Nuclear dynamics of PCNA in DNA replication and repair

The DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA) is central to both DNA replication and repair. The ring-shaped homotrimeric PCNA encircles and slides along double-stranded DNA, acting as a "sliding clamp" that localizes proteins to DNA. We determined the behavior of green fluorescent protein-tagged human PCNA (GFP-hPCNA) in living cells to analyze its different engagements in DNA replication and repair. Photobleaching and tracking of replication foci revealed a dynamic equilibrium between two kinetic pools of PCNA, i.e., bound to replication foci and as a free mobile fraction. To simultaneously monitor PCNA action in DNA replication and repair, we locally inflicted UV-induced DNA damage. A surprisingly longer residence time of PCNA at damaged areas than at replication foci was observed. Using DNA repair mutants, we showed that the initial recruitment of PCNA to damaged sites was dependent on nucleotide excision repair. Local accumulation of PCNA at damaged regions was observed during all cell cycle stages but temporarily disappeared during early S phase. The reappearance of PCNA accumulation in discrete foci at later stages of S phase likely reflects engagements of PCNA in distinct genome maintenance processes dealing with stalled replication forks, such as translesion synthesis (TLS). Using a ubiquitination mutant of GFP-hPCNA that is unable to participate in TLS, we noticed a significantly shorter residence time in damaged areas. Our results show that changes in the position of PCNA result from de novo assembly of freely mobile replication factors in the nucleoplasmic pool and indicate different binding affinities for PCNA in DNA replication and repair.     

Distinct populations of human PCNA are required for initiation of chromosomal DNA replication and concurrent DNA repair

The integrity of genomic DNA during the cell division cycle in eukaryotic cells is maintained by regulated chromosomal DNA replication and repair of damaged DNA. We have used fractionation and reconstitution experiments to purify essential factors for the initiation of human chromosomal DNA replication in late G1 phase template nuclei from human cells. Here, we report the identification of soluble PCNA as an essential initiation factor in this system. Recombinant histidine-tagged human PCNA can substitute for purified endogenous human PCNA to initiate human chromosomal DNA replication. It is recruited specifically to discrete DNA replication foci formed during initiation in vitro. The template nuclei also contain DNA breaks as result of the synchronisation procedure. A separate population of chromatin-bound PCNA is already present in these template nuclei at discrete DNA damage foci, co-localising with gamma-H2AX, RPA and Rad51. This DNA damage-associated PCNA population is marked by mono-ubiquitination, suggesting that it is involved in DNA repair. Importantly, the population of damage focus-associated PCNA is neither involved in, nor required for, the initiation of chromosomal DNA replication in the same nuclei.     

Dynamics of some postreplication DNA repair proteins in carcinogen-damaged mammalian cells

Many types of DNA lesions in template strands block DNA replication and lead to a stalling of replication forks. This block can be overcome (bypassed) by special DNA polymerases (for example, DNA polymerase eta, Pol eta) that perform translesion synthesis on damaged template DNA. The phenomenon of completing DNA replication, while DNA lesions remain in the template strands, has been named post-replication repair (PRR). In yeast Saccharomyces cerevisiae, PRR includes mutagenic and error-free pathways under the regulation of the RAD6/RAD18 complex, which induces ubiquitylation of PCNA. In mammalian cells, Pol eta accumulates in replication foci but the mechanism of this accumulation is not known. Pol eta possesses a conserved PCNA binding motif at the C terminal and phosphorylation of this motif might be essential for its interaction with PCNA. We have shown previously that staurosporine, an inhibitor of protein kinases, inhibits PRR in human cells. In this study we examined whether the accumulation of Pol eta in replication foci after DNA damage is dependent on phosphorylation of the PCNA binding motif. We also studied DNA damage-induced phosphorylation of GFP-tagged human Rad18 (hRad18) and its accumulation in replication foci. Our data indicate that (1) Pol eta is not phosphorylated in response to UV irradiation or MMS treatment, but its diffusional mobility is slightly decreased, and (2) hRad18 accumulates in MMS-treated cells, and considerable amount of the protein co-localizes with detergent insoluble PCNA in replication foci; these responses are sensitive to staurosporine. Our data suggest that hRad18 phosphorylation is the staurosporine-sensitive PRR step.     

DNA damage-induced RPA focalization is independent of gamma-H2AX and RPA hyper-phosphorylation

Replication protein A (RPA) is the major eukaryotic single stranded DNA binding protein that plays a central role in DNA replication, repair and recombination. Like many DNA repair proteins RPA is heavily phosphorylated (specifically on its 32 kDa subunit) in response to DNA damage. Phosphorylation of many repair proteins has been shown to be important for their recruitment to DNA damage-induced intra-nuclear foci. Further, phosphorylation of H2AX (gamma-H2AX) has been shown to be important for either the recruitment or stable retention of DNA repair proteins to these intra-nuclear foci. We address here the relationship between DNA damage-induced hyper-phosphorylation of RPA and its intra-nuclear focalization, and whether gamma-H2AX is required for RPA's presence at these foci. Using GFP-conjugated RPA, we demonstrate the formation of extraction-resistant RPA foci induced by DNA damage or stalled replication forks. The strong DNA damage-induced RPA foci appear after phosphorylated histone H2AX and Chk1, but earlier than the appearance of hyper-phosphorylated RPA. We demonstrate that while the functions of phosphoinositol-3-kinase-related protein kinases are essential for DNA damage-induced H2AX phosphorylation and RPA hyper-phosphorylation, they are dispensable for the induction of extraction-resistant RPA and RPA foci. Furthermore, in mouse cells genetically devoid of H2AX, DNA damage-induced extraction-resistant RPA appears with the same kinetics as in normal mouse cells. These results demonstrate that neither RPA hyper-phosphorylation nor H2AX are required for the formation in RPA intra-nuclear foci in response to DNA damage/replicational stress and are consistent with a role for RPA as a DNA damage sensor involved in the initial recognition of damaged DNA or blocked replication forks.     

UV-induced RPA phosphorylation is increased in the absence of DNA polymerase eta and requires DNA-PK

Signaling from arrested replication forks plays a role in maintaining genome stability. We have investigated this process in xeroderma pigmentosum variant cells that carry a mutation in the POLH gene and lack functional DNA polymerase eta (poleta). Poleta is required for error-free bypass of UV-induced cyclobutane pyrimidine dimers; in the absence of poleta in XPV cells, DNA replication is arrested at sites of UV-induced DNA damage, and mutagenic bypass of lesions is ultimately carried out by other, error-prone, DNA polymerases. The present study investigates whether poleta expression influences the activation of a number of UV-induced DNA damage responses. In a stably transfected XPV cell line (TR30-9) in which active poleta can be induced by addition of tetracycline, expression of poleta determines the extent of DNA double-strand break formation following UV-irradiation. UV-induced phosphorylation of replication protein A (RPA), a key DNA-binding protein involved in DNA replication, repair and recombination, is increased in cells lacking poleta compared to when poleta is expressed in the same cell line. To identify the protein kinase responsible for increased UV-induced hyperphosphorylation of the p34 subunit of RPA, we have used NU7441, a specific small molecule inhibitor of DNA-PK. DNA-PK is necessary for RPA p34 hyperphosphorylation, but DNA-PK-mediated phosphorylation is not required for recruitment of RPA p34 into nuclear foci in response to UV-irradiation. The results demonstrate that activation of a UV-induced DNA damage response pathway, involving phosphorylation of RPA p34 by DNA-PK, is enhanced in cells lacking poleta.     

Ionizing radiation-dependent gamma-H2AX focus formation requires ataxia telangiectasia mutated and ataxia telangiectasia mutated and Rad3-related

The histone variant H2AX is rapidly phosphorylated at the sites of DNA double-strand breaks (DSBs). This phosphorylated H2AX (gamma-H2AX) is involved in the retention of repair and signaling factor complexes at sites of DNA damage. The dependency of this phosphorylation on the various PI3K-related protein kinases (in mammals, ataxia telangiectasia mutated and Rad3-related [ATR], ataxia telangiectasia mutated [ATM], and DNA-PKCs) has been a subject of debate; it has been suggested that ATM is required for the induction of foci at DSBs, whereas ATR is involved in the recognition of stalled replication forks. In this study, using Arabidopsis as a model system, we investigated the ATR and ATM dependency of the formation of gamma-H2AX foci in M-phase cells exposed to ionizing radiation (IR). We find that although the majority of these foci are ATM-dependent, approximately 10% of IR-induced gamma-H2AX foci require, instead, functional ATR. This indicates that even in the absence of DNA replication, a distinct subset of IR-induced damage is recognized by ATR. In addition, we find that in plants, gamma-H2AX foci are induced at only one-third the rate observed in yeasts and mammals. This result may partly account for the relatively high radioresistance of plants versus yeast and mammals.     

Replication-dependent and -independent responses of RAD18 to DNA damage in human cells

Postreplication repair facilitates tolerance of DNA damage during replication, overcoming termination of replication at sites of DNA damage. A major post-replication repair pathway in mammalian cells is translesion synthesis, which is carried out by specialized polymerase(s), such as polymerase eta, and is identified by focus formation by the polymerase after irradiation with UVC light. The formation of these foci depends on RAD18, which ubiquitinates PCNA for the exchange of polymerases. To understand the initial processes in translesion synthesis, we have here analyzed the response to damage of RAD18 in human cells. We find that human RAD18 accumulates very rapidly and remains for a long period of time at sites of different types of DNA damage, including UVC light-induced lesions, and x-ray microbeam- and laser-induced single-strand breaks, in a cell cycle-independent manner. The accumulation of RAD18 at DNA damage is observed even when DNA replication is inhibited, and a small region containing a zinc finger motif located in the middle of RAD18 is essential and sufficient for the replication-independent damage accumulation. The zinc finger motif of RAD18 is not necessary for UV-induced polymerase eta focus formation, but another SAP (SAF-A/B, Acinus and PIAS) motif near the zinc finger is required. These data indicate that RAD18 responds to DNA damage in two distinct ways, one replication-dependent and one replication-independent, involving the SAP and zinc finger motifs, respectively.     

Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells

The eukaryotic sliding DNA clamp, proliferating cell nuclear antigen (PCNA), is essential for DNA replication and repair synthesis. In order to load the ring-shaped, homotrimeric PCNA onto the DNA double helix, the ATPase activity of the replication factor C (RFC) clamp loader complex is required. Although the recruitment of PCNA by RFC to DNA replication sites has well been documented, our understanding of its recruitment during DNA repair synthesis is limited. In this study, we analyzed the accumulation of endogenous and fluorescent-tagged proteins for DNA repair synthesis at the sites of DNA damage produced locally by UVA-laser micro-irradiation in HeLa cells. Accumulation kinetics and in vitro pull-down assays of the large subunit of RFC (RFC140) revealed that there are two distinct modes of recruitment of RFC to DNA damage, a simultaneous accumulation of RFC140 and PCNA caused by interaction between PCNA and the extreme N-terminus of RFC140 and a much faster accumulation of RFC140 than PCNA at the damaged site. Furthermore, RFC140 knock-down experiments showed that PCNA can accumulate at DNA damage independently of RFC. These results suggest that immediate accumulation of RFC and PCNA at DNA damage is only partly interdependent.     

Dynamics of DNA replication factories in living cells

DNA replication occurs in microscopically visible complexes at discrete sites (replication foci) in the nucleus. These foci consist of DNA associated with replication machineries, i.e., large protein complexes involved in DNA replication. To study the dynamics of these nuclear replication foci in living cells, we fused proliferating cell nuclear antigen (PCNA), a central component of the replication machinery, with the green fluorescent protein (GFP). Imaging of stable cell lines expressing low levels of GFP-PCNA showed that replication foci are heterogeneous in size and lifetime. Time-lapse studies revealed that replication foci clearly differ from nuclear speckles and coiled bodies as they neither show directional movements, nor do they seem to merge or divide. These four dimensional analyses suggested that replication factories are stably anchored in the nucleus and that changes in the pattern occur through gradual, coordinated, but asynchronous, assembly and disassembly throughout S phase.     

Bi-directional routing of DNA mismatch repair protein human exonuclease 1 to replication foci and DNA double strand breaks

Human exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the sub-nuclear localization of hEXO1 during S-phase progression and in response to laser-induced DNA double strand breaks (DSBs). We show that hEXO1 and PCNA co-localize in replication foci. This apparent interaction is sustained throughout S-phase. We also demonstrate that hEXO1 is rapidly recruited to DNA DSBs. We have identified a PCNA interacting protein (PIP-box) region on hEXO1 located in its COOH-terminal ((788)QIKLNELW(795)). This motif is essential for PCNA binding and co-localization during S-phase. Recruitment of hEXO1 to DNA DSB sites is dependent on the MMR protein hMLH1. We show that two distinct hMLH1 interaction regions of hEXO1 (residues 390-490 and 787-846) are required to direct the protein to the DNA damage site. Our results reveal that protein domains in hEXO1 in conjunction with specific protein interactions control bi-directional routing of hEXO1 between on-going DNA replication and repair processes in living cells.     

PCNA-dependent accumulation of CDKN1A into nuclear foci after ionizing irradiation

The cyclin-dependent kinase inhibitor CDKN1A/p21 confers cell-cycle arrest in response to DNA damage and inhibits DNA replication through its direct interaction with the proliferating cell nuclear antigen (PCNA) and cyclin/cyclin-dependent kinase complexes. Previously, we reported that in response to densely ionizing radiation CDKN1A rapidly is recruited to the sites of particle traversal, and that CDKN1A foci formation in response to heavy ions is independent of its transactivation by TP53. Here, we show that exposure of normal human fibroblasts to X-rays or to H2O2 also induces nuclear accumulations of CDKN1A. We find that CDKN1A foci formation in response to radiation damage is dependent on its dephosphorylation and on its direct physical interaction with PCNA. Live cell imaging analyses of ectopically expressed EGFP-CDKN1A and dsRed-PCNA show rapid recruitment of both proteins into foci after radiation damage. Detailed dynamic measurements reveal a slightly delayed recruitment of CDKN1A compared to PCNA, which is best described by bi-exponential curve fitting, taking the preceding binding of PCNA to DNA into account. We propose a regulatory role for CDKN1A in mediating PCNA function after radiation damage, and provide evidence that this role is distinct from its involvement in nucleotide excision repair and unrelated to double-strand break repair.     

Human DNA polymerase epsilon colocalizes with proliferating cell nuclear antigen and DNA replication late, but not early, in S phase.

DNA polymerase epsilon (pol epsilon) has been implicated in DNA replication, DNA repair, and cell cycle control, but its precise roles are unclear. When the subcellular localization of human pol epsilon was examined by indirect immunofluorescence, pol epsilon appeared in discrete nuclear foci that colocalized with proliferating cell nuclear antigen (PCNA) foci and sites of DNA synthesis only late in S phase. Early in S phase, pol epsilon foci were adjacent to PCNA foci. In contrast to PCNA foci that were only present in S phase, pol epsilon foci were present throughout mitosis and the G(1) phase of cycling cells. It is hypothesized from these observations that pol epsilon and PCNA have separate but associated functions early in S phase and that pol epsilon participates with PCNA in DNA replication late in S phase.     

Retention but not recruitment of Crb2 at double-strand breaks requires Rad1 and Rad3 complexes

The fission yeast checkpoint protein Crb2, related to budding yeast Rad9 and human 53BP1 and BRCA1, has been suggested to act as an adapter protein facilitating the phosphorylation of specific substrates by Rad3-Rad26 kinase. To further understand its role in checkpoint signaling, we examined its localization in live cells by using fluorescence microscopy. In response to DNA damage, Crb2 localizes to distinct nuclear foci, which represent sites of DNA double-strand breaks (DSBs). Crb2 colocalizes with Rad22 at persistent foci, suggesting that Crb2 is retained at sites of DNA damage during repair. Damage-induced Crb2 foci still form in cells defective in Rad1, Rad3, and Rad17 complexes, but these foci do not persist as long as in wild-type cells. Our results suggest that Crb2 functions at the sites of DNA damage, and its regulated persistent localization at damage sites may be involved in facilitating DNA repair and/or maintaining the checkpoint arrest while DNA repair is under way.     

14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif

Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins are regulatory phosphorserine/threonine binding proteins involved in the control of diverse cellular events, including cell cycle checkpoint and apoptosis signaling. hEXO1 is regulated by post-translation Ser/Thr phosphorylation in a yet not fully clarified manner, but evidently three phosphorylation sites are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding experiments reveal weak affinity of the more selective isoform 14-3-3σ but both 14-3-3 isoforms η and σ significantly stimulate hEXO1 activity, indicating that these regulatory proteins exert a common regulation mode on hEXO1. Results demonstrate that binding involves the phosphorable amino acid S746 in hEXO1 and most likely a second unidentified binding motif. 14-3-3 associations do not appear to directly influence hEXO1 in vitro nuclease activity or in vitro DNA replication initiation. Moreover, specific phosphorylation variants, including hEXO1 S746A, are efficiently imported to the nucleus; to associate with PCNA in distinct replication foci and respond to DNA double strand breaks (DSBs), indicating that 14-3-3 binding does not involve regulating the subcellular distribution of hEXO1. Altogether, these results suggest that association may be related to regulation of hEXO1 availability during the DNA damage response to plausibly prevent extensive DNA resection at the damage site, as supported by recent studies.     

Replication Protein A is Required for Etoposide-Induced Assembly of MRE11/RAD50/NBS1 Complex Repair Foci

The presence of DNA damage activates a specific response cascade culminating in DNA repair activity and cell cycle checkpoints. Although the type of lesion dictates what proteins are involved in the response, replication protein A (RPA) and the Mre11/Rad50/Nbs1 complex (MRN) respond to most types of lesions. To examine the relationship of RPA and the MRN complex in DNA damage responses, we used siRNA-mediated protein depletion of RPA-p70 and Mre11. Depletion of RPA-p70 decreased the ability of cells to form phospho-Nbs1 foci and increased levels of DNA double-strand breaks (DSBs) following treatment with etoposide (ETOP). In contrast, depletion of Mre11 led to increased levels of RPA-p34 foci formation, but abrogated phospho-RPA-p34 foci formation. These data support a role for RPA as an initial signal/sensor for DNA damage that facilitates recruitment of MRN and ATM/ATR to sites of damage, where they then work together to fully activate the DNA damage response.     

A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage

BACKGROUND: The response of eukaryotic cells to double-strand breaks in genomic DNA includes the sequestration of many factors into nuclear foci. Recently it has been reported that a member of the histone H2A family, H2AX, becomes extensively phosphorylated within 1-3 minutes of DNA damage and forms foci at break sites. RESULTS: In this work, we examine the role of H2AX phosphorylation in focus formation by several repair-related complexes, and investigate what factors may be involved in initiating this response. Using two different methods to create DNA double-strand breaks in human cells, we found that the repair factors Rad50 and Rad51 each colocalized with phosphorylated H2AX (gamma-H2AX) foci after DNA damage. The product of the tumor suppressor gene BRCA1 also colocalized with gamma-H2AX and was recruited to these sites before Rad50 or Rad51. Exposure of cells to the fungal inhibitor wortmannin eliminated focus formation by all repair factors examined, suggesting a role for the phosphoinositide (PI)-3 family of protein kinases in mediating this response. Wortmannin treatment was effective only when it was added early enough to prevent gamma-H2AX formation, indicating that gamma-H2AX is necessary for the recruitment of other factors to the sites of DNA damage. DNA repair-deficient cells exhibit a substantially reduced ability to increase the phosphorylation of H2AX in response to ionizing radiation, consistent with a role for gamma-H2AX in DNA repair. CONCLUSIONS: The pattern of gamma-H2AX foci that is established within a few minutes of DNA damage accounts for the patterns of Rad50, Rad51, and Brca1 foci seen much later during recovery from damage. The evidence presented strongly supports a role for the gamma-H2AX and the PI-3 protein kinase family in focus formation at sites of double-strand breaks and suggests the possibility of a change in chromatin structure accompanying double-strand break repair.     

A novel role for the mono-ADP-ribosyltransferase PARP14/ARTD8 in promoting homologous recombination and protecting against replication stress

Genomic instability, a major hallmark of cancer cells, is caused by incorrect or ineffective DNA repair. Many DNA repair mechanisms cooperate in cells to fight DNA damage, and are generally regulated by post-translational modification of key factors. Poly-ADP-ribosylation, catalyzed by PARP1, is a post-translational modification playing a prominent role in DNA repair, but much less is known about mono-ADP-ribosylation. Here we report that mono-ADP-ribosylation plays an important role in homologous recombination DNA repair, a mechanism essential for replication fork stability and double strand break repair. We show that the mono-ADP-ribosyltransferase PARP14 interacts with the DNA replication machinery component PCNA and promotes replication of DNA lesions and common fragile sites. PARP14 depletion results in reduced homologous recombination, persistent RAD51 foci, hypersensitivity to DNA damaging agents and accumulation of DNA strand breaks. Our work uncovered PARP14 as a novel factor required for mitigating replication stress and promoting genomic stability.     

An analysis of the regulation of DNA synthesis by cdk2, Cip1, and licensing factor

The activation of DNA replication appears to involve at least four steps. These include origin recognition, origin unwinding, primer synthesis, and a switching step to a continuous elongation mode. Moreover, in higher eukaryotes a number of studies have shown that much of the DNA replication which occurs is restricted to specific sites within the nuclei. It has been proposed that these replication foci are composed of a large number of origin sites which are clustered together into an aggregate. The molecular basis for this aggregation is currently not well understood. Regulation of the activation of DNA replication is a complicated process. The G1-S kinase cdk2 is a positive regulator of replication. The p21 protein is a negative regulator of replication both by inhibiting cdk2 kinase and the replication protein PCNA. Moreover, it has been proposed that origin usage is restricted to a single firing per cell cycle by a "licensing factor." Using a cell-free replication system derived from Xenopus eggs we have investigated at what step in the replication process these regulators participate. We present evidence that the clustered organization of DNA into foci is not a transient arrangement, but rather, it persists following DNA replication. We also find that foci form on both sperm chromatin and bacteriophage lambda DNA incubated in extracts depleted of cdk2 kinase. Therefore, our data support the conclusion that organization of chromatin into foci is an early event in the replication pathway preceding activation of cdk2 kinase. With respect to the role of cdk2 during activation of DNA replication we find that in cdk2-depleted extracts primer synthesis does not occur and RP-A remains tightly associated with foci. This strongly suggests that cdk2 kinase is required for activating the origin unwinding step of the replication process. Consistent with this interpretation we find that addition of rate limiting quantities of the cdk2 inhibitor p21 protein to an extract delays primer synthesis. Interestingly, in the presence of p21 primer synthesis does occur after a delay and then replication arrests. This is consistent with the published demonstration that p21 can inhibit PCNA, a protein required for replication beyond the priming step. Therefore, our results provide additional support to the proposal that the post-priming switching step is a key regulatory step in replication. With respect to the role of licensing factor during DNA replication it has recently been shown that treatment of mitotic extracts with kinase inhibitor DMAP inactivates "licensing factor."(ABSTRACT TRUNCATED AT 400 WORDS)     

Heat shock protein 90α (Hsp90α) is phosphorylated in response to DNA damage and accumulates in repair foci

DNA damage triggers a complex signaling cascade involving a multitude of phosphorylation events. We found that the threonine 7 (Thr-7) residue of heat shock protein 90α (Hsp90α) was phosphorylated immediately after DNA damage. The phosphorylated Hsp90α then accumulated at sites of DNA double strand breaks and formed repair foci with slow kinetics, matching the repair kinetics of complex DNA damage. The phosphorylation of Hsp90α was dependent on phosphatidylinositol 3-kinase-like kinases, including the DNA-dependent protein kinase (DNA-PK) in particular. DNA-PK plays an essential role in the repair of DNA double strand breaks by nonhomologous end-joining and in the signaling of DNA damage. It is also present in the cytoplasm of the cell and has been suggested to play a role in cytoplasmic signaling pathways. Using stabilized double-stranded DNA molecules to activate DNA-PK, we showed that an active DNA-PK complex could be assembled in the cytoplasm, resulting in phosphorylation of the cytoplasmic pool of Hsp90α. In vivo, reverse phase protein array data for tumors revealed that basal levels of Thr-7-phosphorylated Hsp90α were correlated with phosphorylated histone H2AX levels. The Thr-7 phosphorylation of the ubiquitously produced and secreted Hsp90α may therefore serve as a surrogate biomarker of DNA damage. These findings shed light on the interplay between central DNA repair enzymes and an essential molecular chaperone.