombination, was nearly the same as the number of cells integrating these recombinant molecules. Furthermore, when a predefined DSB was created at a chromosomal site, the extrachromosomal recombinant DNA molecules were shown to integrate preferentially at that site by Southern and fiber-FISH (fluorescence in situ hybridization) analysis. Together these data indicate that the initial recombination event can potentiate or commit extrachromosomal DNA to integration in the genome at the site of a chromosomal DSB. The efficiency at which extrachromosomal recombinant molecules are used as substrates in chromosomal DSB repair suggests extrachromosomal DSB repair can be coupled to the repair of chromosomal DSBs in mammalian cells.     

Repair at single targeted DNA double-strand breaks in pluripotent and differentiated human cells

Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical, environmental, and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB) in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). For the most part, previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation, which are highly nonphysiologic, or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair, we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs, compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus, the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny.     

A non-radioactive, PFGE-based assay for low levels of DNA double-strand breaks in mammalian cells

A PFGE method was adapted to measure DNA double-strand breaks (DSBs) in mammalian cells after low (0-25 Gy) doses of ionising radiation. Instead of radionuclide incorporation, DNA staining in the gel by SYBR-Gold was used, which lowered the background of DNA damage and could be applied to non-cycling cells. DSB level was defined as a product of a fraction of DNA released to the gel (FR) and a number of DNA fragments in the gel (DNA(fragm)) and expressed as a percentage above control value. The slope of the dose-response curve was two-fold higher compared to that with FR alone as DSB level indicator (31.4 versus 15.6% per Gy). Two alternative ways were proposed to determine the total amount of DNA, used for FR calculation: measurement of DNA content in a plug not subjected to electrophoresis, with the use of Pico-Green, or estimation of DNA released to the gel from a plug irradiated with 600 Gy of gamma-rays. The limit of DSB detection was 0.25 Gy for human G1-lymphocytes and 0.5-1 Gy for asynchronous cultures of human glioma M059 K and J or mouse lymphoma L5178Y-R and -S cells. Specificity of our PFGE assay to DSB was confirmed by the fact that no damage was detected after treatment of the cells with H(2)O(2), an inducer of single-strand DNA breaks (SSBs). On the contrary, the H(2)O(2) inflicted damage was detected by neutral comet assay, attaining 160% above control (equivalent to 2.5 Gy of X-radiation). DSB rejoining, measured in cells after X-irradiation with a dose of 10 Gy, generally proceeded faster than that measured previously after higher (30-50 Gy) doses of ionising radiation. Clearly seen were defects in DSB rejoining in radiosensitive M059 J and L5178Y-S cells compared to their radioresistant counterparts, M059 K and L5178Y-R. In some cell lines, a secondary post-irradiation increase in DSB levels was observed. The possibility is considered that these additional DSBs may accumulate during processing of non-DSB clustered DNA damage or/and represent early apoptotic events.     

Multiple pathways for repair of DNA double-strand breaks in mammalian chromosomes

To study repair of DNA double-strand breaks (DSBs) in mammalian chromosomes, we designed DNA substrates containing a thymidine kinase (TK) gene disrupted by the 18-bp recognition site for yeast endonuclease I-SceI. Some substrates also contained a second defective TK gene sequence to serve as a genetic donor in recombinational repair. A genomic DSB was induced by introducing endonuclease I-SceI into cells containing a stably integrated DNA substrate. DSB repair was monitored by selection for TK-positive segregants. We observed that intrachromosomal DSB repair is accomplished with nearly equal efficiencies in either the presence or absence of a homologous donor sequence. DSB repair is achieved by nonhomologous end-joining or homologous recombination, but rarely by nonconservative single-strand annealing. Repair of a chromosomal DSB by homologous recombination occurs mainly by gene conversion and appears to require a donor sequence greater than a few hundred base pairs in length. Nonhomologous end-joining events typically involve loss of very few nucleotides, and some events are associated with gene amplification at the repaired locus. Additional studies revealed that precise religation of DNA ends with no other concomitant sequence alteration is a viable mode for repair of DSBs in a mammalian genome.     

Studies on mammalian mutants defective in rejoining double-strand breaks in DNA

Mutants with defects in the rejoining of DNA double-strand breaks (dsbs) have been identified and characterised from E. coli and the yeast, Saccharomyces cerevisiae. More recently, 3 mammalian cell mutants with defective dsb rejoining have also been described. These mutants are xrs, XR-1 and L5178Y/S, and they are derived from at least two distinct complementation groups. The aim of this article is to review the current status of the studies with these mammalian cell mutants which are defective in dsb rejoining and, in particular, to compare their properties with those mutants identified from lower organisms. Possible mechanistic differences in the process of dsb rejoining between prokaryotes and lower and higher eukaryotes are discussed. All the mammalian mutants defective in dsb rejoining, are sensitive primarily to ionising radiation with little cross-sensitivity to UV-radiation. This is similar to the rad52 mutants of S. cerevisiae but contrasts to the majority of the E. coli mutants with defective dsb rejoining. Where studied, the mammalian cell mutants show enhanced resistance to ionizing radiation in late S/G2 phase, which, in one case, correlates with an enhanced ability to rejoin dsbs. This, together with other evidence, suggests that two mechanisms of dsb rejoining may exist in higher eukaryotes, one which operates uniquely in S/G2 phase and a second mechanism operating throughout the cell cycle and dependent upon the xrs and XR-1 gene products (although whether the xrs and XR-1 dependent pathways are distinct cannot at present be ascertained). Since duplicate homologues will be present in late S/G2 phase cells, this pathway may involve a recombinational mechanism. The xrs-dependent pathway might involve illegitimate recombination, but the xrs mutants do not appear to have a major defect in homologous recombination (involving plasmid DNA) and in this respect are distinct from rad52 mutants.     

Homologous recombination protects mammalian cells from replication-associated DNA double-strand breaks arising in response to methyl methanesulfonate

DNA-methylating agents of the S_N2 type target DNA mostly at ring nitrogens, producing predominantly N-methylated purines. These adducts are repaired by base excision repair (BER). Since defects in BER cause accumulation of DNA single-strand breaks (SSBs) and sensitize cells to the agents, it has been suggested that some of the lesions on their own or BER intermediates (e.g. apurinic sites) are cytotoxic, blocking DNA replication and inducing replication-mediated DNA double-strand breaks (DSBs). Here, we addressed the question of whether homologous recombination (HR) or non-homologous end-joining (NHEJ) or both are involved in the repair of DSBs formed following treatment of cells with methyl methanesulfonate (MMS). We show that HR defective cells (BRCA2, Rad51D and XRCC3 mutants) are dramatically more sensitive to MMS-induced DNA damage as measured by colony formation, apoptosis and chromosomal aberrations, while NHEJ defective cells (Ku80 and DNA-PK_CS mutants) are only mildly sensitive to the killing, apoptosis-inducing and clastogenic effects of MMS. On the other hand, the HR mutants were almost completely refractory to the formation of sister chromatid exchanges (SCEs) following MMS treatment. Since DSBs are expected to be formed specifically in the S-phase, we assessed the formation and kinetics of repair of DSBs by γH2AX quantification in a cell cycle specific manner. In the cytotoxic dose range of MMS a significant amount of γH2AX foci was induced in S, but not G1- and G2-phase cells. A major fraction of γH2AX foci colocalized with 53BP1 and phosphorylated ATM, indicating they are representative of DSBs. DSB formation following MMS treatment was also demonstrated by the neutral comet assay. Repair kinetics revealed that HR mutants exhibit a significant delay in DSB repair, while NHEJ mutants completed S-phase specific DSB repair with a kinetic similar to the wildtype. Moreover, DNA-PKcs inhibition in HR mutants did not affect the repair kinetics after MMS treatment. Overall, the data indicate that agents producing N-alkylpurines in the DNA induce replication-dependent DSBs. Further, they show that HR is the major pathway of protection of cells against DSB formation, killing and genotoxicity following S_N2-alkylating agents.     

The joining of non-complementary DNA double-strand breaks by mammalian extracts.

We have developed a high efficiency system in which mammalian extracts join DNA double-strand breaks with non-complementary termini. This system has been used to obtain a large number of junction sequences from a range of different break-end combinations, allowing the elucidation of the joining mechanisms. Using an extract of calf thymus it was found that the major mechanism of joining was by blunt-end ligation following removal or fill-in of the single-stranded bases. However, some break-end combinations were joined through an efficient mechanism using short repeat sequences and we have succeeded in separating this mechanism from blunt-end joining by the biochemical fractionation of extracts. Characterization of activities and sequence data in an extensively purified fraction that will join ends by the repeat mechanism led to a model where joining is initiated by 3' strand invasion followed by pairing to short repeat sequences close to the break site. Thus the joining of double-strand breaks by mammalian extracts is achieved by several mechanisms and this system will allow the purification of the factors involved in each by the judicial choice of the non-complementary ends used in the assay.     

Translesion DNA synthesis-assisted non-homologous end-joining of complex double-strand breaks prevents loss of DNA sequences in mammalian cells

Double strand breaks (DSB) are severe DNA lesions, and if not properly repaired, may lead to cell death or cancer. While there is considerable data on the repair of simple DSB (sDSB) by non-homologous end-joining (NHEJ), little is known about the repair of complex DSBs (cDSB), namely breaks with a nearby modification, which precludes ligation without prior processing. To study the mechanism of cDSB repair we developed a plasmid-based shuttle assay for the repair of a defined site-specific cDSB in cultured mammalian cells. Using this assay we found that repair efficiency and accuracy of a cDSB with an abasic site in a 5′ overhang was reduced compared with a sDSB. Translesion DNA synthesis (TLS) across the abasic site located at the break prevented loss of DNA sequences, but was highly mutagenic also at the template base next to the abasic site. Similar to sDSB repair, cDSB repair was totally dependent on XrccIV, and altered in the absence of Ku80. In contrast, Artemis appears to be specifically involved in cDSB repair. These results may indicate that mammalian cells have a damage control strategy, whereby severe deletions are prevented at the expense of the less deleterious point mutations during NHEJ.     

Influence of chromatin structure on induction of double-strand breaks in mammalian cells irradiated with DNA-incorporated 125I

In this study the induction of double-strand breaks (DSBs) was investigated in Chinese hamster V79-379A cells irradiated with the Auger-electron emitter (125)I incorporated into DNA. The role of chromatin organization was studied by pulse-labeling synchronized cells with (125)IdU before decay accumulation in early or late S phase. Pulsed-field gel electrophoresis and fragment-size analysis were used to quantify the distribution of DNA fragments in irradiated intact cells and naked DNA as well as in DNA from asynchronously labeled cultures in a different scavenging environment. The results show that in intact cells, after accumulation of decays at -70 degrees C in the presence of 10% DMSO, almost four times more DSBs were induced in late S phase compared with early S phase and the fragment distribution was clearly non-random with an excess of fragments <0.2 Mbp. The DSB yield was 0.6 DSB/cell and decay for cells irradiated in early S phase and 2.3 DSBs/cell and decay for cells irradiated in late S phase. When similar experiments were performed on naked genomic DNA or intact cells irradiated with gamma rays, the difference in yield was not as prominent. These data imply a role of chromatin organization in the induction of DSBs by DNA-incorporated (125)I. In summary, the results presented here suggest that the yield of DSBs as well as the fragment distribution induced by (125)IdU decay may vary significantly depending on the chromatin organization during S phase and the labeling procedure used.     

Illegitimate recombination induced by DNA double-strand breaks in a mammalian chromosome.

We examined DNA double-strand-break-induced mutations in the endogenous adenine phosphoribosyl-transferase (APRT) gene in cultured Chinese hamster ovary cells after exposure to restriction endonucleases. PvuII, EcoRV, and StuI, all of which produce blunt-end DNA double-strand breaks, were electroporated into CHO-AT3-2 cells hemizygous at the APRT locus. Colonies of viable cells containing mutations at APRT were expanded, and the mutations that occurred during break repair were analyzed at the DNA sequence level. Restriction enzyme-induced mutations consisted of small deletions of 1 to 36 bp, insertions, and combinations of insertions and deletions at the cleavage sites. Most of the small deletions involved overlaps of one to four complementary bases at the recombination junctions. Southern blot analysis revealed more complex mutations, suggesting translocation, inversion, or insertion of larger chromosomal fragments. These results indicate that blunt-end DNA double-strand breaks can induce illegitimate (nonhomologous) recombination in mammalian chromosomes and that they play an important role in mutagenesis.     

H2AX phosphorylation at the sites of DNA double-strand breaks in cultivated mammalian cells and tissues

A sequence variant of histone H2A called H2AX is one of the key components of chromatin involved in DNA damage response induced by different genotoxic stresses. Phosphorylated H2AX (γH2AX) is rapidly concentrated in chromatin domains around DNA double-strand breaks (DSBs) after the action of ionizing radiation or chemical agents and at stalled replication forks during replication stress. γH2AX foci could be easily detected in cell nuclei using immunofluorescence microscopy that allows to use γH2AX as a quantitative marker of DSBs in various applications. H2AX is phosphorylated in situ by ATM, ATR, and DNA-PK kinases that have distinct roles in different pathways of DSB repair. The γH2AX serves as a docking site for the accumulation of DNA repair proteins, and after rejoining of DSBs, it is released from chromatin. The molecular mechanism of γH2AX dephosphorylation is not clear. It is complicated and requires the activity of different proteins including phosphatases and chromatin-remodeling complexes. In this review, we summarize recently published data concerning the mechanisms and kinetics of γH2AX loss in normal cells and tissues as well as in those deficient in ATM, DNA-PK, and DSB repair proteins activity. The results of the latest scientific research of the low-dose irradiation phenomenon are presented including the bystander effect and the adaptive response estimated by γH2AX detection in cells and tissues.     

An xrcc4 defect or Wortmannin stimulates homologous recombination specifically induced by double-strand breaks in mammalian cells

Non-homologous end joining (NHEJ) and homologous recombination (HR) are two alternative/competitor pathways for the repair of DNA double-strand breaks (DSBs). To gain further insights into the regulation of DSB repair, we detail here the different HR pathways affected by (i) the inactivation of DNA-PK activity, by treatment with Wortmannin, and (ii) a mutation in the xrcc4 gene, involved in a late NHEJ step, using the XR-1 cell line. Here we have analyzed not only the impact of NHEJ inactivation on recombination induced by a single DSB targeted to the recombination substrate (using I-SceI endonuclease) but also on γ-ray- and UV-C-induced and spontaneous recombination and finally on Rad51 foci formation, i.e. on the assembly of the homologous recombination complex, at the molecular level. The results presented here show that in contrast to embryonic stem cells, the xrcc4 mutation strongly stimulates I-SceI-induced HR in adult hamster cells. More precisely, we show here that both single strand annealing and gene conversion are stimulated. In contrast, Wortmannin does not affect I-SceI-induced HR. In addition, γ-ray-induced recombination is stimulated by both xrcc4 mutation and Wortmannin treatment in an epistatic-like manner. In contrast, neither spontaneous nor UV-C-induced recombination was affected by xrcc4 mutation, showing that the channeling from NHEJ to HR is specific to DSBs. Finally, we show here that xrcc4 mutation or Wortmannin treatment results in a stimulation of Rad51 foci assembly, thus that a late NHEJ step is able to affect Rad51 recombination complex assembly. The present data suggest a model according to which NHEJ and HR do not simply compete for DSB repair but can act sequentially: a defect in a late NHEJ step is not a dead end and can make DSB available for subsequent Rad51 recombination complex assembly.     

Asymmetric field inversion gel electrophoresis: a new method for detecting DNA double-strand breaks in mammalian cells

A new method is described for detecting DNA double-strand breaks (DSBs) that utilizes asymmetric field inversion gel electrophoresis (AFIGE). DNA purified from cells in agarose plugs is subjected to AFIGE and DNA breakage quantitated by the fraction of DNA released from the plug. To test the specificity of the method for DNA DSBs, purified DNA in agarose plugs was treated for increasing times with restriction endonuclease, XhoI. After an initial time period, the fraction of DNA released increased in direct proportion to time. This correlates with the expected response for a randomly broken DNA molecule. In contrast, treatment with the single-strand breaking agent, hydrogen peroxide, over a 1000-fold range produced no release of DNA from the plug. Thus the assay appears to be specific for DNA DSBs and was used to measure DNA breaks induced by gamma radiation. Purified DNA, irradiated in agarose plugs, exhibited a log-linear dose response up to doses that release greater than 90% DNA from the plug. When live cells were irradiated in agarose, a similar linear dose response was observed up to 40 Gy and a significant signal as low as 2.5 Gy. Also in live cells, a threefold lower percentage of DNA was released from the plug over the same dose range. However, less DNA per gray is released at doses above 40 Gy and may reflect a crosslinking effect produced by the irradiation of DNA in live cells. DNA which was "pulse-labeled" was used to test the effect of DNA replication on the ability of AFIGE to detect DNA DSBs. Replicating DNA irradiated in the cell or after purification exhibited a reduced rate of release from the plug per dose of irradiation. Overall, the above results indicate that AFIGE is a sensitive method for detecting DSBs in DNA.