Base excision repair defects invoke hypersensitivity to PARP inhibition.

UNLABELLED: PARP-1 is important for the recognition of both endogenous and exogenous DNA damage, and binds to DNA strand breaks including intermediates of base excision repair (BER). Once DNA-bound, PARP-1 becomes catalytically activated synthesizing PAR polymers onto itself and other repair factors (PARylation). As a result, BER repair proteins such as XRCC1 and DNA polymerase ß (pol ß) are more efficiently and rapidly recruited to sites of DNA damage. In the presence of an inhibitor of PARP activity (PARPi), PARP-1 binds to sites of DNA damage, but PARylation is prevented. BER enzyme recruitment is hindered, but binding of PARP-1 to DNA is stabilized, impeding DNA repair and leading to double-strand DNA breaks (DSB). Deficiencies in pol ß(-/-) and Xrcc1(-/-) cells resulted in hypersensitivity to the PARP inhibitor 4-AN and reexpression of pol ß or XRCC1, in these contexts, reversed the 4-AN hypersensitivity phenotype. BER deficiencies also showed evidence of replication defects that lead to DSB-induced apoptosis upon PARPi treatment. Finally, the clinically relevant PARP inhibitors olaparib and veliparib also exhibited hypersensitivity in both pol ß(-/-) and Xrcc1(-/-) BER-deficient cells. These results reveal heightened sensitivity to PARPi as a function of BER deficiency. IMPLICATIONS: BER deficiency represents a new therapeutic opportunity to enhance PARPi efficacy.

Interaction between DNA Polymerase ß and BRCA1.

The breast cancer 1 (BRCA1) protein is a tumor suppressor playing roles in DNA repair and cell cycle regulation. Studies of DNA repair functions of BRCA1 have focused on double-strand break (DSB) repair pathways and have recently included base excision repair (BER). However, the function of BRCA1 in BER is not well defined. Here, we examined a BRCA1 role in BER, first in relation to alkylating agent (MMS) treatment of cells and the BER enzyme DNA polymerase ß (pol ß). MMS treatment of BRCA1 negative human ovarian and chicken DT40 cells revealed hypersensitivity, and the combined gene deletion of BRCA1 and pol ß in DT40 cells was consistent with these factors acting in the same repair pathway, possibly BER. Using cell extracts and purified proteins, BRCA1 and pol ß were found to interact in immunoprecipitation assays, yet in vivo and in vitro assays for a BER role of BRCA1 were negative. An alternate approach with the human cells of immunofluorescence imaging and laser-induced DNA damage revealed negligible BRCA1 recruitment during the first 60 s after irradiation, the period typical of recruitment of pol ß and other BER factors. Instead, 15 min after irradiation, BRCA1 recruitment was strong and there was γ-H2AX co-localization, consistent with DSBs and repair. The rapid recruitment of pol ß was similar in BRCA1 positive and negative cells. However, a fraction of pol ß initially recruited remained associated with damage sites much longer in BRCA1 positive than negative cells. Interestingly, pol ß expression was required for BRCA1 recruitment, suggesting a partnership between these repair factors in DSB repair.

Preventing oxidation of cellular XRCC1 affects PARP-mediated DNA damage responses.

Poly(ADP-ribose) polymerase-1 (PARP-1) binds intermediates of base excision repair (BER) and becomes activated for poly(ADP-ribose) (PAR) synthesis. PAR mediates recruitment and functions of the key BER factors XRCC1 and DNA polymerase ß (pol ß) that in turn regulate PAR. Yet, the molecular mechanism and implications of coordination between XRCC1 and pol ß in regulating the level of PAR are poorly understood. A complex of PARP-1, XRCC1 and pol ß is found in vivo, and it is known that pol ß and XRCC1 interact through a redox-sensitive binding interface in the N-terminal domain of XRCC1. We confirmed here that both oxidized and reduced forms of XRCC1 are present in mouse fibroblasts. To further understand the importance of the C12-C20 oxidized form of XRCC1 and the interaction with pol ß, we characterized cell lines representing stable transfectants in Xrcc1(-/-) mouse fibroblasts of wild-type XRCC1 and two mutants of XRCC1, a novel reduced form with the C12-C20 disulfide bond blocked (C12A) and a reference mutant that is unable to bind pol ß (V88R). XRCC1-deficient mouse fibroblasts are extremely hypersensitive to methyl methanesulfonate (MMS), and transfected wild-type and C12A mutant XRCC1 proteins similarly reversed MMS hypersensitivity. However, after MMS exposure the cellular PAR level was found to increase to a much greater extent in cells expressing the C12A mutant than in cells expressing wild-type XRCC1. PARP inhibition resulted in very strong MMS sensitization in cells expressing wild-type XRCC1, but this sensitization was much less in cells expressing the C12A mutant. The results suggest a role for the oxidized form of XRCC1 in the interaction with pol ß in (1) controlling the PAR level after MMS exposure and (2) enabling the extreme cytotoxicity of PARP inhibition during the MMS DNA damage response.

Hyperactivation of PARP triggers nonhomologous end-joining in repair-deficient mouse fibroblasts.

Regulation of poly(ADP-ribose) (PAR) synthesis and turnover is critical to determining cell fate after genotoxic stress. Hyperactivation of PAR synthesis by poly(ADP-ribose) polymerase-1 (PARP-1) occurs when cells deficient in DNA repair are exposed to genotoxic agents; however, the function of this hyperactivation has not been adequately explained. Here, we examine PAR synthesis in mouse fibroblasts deficient in the base excision repair enzyme DNA polymerase ß (pol ß). The extent and duration of PARP-1 activation was measured after exposure to either the DNA alkylating agent, methyl methanesulfonate (MMS), or to low energy laser-induced DNA damage. There was strong DNA damage-induced hyperactivation of PARP-1 in pol ß nullcells, but not in wild-type cells. In the case of MMS treatment, PAR synthesis did not lead to cell death in the pol ß null cells, but instead resulted in increased PARylation of the nonhomologous end-joining (NHEJ) protein Ku70 and increased association of Ku70 with PARP-1. Inhibition of the NHEJ factor DNA-PK, under conditions of MMS-induced PARP-1 hyperactivation, enhanced necrotic cell death. These data suggest that PARP-1 hyperactivation is a protective mechanism triggering the classical-NHEJ DNA repair pathway when the primary alkylated base damage repair pathway is compromised.

HMGN1 protein regulates poly(ADP-ribose) polymerase-1 (PARP-1) self-PARylation in mouse fibroblasts.

In mammalian cells, the nucleosome-binding protein HMGN1 (high mobility group N1) affects the structure and function of chromatin and plays a role in repair of damaged DNA. HMGN1 affects the interaction of DNA repair factors with chromatin and their access to damaged DNA; however, not all of the repair factors affected have been identified. Here, we report that HMGN1 affects the self-poly(ADP-ribosyl)ation (i.e., PARylation) of poly(ADP-ribose) polymerase-1 (PARP-1), a multifunctional and abundant nuclear enzyme known to recognize DNA lesions and promote chromatin remodeling, DNA repair, and other nucleic acid transactions. The catalytic activity of PARP-1 is activated by DNA with a strand break, and this results in self-PARylation and PARylation of other chromatin proteins. Using cells obtained from Hmgn1(-/-) and Hmgn1(+/+) littermate mice, we find that in untreated cells, loss of HMGN1 protein reduces PARP-1 self-PARylation. A similar result was obtained after MMS treatment of these cells. In imaging experiments after low energy laser-induced DNA damage, less PARylation at lesion sites was observed in Hmgn1(-/-) than in Hmgn1(+/+) cells. The HMGN1 regulation of PARP-1 activity could be mediated by direct protein-protein interaction as HMGN1 and PARP-1 were found to interact in binding assays. Purified HMGN1 was able to stimulate self-PARylation of purified PARP-1, and in experiments with cell extracts, self-PARylation was greater in Hmgn1(+/+) than in Hmgn1(-/-) extract. The results suggest a regulatory role for HMGN1 in PARP-1 activation.

Increased PARP-1 association with DNA in alkylation damaged, PARP-inhibited mouse fibroblasts.

Treatment of base excision repair-proficient mouse fibroblasts with the DNA alkylating agent methyl methanesulfonate (MMS) and a small molecule inhibitor of PARP-1 results in a striking cell killing phenotype, as previously reported. Earlier studies showed that the mechanism of cell death is apoptosis and requires DNA replication, expression of PARP-1, and an intact S-phase checkpoint cell signaling system. It is proposed that activity-inhibited PARP-1 becomes immobilized at DNA repair intermediates, and that this blocks DNA repair and interferes with DNA replication, eventually promoting an S-phase checkpoint and G(2)-M block. Here we report studies designed to evaluate the prediction that inhibited PARP-1 remains DNA associated in cells undergoing repair of alkylation-induced damage. Using chromatin immunoprecipitation with anti-PARP-1 antibody and qPCR for DNA quantification, a higher level of DNA was found associated with PARP-1 in cells treated with MMS plus PARP inhibitor than in cells without inhibitor treatment. These results have implications for explaining the extreme hypersensitivity phenotype after combination treatment with MMS and a PARP inhibitor.

DNA polymerases beta and lambda mediate overlapping and independent roles in base excision repair in mouse embryonic fibroblasts.

Base excision repair (BER) is a DNA repair pathway designed to correct small base lesions in genomic DNA. While DNA polymerase beta (pol beta) is known to be the main polymerase in the BER pathway, various studies have implicated other DNA polymerases in back-up roles. One such polymerase, DNA polymerase lambda (pol lambda), was shown to be important in BER of oxidative DNA damage. To further explore roles of the X-family DNA polymerases lambda and beta in BER, we prepared a mouse embryonic fibroblast cell line with deletions in the genes for both pol beta and pol lambda. Neutral red viability assays demonstrated that pol lambda and pol beta double null cells were hypersensitive to alkylating and oxidizing DNA damaging agents. In vitro BER assays revealed a modest contribution of pol lambda to single-nucleotide BER of base lesions. Additionally, using co-immunoprecipitation experiments with purified enzymes and whole cell extracts, we found that both pol lambda and pol beta interact with the upstream DNA glycosylases for repair of alkylated and oxidized DNA bases. Such interactions could be important in coordinating roles of these polymerases during BER.

Base excision repair and design of small molecule inhibitors of human DNA polymerase ß.

Base excision repair (BER) can protect a cell after endogenous or exogenous genotoxic stress, and a deficiency in BER can render a cell hypersensitive to stress-induced apoptotic and necrotic cell death, mutagenesis, and chromosomal rearrangements. However, understanding of the mammalian BER system is not yet complete as it is extraordinarily complex and has many back-up processes that complement a deficiency in any one step. Due of this lack of information, we are unable to make accurate predictions on therapeutic approaches targeting BER. A deeper understanding of BER will eventually allow us to conduct more meaningful clinical interventions. In this review, we will cover historical and recent information on mammalian BER and DNA polymerase ß and discuss approaches toward development and use of small molecule inhibitors to manipulate BER. With apologies to others, we will emphasize results obtained in our laboratory and those of our collaborators.

PARP inhibition during alkylation-induced genotoxic stress signals a cell cycle checkpoint response mediated by ATM.

By limiting cell cycle progression following detection of DNA damage, checkpoints are critical for cell survival and genome stability. Methylated DNA damage, when combined with inhibition of PARP activity, results in an ATR-dependent S phase delay of the cell cycle. Here, we demonstrate that another checkpoint kinase, ATM, also is involved in the DNA damage response following treatment with a sub-lethal concentration of MMS combined with the PARP inhibitor 4-AN. Both ATM and PARP activities are important for moderating cellular sensitivity to MMS. Loss of ATM activity, or that of its downstream effector Chk2, limited the duration of the S phase delay. The combination of MMS and 4-AN resulted in ATM and Chk2 phosphorylation and the time course of phosphorylation for both kinases correlated with the S phase delay. Chk2 phosphorylation was reduced in the absence of ATM activity. The Chk2 phosphorylation that remained in the absence of ATM appeared to be dependent on ATR and DNA-PK. The results demonstrate that, following initiation of base excision repair and inhibition of PARP activity, ATM activation is critical for preventing the cell from progressing through S phase, and for protection against MMS-induced cytotoxicity.

Interaction between PARP-1 and ATR in mouse fibroblasts is blocked by PARP inhibition.

Inhibition of PARP activity results in extreme sensitization to MMS-induced cell killing in cultured mouse fibroblasts. In these MMS-treated cells, PARP inhibition is accompanied by an accumulation of S-phase cells that requires signaling by the checkpoint kinase ATR [J.K. Horton, D.F. Stefanick, J.M. Naron, P.S. Kedar, S.H. Wilson, Poly(ADP-ribose) polymerase activity prevents signaling pathways for cell cycle arrest following DNA methylating agent exposure, J. Biol. Chem. 280 (2005) 15773-15785]. Here, we examined mouse fibroblast extracts for formation of a complex that may reflect association between the damage responsive proteins PARP-1 and ATR. Co-immunoprecipitation of PARP-1 and ATR was observed in extracts prepared from MMS-treated cells, but not under conditions of PARP inhibition. Further, our experiments demonstrated PAR-adduction of ATR in extracts from control and MMS-treated cells. An interaction between purified ATR and PARP-1 was similarly demonstrated, suggesting that the observed co-immunoprecipitation of ATR and PARP-1 from cell extracts may be due to a direct interaction between the two enzymes. In addition, purified recombinant ATR is a substrate for poly(ADP-ribosyl)ation by PARP-1, and poly(ADP-ribose) adduction of PARP-1 and ATR resulted in an increase in PARP-1 and ATR co-immunoprecipitation.

HMGB1 is a cofactor in mammalian base excision repair.

Deoxyribose phosphate (dRP) removal by DNA polymerase beta (Pol beta) is a pivotal step in base excision repair (BER). To identify BER cofactors, especially those with dRP lyase activity, we used a Pol beta null cell extract and BER intermediate as bait for sodium borohydride crosslinking. Mass spectrometry identified the high-mobility group box 1 protein (HMGB1) as specifically interacting with the BER intermediate. Purified HMGB1 was found to have weak dRP lyase activity and to stimulate AP endonuclease and FEN1 activities on BER substrates. Coimmunoprecipitation experiments revealed interactions of HMGB1 with known BER enzymes, and GFP-tagged HMGB1 was found to accumulate at sites of oxidative DNA damage in living cells. HMGB1(-/-) mouse cells were slightly more resistant to MMS than wild-type cells, probably due to the production of fewer strand-break BER intermediates. The results suggest HMGB1 is a BER cofactor capable of modulating BER capacity in cells.

Coordination of steps in single-nucleotide base excision repair mediated by apurinic/apyrimidinic endonuclease 1 and DNA polymerase beta.

The individual steps in single-nucleotide base excision repair (SN-BER) are coordinated to enable efficient repair without accumulation of cytotoxic DNA intermediates. The DNA transactions and various proteins involved in SN-BER of abasic sites are well known in mammalian systems. Yet, despite a wealth of information on SN-BER, the mechanism of step-by-step coordination is poorly understood. In this study we conducted experiments toward understanding step-by-step coordination during BER by comparing DNA binding specificities of two major human SN-BER enzymes, apurinic/aprymidinic endonuclease 1 (APE) and DNA polymerase beta (Pol beta). It is known that these enzymes do not form a stable complex in solution. For each enzyme, we found that DNA binding specificity appeared sufficient to explain the sequential processing of BER intermediates. In addition, however, we identified at higher enzyme concentrations a ternary complex of APE.Pol beta.DNA that formed specifically at BER intermediates containing a 5'-deoxyribose phosphate group. Formation of this ternary complex was associated with slightly stronger Pol beta gap-filling and much stronger 5'-deoxyribose phosphate lyase activities than was observed with the Pol beta.DNA binary complex. These results indicate that step-by-step coordination in SN-BER can rely on DNA binding specificity inherent in APE and Pol beta, although coordination also may be facilitated by APE.Pol beta.DNA ternary complex formation with appropriate enzyme expression levels or enzyme recruitment to sites of repair.

ATR signaling mediates an S-phase checkpoint after inhibition of poly(ADP-ribose) polymerase activity.

Human fibroblasts, capable of expressing a kinase-dead form of ATR (ATRkd), can be sensitized to the cytotoxic effects of methyl methanesulfonate (MMS) by the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN). The combination of MMS+4-AN results in accumulation of cells in S-phase of the cell cycle and activation of Chk1. Inhibition of ATR activity by expression of ATRkd suppresses the S-phase accumulation and partially reverses the Chk1 phosphorylation. The results confirm involvement of an ATR-mediated damage response pathway in the MMS+4-AN-induced S-phase cell cycle checkpoint in human fibroblasts. Consistent with this hypothesis, the inhibitors caffeine and UCN-01 also abrogate the ATR- and Chk1-mediated delay in progression through S-phase. In the absence of ATR-mediated signaling, MMS+4-AN exposure results in a G(2)/M arrest, rather than an S-phase checkpoint. Thus, whereas ATR mediates the S-phase response, it is not critical for arrest of cells in G(2)/M.

NEIL2-initiated, APE-independent repair of oxidized bases in DNA: Evidence for a repair complex in human cells.

DNA glycosylases/AP lyases initiate repair of oxidized bases in the genomes of all organisms by excising these lesions and then cleaving the DNA strand at the resulting abasic (AP) sites and generate 3' phospho alpha,beta-unsaturated aldehyde (3' PUA) or 3' phosphate (3' P) terminus. In Escherichia coli, the AP-endonucleases (APEs) hydrolyze both 3' blocking groups (3' PUA and 3' P) to generate the 3'-OH termini needed for repair synthesis. In mammalian cells, the previously characterized DNA glycosylases, NTH1 and OGG1, produce 3' PUA, which is removed by the only AP-endonuclease, APE1. However, APE1 is barely active in removing 3' phosphate generated by the recently discovered mammalian DNA glycosylases NEIL1 and NEIL2. We showed earlier that the 3' phosphate generated by NEIL1 is efficiently removed by polynucleotide kinase (PNK) and not APE1. Here we show that the NEIL2-initiated repair of 5-hydroxyuracil (5-OHU) similarly requires PNK. We have also observed stable interaction between NEIL2 and other BER proteins DNA polymerase beta (Pol beta), DNA ligase IIIalpha (Lig IIIalpha) and XRCC1. In spite of their limited sequence homology, NEIL1 and NEIL2 interact with the same domains of Pol beta and Lig IIIalpha. Surprisingly, while the catalytically dispensable C-terminal region of NEIL1 is the common interacting domain, the essential N-terminal segment of NEIL2 is involved in analogous interaction. The BER proteins including NEIL2, PNK, Pol beta, Lig IIIalpha and XRCC1 (but not APE1) could be isolated as a complex from human cells, competent for repair of 5-OHU in plasmid DNA.

DNA polymerase lambda protects mouse fibroblasts against oxidative DNA damage and is recruited to sites of DNA damage/repair.

DNA polymerase lambda (pol lambda) is a member of the X family of DNA polymerases that has been implicated in both base excision repair and non-homologous end joining through in vitro studies. However, to date, no phenotype has been associated with cells deficient in this DNA polymerase. Here we show that pol lambda null mouse fibroblasts are hypersensitive to oxidative DNA damaging agents, suggesting a role of pol lambda in protection of cells against the cytotoxic effects of oxidized DNA. Additionally, pol lambda co-immunoprecipitates with an oxidized base DNA glycosylase, single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), and localizes to oxidative DNA lesions in situ. From these data, we conclude that pol lambda protects cells against oxidative stress and suggest that it participates in oxidative DNA damage base excision repair.

Poly(ADP-ribose) polymerase activity prevents signaling pathways for cell cycle arrest after DNA methylating agent exposure.

Mouse fibroblasts, deficient in DNA polymerase beta, are hypersensitive to monofunctional DNA methylating agents such as methyl methanesulfonate (MMS). Both wild-type and, in particular, repair-deficient DNA polymerase beta null cells are highly sensitized to the cytotoxic effects of MMS by 4-amino-1,8-naphthalimide (4-AN), an inhibitor of poly(ADP-ribose) polymerase (PARP) activity. Experiments with synchronized cells suggest that exposure during S-phase of the cell cycle is required for the 4-AN effect. 4-AN elicits a similar extreme sensitization to the thymidine analog, 5-hydroxymethyl-2'-deoxyuridine, implicating the requirement for an intermediate of DNA repair. In PARP-1-expressing fibroblasts treated with a combination of MMS and 4-AN, a complete inhibition of DNA synthesis is apparent after 4 h, and by 24 h, all cells are arrested in S-phase of the cell cycle. Continuous incubation with 4-AN is required to maintain the cell cycle arrest. Caffeine, an inhibitor of the upstream checkpoint kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related), has no effect on the early inhibition of DNA synthesis, but cells are no longer able to maintain the block after 8 h. Instead, the addition of caffeine leads to arrest of cells in G(2)/M rather than S-phase after 24 h. Analysis of signaling pathways in cell extracts reveals an activation of Chk1 after treatment with MMS and 4-AN, which can be suppressed by caffeine. Our results suggest that inhibition of PARP activity results in sensitization to MMS through maintenance of an ATR and Chk1-dependent S-phase checkpoint.

AP endonuclease-independent DNA base excision repair in human cells.

The paradigm for repair of oxidized base lesions in genomes via the base excision repair (BER) pathway is based on studies in Escherichia coli, in which AP endonuclease (APE) removes all 3' blocking groups (including 3' phosphate) generated by DNA glycosylase/AP lyases after base excision. The recently discovered mammalian DNA glycosylase/AP lyases, NEIL1 and NEIL2, unlike the previously characterized OGG1 and NTH1, generate DNA strand breaks with 3' phosphate termini. Here we show that in mammalian cells, removal of the 3' phosphate is dependent on polynucleotide kinase (PNK), and not APE. NEIL1 stably interacts with other BER proteins, DNA polymerase beta (pol beta) and DNA ligase IIIalpha. The complex of NEIL1, pol beta, and DNA ligase IIIalpha together with PNK suggests coordination of NEIL1-initiated repair. That NEIL1/PNK could also repair the products of other DNA glycosylases suggests a broad role for this APE-independent BER pathway in mammals.

The Werner syndrome protein stimulates DNA polymerase beta strand displacement synthesis via its helicase activity.

Werner syndrome is a hereditary premature aging disorder characterized by genomic instability. Genetic analysis and protein interaction studies indicate that the defective gene product (WRN) may play an important role in DNA replication, recombination, and repair. DNA polymerase beta (pol beta) is a central participant in both short and long-patch base excision repair (BER) pathways, which function to process most spontaneous, alkylated, and oxidative DNA damage. We report here a physical interaction between WRN and pol beta, and using purified proteins reconstitute of a portion of the long-patch BER pathway to examine a potential role for WRN in this repair response. We demonstrate that WRN stimulates pol beta strand displacement DNA synthesis and that this stimulation is dependent on the helicase activity of WRN. In addition, a truncated WRN protein, containing primarily the helicase domain, retains helicase activity and is sufficient to mediate the stimulation of pol beta. The WRN helicase also unwinds a BER substrate, providing evidence that WRN plays a role in unwinding DNA repair intermediates. Based on these findings, we propose a novel mechanism by which WRN may mediate pol beta-directed long-patch BER.

Direct interaction between mammalian DNA polymerase beta and proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the DNA replication and DNA repair machinery. As such, PCNA interacts with many proteins that have a sequence motif termed the PCNA interacting motif (PIM) and also with proteins lacking a PIM. Three regions in human and rat DNA polymerases beta (beta-pol) that resemble the consensus PIM were identified, and we show here that beta-polymerase and PCNA can form a complex both in vitro and in vivo. Immunoprecipitation experiments, yeast two-hybrid analysis, and overlay binding assays were used to examine the interaction between the two proteins. Competition experiments with synthetic PIM-containing peptides suggested the importance of a PIM in the interaction, and studies of a beta-polymerase PIM mutant, H222A/F223A, demonstrated that this alteration blocked the interaction with PCNA. The results indicate that at least one of the PIM-like sequences in beta-polymerase appears to be a functional PIM and was required in the interaction between beta-polymerase and PCNA.

Functional link between BLM defective in Bloom's syndrome and the ataxia-telangiectasia-mutated protein, ATM.

Chromosome aberrations, genomic instability, and cancer predisposition are hallmarks of a number of syndromes in which the defective genes recognize and/or repair DNA damage or are involved in some aspect of DNA processing. We report here direct interaction between BLM, mutated in Bloom's Syndrome (BS), and ATM, mutated is ataxia-telangiectasia, and we have mapped the sites of interaction. Full-length BLM cDNA corrected sister chromatid exchange (SCE) and radiosensitivity in BS cells. Mitotic phosphorylation of BLM was partially dependent on ATM, and phosphorylation sites on BLM were identified. A phosphospecific antibody against one of these sites (Thr-99) revealed radiation-induced phosphorylation, which was defective in ataxia-telangiectasia cells. Stable cell lines expressing phosphorylation site mutants failed to correct radiosensitivity in BS cells but corrected SCE. These mutants also sensitized normal control cells to radiation and increased radiation-induced chromosome aberrations but did not cause SCE numbers to increase. These data suggest that ATM and BLM function together in recognizing abnormal DNA structures by direct interaction and that these phosphorylation sites in BLM are important for radiosensitivity status but not for SCE frequency.