Spatiotemporal Dynamics of Early DNA Damage Response Proteins on Complex DNA Lesions

The response of cells to ionizing radiation-induced DNA double-strand breaks (DSB) is determined by the activation of multiple pathways aimed at repairing the injury and maintaining genomic integrity. Densely ionizing radiation induces complex damage consisting of different types of DNA lesions in close proximity that are difficult to repair and may promote carcinogenesis. Little is known about the dynamic behavior of repair proteins on complex lesions. In this study we use live-cell imaging for the spatio-temporal characterization of early protein interactions at damage sites of increasing complexity. Beamline microscopy was used to image living cells expressing fluorescently-tagged proteins during and immediately after charged particle irradiation to reveal protein accumulation at damaged sites in real time. Information on the mobility and binding rates of the recruited proteins was obtained from fluorescence recovery after photobleaching (FRAP). Recruitment of the DNA damage sensor protein NBS1 accelerates with increasing lesion density and saturates at very high damage levels. FRAP measurements revealed two different binding modalities of NBS1 to damage sites and a direct impact of lesion complexity on the binding. Faster recruitment with increasing lesion complexity was also observed for the mediator MDC1, but mobility was limited at very high damage densities due to nuclear-wide binding. We constructed a minimal computer model of the initial response to DSB based on known protein interactions only. By fitting all measured data using the same set of parameters, we can reproduce the experimentally characterized steps of the DNA damage response over a wide range of damage densities. The model suggests that the influence of increasing lesion density accelerating NBS1 recruitment is only dependent on the different binding modes of NBS1, directly to DSB and to the surrounding chromatin via MDC1. This elucidates an impact of damage clustering on repair without the need of invoking extra processing steps.     

Zinc finger protein 668 interacts with Tip60 to promote H2AX acetylation after DNA damage

Many tumor suppressors play an important role in the DNA damage pathway. Zinc finger protein 668 (ZNF668) has recently been identified as one of the potential tumor suppressors in breast cancer, but its function in DNA damage response is unknown. Herein, we report that ZNF668 is a regulator of DNA repair. ZNF668 knockdown impairs cell survival after DNA damage without affecting the ATM/ATR DNA-damage signaling cascade. However, recruitment of repair proteins to DNA lesions is decreased. In response to IR, ZNF668 knockdown reduces Tip60-H2AX interaction and impairs IR-induced histone H2AX hyperacetylation, thus impairing chromatin relaxation. Impaired chromatin relaxation causes decreased recruitment of repair proteins to DNA lesions, defective homologous recombination (HR) repair and impaired cell survival after IR. In addition, ZNF668 knockdown decreased RPA phosphorylation and its recruitment to DNA damage foci in response to UV. In both IR and UV damage responses, chromatin relaxation counteracted the impaired loading of repair proteins and DNA repair defects in ZNF668-deficient U2OS cells, indicating that impeded chromatin accessibility at sites of DNA breaks caused the DNA repair defects observed in the absence of ZNF668. Our findings suggest that ZNF668 is a key molecule that links chromatin relaxation with DNA damage response in DNA repair control.     

DNA double-strand breaks in heterochromatin elicit fast repair protein recruitment, histone H2AX phosphorylation and relocation to euchromatin

DNA double-strand breaks (DSBs) can induce chromosomal aberrations and carcinogenesis and their correct repair is crucial for genetic stability. The cellular response to DSBs depends on damage signaling including the phosphorylation of the histone H2AX (γH2AX). However, a lack of γH2AX formation in heterochromatin (HC) is generally observed after DNA damage induction. Here, we examine γH2AX and repair protein foci along linear ion tracks traversing heterochromatic regions in human or murine cells and find the DSBs and damage signal streaks bending around highly compacted DNA. Given the linear particle path, such bending indicates a relocation of damage from the initial induction site to the periphery of HC. Real-time imaging of the repair protein GFP-XRCC1 confirms fast recruitment to heterochromatic lesions inside murine chromocenters. Using single-ion microirradiation to induce localized DSBs directly within chromocenters, we demonstrate that H2AX is early phosphorylated within HC, but the damage site is subsequently expelled from the center to the periphery of chromocenters within ～ 20 min. While this process can occur in the absence of ATM kinase, the repair of DSBs bordering HC requires the protein. Finally, we describe a local decondensation of HC at the sites of ion hits, potentially allowing for DSB movement via physical forces.     

The chromatin remodeling factor BRG1 stimulates nucleotide excision repair by facilitating recruitment of XPC to sites of DNA damage

BRG1 is a catalytic subunit of the human SWI/SNF-like BAF chromatin remodeling complexes. Recent findings have shown that inactivation of BRG1 sensitizes mammalian cells to various DNA damaging agents, including ultraviolet (UV) and ionizing radiation. However, it is unclear whether BRG1 facilitates nucleotide excision repair (NER). Here we show that re-expression of BRG1 in cells lacking endogenous BRG1 expression stimulates nucleotide excision repair of UV induced DNA damage. Using a micropore UV radiation technique, we demonstrate that recruitment of the DNA damage recognition protein XPC to sites of UV lesions is significantly disrupted when BRG1 is depleted. Chromatin immunoprecipitation of the endogenous DDB2 protein, which is involved in recruiting XPC to UV-induced CPDs (cyclobutane pyrimidine dimers), shows that elevated levels of BRG1 are associated with DDB2 in chromatin in response to UV radiation. Additionally, we detected slow BRG1 accumulation at sites of UV lesions. Our findings suggest that the chromatin remodeling factor BRG1 is recruited to sites of UV lesions to facilitate NER in human chromatin.     

Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo

DNA damage and its repair can cause both local and global rearrangements of chromatin structure. In each case, the epigenetic information contained within this structure must be maintained. Using the recently developed method for the localized UV irradiation of cells, we analysed responses that occur locally to damage sites and global events triggered by local damage recognition. We thus demonstrate that, within a single cell, the recruitment of chromatin assembly factor 1 (CAF-1) to UV-induced DNA damage is a strictly local phenomenon, restricted to damage sites. Concomitantly, proliferating cell nuclear antigen (PCNA) locates to the same sites. This localized recruitment suggests that CAF-1 participates directly in chromatin structural rearrangements that occur in the vicinity of the damage. Use of nucleotide excision repair (NER)-deficient cells shows that the NER pathway--specifically dual incision--is required for recruitment of CAF-1 and PCNA. This in vivo demonstration of the local role of CAF-1, depending directly on NER, supports the hypothesis that CAF-1 ensures the maintenance of epigenetic information by acting locally at repair sites.     

MU2 and HP1a regulate the recognition of double strand breaks in Drosophila melanogaster

Chromatin structure regulates the dynamics of the recognition and repair of DNA double strand breaks; open chromatin enhances the recruitment of DNA damage response factors, while compact chromatin is refractory to the assembly of radiation-induced repair foci. MU2, an orthologue of human MDC1, a scaffold for ionizing radiation-induced repair foci, is a widely distributed chromosomal protein in Drosophila melanogaster that moves to DNA repair foci after irradiation. Here we show using yeast 2 hybrid screens and co-immunoprecipitation that MU2 binds the chromoshadow domain of the heterochromatin protein HP1 in untreated cells. We asked what role HP1 plays in the formation of repair foci and cell cycle control in response to DNA damage. After irradiation repair foci form in heterochromatin but are shunted to the edge of heterochromatic regions an HP1-dependent manner, suggesting compartmentalized repair. Hydroxyurea-induced repair foci that form at collapsed replication forks, however, remain in the heterochromatic compartment. HP1a depletion in irradiated imaginal disc cells increases apoptosis and disrupts G2/M arrest. Further, cells irradiated in mitosis produced more and brighter repair foci than to cells irradiated during interphase. Thus, the interplay between MU2 and HP1a is dynamic and may be different in euchromatin and heterochromatin during DNA break recognition and repair.     

Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair

Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3′ side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4^Cdt2. Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4^Cdt2 for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER.     

Nuclear envelope defects impede a proper response to micronuclear DNA lesions

When damage is inflicted in nuclear DNA, cells activate a hierarchical plethora of proteins that constitute the DNA damage response machinery. In contrast to the cell nucleus, the ability of micronuclear DNA lesions to activate this complex network is controversial. In order to determine whether the DNA contained in micronuclei is protected by the cellular damage response system, we studied the recruitment of excision repair factors to photolesions inflicted in the DNA of radiation-induced micronuclei. To perform this analysis, primary human dermal fibroblasts were exposed to UV-C light to induce photolesions in nuclear and micronuclear DNA. By means of immunofluorescence techniques, we observed that most micronuclei were devoid of NER factors. We conclude that UV photoproducts in micronuclei are mostly unable to generate an effective DNA damage response. We observed that the micronuclear envelope structure is a determinant factor that influences the repair of the DNA lesions inside micronuclei. Therefore, our results allow us to conclude that photolesions in radiation-induced micronuclei are poorly processed because the repair factors are unable to reach the micronuclear chromatin when a micronucleus is formed or after a genotoxic insult.     

Spatiotemporal regulation of posttranslational modifications in the DNA damage response

A timely and accurate cellular response to DNA damage requires tight regulation of the action of DNA damage response (DDR) proteins at lesions. A multitude of posttranslational modifications (PTMs) of chromatin and chromatin‐associated proteins coordinates the recruitment of critical proteins that dictate the appropriate DNA repair pathway and enable the actual repair of lesions. Phosphorylation, ubiquitylation, SUMOylation, neddylation, poly(ADP‐ribosyl)ation, acetylation, and methylation are among the DNA damage‐induced PTMs that have taken center stage as important DDR regulators. Redundant and multivalent interactions of DDR proteins with PTMs may not only be a means to facilitate efficient relocalization, but also a feature that allows high temporal and spatial resolution of protein recruitment to, and extraction from, DNA damage sites. In this review, we will focus on the complex interplay between such PTMs, and discuss the importance of their interconnectivity in coding DNA lesions and maintaining the integrity of the genome.     

HP1alpha recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair

Heterochromatin protein 1 (HP1), a major component of constitutive heterochromatin, is recruited to DNA damage sites. However, the mechanism involved in this recruitment and its functional importance during DNA repair remain major unresolved issues. Here, by characterizing HP1α dynamics at laser-induced damage sites in mammalian cells, we show that the de novo accumulation of HP1α occurs within both euchromatin and heterochromatin as a rapid and transient event after DNA damage. This recruitment is strictly dependent on p150CAF-1, the largest subunit of chromatin assembly factor 1 (CAF-1), and its ability to interact with HP1α. We find that HP1α depletion severely compromises the recruitment of the DNA damage response (DDR) proteins 53BP1 and RAD51. Moreover, HP1α depletion leads to defects in homologous recombination-mediated repair and reduces cell survival after DNA damage. Collectively, our data reveal that HP1α recruitment at early stages of the DDR involves p150CAF-1 and is critical for proper DNA damage signaling and repair.     

Direct measurement of protein–protein interactions by FLIM-FRET at UV laser-induced DNA damage sites in living cells

Protein–protein interactions are essential to ensure timely and precise recruitment of chromatin remodellers and repair factors to DNA damage sites. Conventional analyses of protein–protein interactions at a population level may mask the complexity of interaction dynamics, highlighting the need for a method that enables quantification of DNA damage-dependent interactions at a single-cell level. To this end, we integrated a pulsed UV laser on a confocal fluorescence lifetime imaging (FLIM) microscope to induce localized DNA damage. To quantify protein–protein interactions in live cells, we measured Förster resonance energy transfer (FRET) between mEGFP- and mCherry-tagged proteins, based on the fluorescence lifetime reduction of the mEGFP donor protein. The UV-FLIM-FRET system offers a unique combination of real-time and single-cell quantification of DNA damage-dependent interactions, and can distinguish between direct protein–protein interactions, as opposed to those mediated by chromatin proximity. Using the UV-FLIM-FRET system, we show the dynamic changes in the interaction between poly(ADP-ribose) polymerase 1, amplified in liver cancer 1, X-ray repair cross-complementing protein 1 and tripartite motif containing 33 after DNA damage. This new set-up complements the toolset for studying DNA damage response by providing single-cell quantitative and dynamic information about protein–protein interactions at DNA damage sites.     

Spatiotemporal dynamics of regulatory protein recruitment at DNA damage sites

Mammalian cells are constantly threatened by multiple types of DNA lesions arising from various sources like irradiation, environmental agents, replication errors or by-products of the normal cellular metabolism. If not readily detected and repaired these lesions can lead to cell death or to the transformation of cells giving rise to life-threatening diseases like cancer. Multiple specialized repair pathways have evolved to preserve the genetic integrity of a cell. The increasing number of DNA damage sensors, checkpoint regulators, and repair factors identified in the numerous interconnected repair pathways raises the question of how DNA repair is coordinated. In the last decade, various methods have been developed that allow the induction of DNA lesions and subsequent real-time analysis of repair factor assembly at DNA repair sites in living cells. This combination of biophysical and molecular cell biology methods has yielded interesting new insights into the order and kinetics of protein recruitment and identified regulatory sequences and selective loading platforms for the efficient restoration of the genetic and epigenetic integrity of mammalian cells.     

Should I stay or should I go: VCP/p97-mediated chromatin extraction in the DNA damage response

The ordered assembly of DNA repair factors on chromatin has been studied in great detail, whereas we are only beginning to realize that selective extraction of proteins from chromatin plays a central role in the DNA damage response. Interestingly, the protein modifier ubiquitin not only regulates the well-documented recruitment of repair proteins, but also governs the temporally and spatially controlled extraction of proteins from DNA lesions. The facilitator of protein extraction is the ubiquitin-dependent ATPase valosin-containing protein (VCP)/p97 complex, which, through its segregase activity, directly extracts ubiquitylated proteins from chromatin. In this review, we summarize recent studies that uncovered this important role of VCP/p97 in the cellular response to genomic insults and discuss how ubiquitin regulates two intuitively counteracting activities at sites of DNA damage.     

Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair

Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3′ side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4^Cdt2. Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4^Cdt2 for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER.     

High turnover and rescue effect of XRCC1 in response to heavy charged particle radiation

The DNA damage response is a highly orchestrated process. The involvement of the DNA damage response factors in DNA damage response depends on their biochemical reactions with each other and with chromatin. Using online live-cell imaging combined with heavy ion microbeam irradiation, we studied the response of the scaffold protein X-ray repair cross-complementary protein 1 (XRCC1) at the localized DNA damage in charged particle irradiated HT1080 cells expressing XRCC1-tagged RFP. The results showed that XRCC1 was recruited to the DNA damage with ultrafast kinetics in a poly ADP-ribose polymerase-dependent manner. The consecutive reaction model well explained the response of XRCC1 at ion hits, and we found that the XRCC1 recruitment was faster and dissociation was slower in the G2 phase than those in the G1 phase. The fractionated irradiation of the same cells resulted in accelerated dissociation at the previous damage sites, and the dissociated XRCC1 immediately recycled with a higher recruitment efficiency. Our data revealed XRCC1’s new rescue mechanism and its high turnover in DNA damage response, which benefits our understanding of the biochemical mechanism in DNA damage response.     

Real-Time Imaging of DNA Damage in Yeast Cells Using Ultra-Short Near-Infrared Pulsed Laser Irradiation

Analysis of accumulation of repair and checkpoint proteins at repair sites in yeast nuclei has conventionally used chemical agents, ionizing radiation or induction of endonucleases to inflict localized damage. In addition to these methods, similar studies in mammalian cells have used laser irradiation, which has the advantage that damage is inflicted at a specific nuclear region and at a precise time, and this allows accurate kinetic analysis of protein accumulation at DNA damage sites. We show here that it is feasible to use short pulses of near-infrared laser irradiation to inflict DNA damage in subnuclear regions of yeast nuclei by multiphoton absorption. In conjunction with use of fluorescently-tagged proteins, this allows quantitative analysis of protein accumulation at damage sites within seconds of damage induction. PCNA accumulated at damage sites rapidly, such that maximum accumulation was seen approximately 50 s after damage, then levels declined linearly over 200–1000 s after irradiation. RPA accumulated with slower kinetics such that hardly any accumulation was detected within 60 s of irradiation, and levels subsequently increased linearly over the next 900 s, after which levels were approximately constant (up to ca. 2700 s) at the damage site. This approach complements existing methodologies to allow analysis of key damage sensors and chromatin modification changes occurring within seconds of damage inception.     

BMI1 is recruited to DNA breaks and contributes to DNA damage-induced H2A ubiquitination and repair

DNA damage activates signaling pathways that lead to modification of local chromatin and recruitment of DNA repair proteins. Multiple DNA repair proteins having ubiquitin ligase activity are recruited to sites of DNA damage, where they ubiquitinate histones and other substrates. This DNA damage-induced histone ubiquitination is thought to play a critical role in mediating the DNA damage response. We now report that the polycomb protein BMI1 is rapidly recruited to sites of DNA damage, where it persists for more than 8 h. The sustained localization of BMI1 to damage sites is dependent on intact ATM and ATR and requires H2AX phosphorylation and recruitment of RNF8. BMI1 is required for DNA damage-induced ubiquitination of histone H2A at lysine 119. Loss of BMI1 leads to impaired repair of DNA double-strand breaks by homologous recombination and the accumulation of cells in G(2)/M. These data support a crucial role for BMI1 in the cellular response to DNA damage.