Involvement of nuclear domains in the cellular response to DNA damage

All cells of human organism are continuously damaged, and a damage of the genetic material can be especially dangerous. The reaction of the cell to DNA damage is a complex process, which includes damage signaling, repair, apoptosis or cell death. It is connected with serious changes in the cell nucleus, which are caused by posttranslational modifications and dynamic relocalizations of proteins as well as alterations in the expression of many genes. These changes are not limited to the sites of DNA damage, but involve whole cell nucleus, including its domains: PML bodies, nucleolus and Cajal bodies.     

Rtt107 BRCT domains act as a targeting module in the DNA damage response

Cells are constantly exposed to assaults that cause DNA damage, which must be detected and repaired to prevent genome instability. The DNA damage response is mediated by key kinases that activate various signaling pathways. In Saccharomyces cerevisiae, one of these kinases is Mec1, which phosphorylates numerous targets, including H2A and the DNA damage protein Rtt107. In addition to being phosphorylated, Rtt107 contains six BRCA1 C-terminal (BRCT) domains, which typically recognize phospho-peptides. Thus Rtt107 represented an opportunity to study complementary aspects of the phosphorylation cascades within one protein. Here we sought to describe the functional roles of the multiple BRCT domains in Rtt107. Rtt107 BRCT5/6 facilitated recruitment to sites of DNA lesions via its interaction with phosphorylated H2A. Rtt107 BRCT3/4 also contributed to Rtt107 recruitment, but BRCT3/4 was not sufficient for recruitment when BRCT5/6 was absent. Intriguingly, both mutations that affected Rtt107 recruitment also abrogated its phosphorylation. Pointing to its modular nature, replacing Rtt107 BRCT5/6 with the BRCT domains from the checkpoint protein Rad9 was able to sustain Rtt107 function. Although Rtt107 physically interacts with both the endonuclease Slx4 and the DNA replication and repair protein Dpb11, only Slx4 was dependent on Rtt107 for its recruitment to DNA lesions. Fusing Rtt107 BRCT5/6 to Slx4, which presumably allows artificial recruitment of Slx4 to DNA lesions, alleviated some phenotypes of rtt107Δ mutants, indicating the functional importance of Slx4 recruitment. Together this data revealed a key function of the Rtt107 BRCT domains for targeting of both itself and its interaction partners to DNA lesions.     

14-3-3 proteins, FHA domains and BRCT domains in the DNA damage response

The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phsophorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G₂/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest.     

The response to DNA damage in heterochromatin domains

Eukaryotic genomes are organized into chromatin, divided into structurally and functionally distinct euchromatin and heterochromatin compartments. The high level of compaction and the abundance of repeated sequences in heterochromatin pose multiple challenges for the maintenance of genome stability. Cells have evolved sophisticated and highly controlled mechanisms to overcome these constraints. Here, we summarize recent findings on how the heterochromatic state influences DNA damage formation, signaling, and repair. By focusing on distinct heterochromatin domains in different eukaryotic species, we highlight the heterochromatin contribution to the compartmentalization of DNA damage repair in the cell nucleus and to the repair pathway choice. We also describe the diverse chromatin alterations associated with the DNA damage response in heterochromatin domains and present our current understanding of their regulatory mechanisms. Finally, we discuss the biological significance and the evolutionary conservation of these processes.     

DNA damage response links calpain to cellular senescence

Senescence represents an important barrier against cellular transformation. Here we show that CAPNS1 depletion impairs senescence induction both in BJ-ET H-Ras^v12 inducible human fibroblasts upon Ras induction and in HT1080 targeted to senescence by treatment with low doses of doxorubicin. We further show that CAPNS1 depletion is coupled to reduced levels of H2AX phosphorylation, not only in Ras^v12 induced BJ-ET fibroblasts, but also in a number of cellular systems upon genotoxic stress. In particular CAPNS1 depletion affects γ-H2AX appearance or persistence in U2OS osteosarcoma cells 24 hours after MMC addition or UV light exposure; HT1080 upon camptothecin treatment for 4 hours and 48 hours after addition of MMC; MDA-MB-231, 24 hours after UV light exposure and 2 hours after bleomycin addition. Overall this study unveils a novel link between calpain, cellular senescence and DNA damage response.     

A wide-ranging cellular response to UV damage of DNA

The kinases ATM and ATR are central to proper function of the DNA damage response. These kinases phosphorylate proteins to coordinate cell cycle progression and DNA damage repair/bypass. We have recently reported a large-scale identification of ATM/ATR substrates phosphorylated in response to UV damage of DNA. Overall 231 sites of phosphorylation were induced by UV damage of DNA or dependent on proper function of ATR. The study expanded the number of phosphorylation sites from protein classes known to be involved in the DNA damage response. Further, many sites were identified from protein types not thought to have a role in damage signaling. This observation suggests that the DNA damage response affects a much wider range of cellular processes than was previously appreciated. This study has also extended the successful use of the PhosphoScan® proteomic method from phospho-tyrosine to serine/threonine motifs, providing a general blueprint to use the method to study signaling pathways underlying a wide range of diseases.     

HTLV-1 Tax oncoprotein subverts the cellular DNA damage response via binding to DNA-dependent protein kinase

Human T-cell leukemia virus type-1 is the causative agent for adult T-cell leukemia. Previous research has established that the viral oncoprotein Tax mediates the transformation process by impairing cell cycle control and cellular response to DNA damage. We showed previously that Tax sequesters huChk2 within chromatin and impairs the response to ionizing radiation. Here we demonstrate that DNA-dependent protein kinase (DNA-PK) is a member of the Tax.Chk2 nuclear complex. The catalytic subunit, DNA-PKcs, and the regulatory subunit, Ku70, were present. Tax-containing nuclear extracts showed increased DNA-PK activity, and specific inhibition of DNA-PK prevented Tax-induced activation of Chk2 kinase activity. Expression of Tax induced foci formation and phosphorylation of H2AX. However, Tax-induced constitutive signaling of the DNA-PK pathway impaired cellular response to new damage, as reflected in suppression of ionizing radiation-induced DNA-PK phosphorylation and gammaH2AX stabilization. Tax co-localized with phospho-DNA-PK into nuclear speckles and a nuclear excluded Tax mutant sequestered endogenous phospho-DNA-PK into the cytoplasm, suggesting that Tax interaction with DNA-PK is an initiating event. We also describe a novel interaction between DNA-PK and Chk2 that requires Tax. We propose that Tax binds to and stabilizes a protein complex with DNA-PK and Chk2, resulting in a saturation of DNA-PK-mediated damage repair response.     

Vertebrate DNA damage tolerance requires the C-terminus but not BRCT or transferase domains of REV1

REV1 is central to the DNA damage response of eukaryotes through an as yet poorly understood role in translesion synthesis. REV1 is a member of the Y-type DNA polymerase family and is capable of in vitro deoxycytidyl transferase activity opposite a range of damaged bases. However, non-catalytic roles for REV1 have been suggested by the Saccharomyces cerevisiae rev1-1 mutant, which carries a point mutation in the N-terminal BRCT domain, and the recently demonstrated ability of the mammalian protein to interact with each of the other translesion polymerases via its extreme C-terminus. Here, we show that a region adjacent to this polymerase interacting domain mediates an interaction with PCNA. These C-terminal domains of REV1 are necessary, although not sufficient, for effective tolerance of DNA damage in the avian cell line DT40, while the BRCT domain and transferase activity are not directly required. Together these data provide strong support for REV1 playing an important non-catalytic role in coordinating translesion synthesis. Further, unlike in budding yeast, rad18 is not epistatic to rev1 for DNA damage tolerance suggesting that REV1 and RAD18 play largely independent roles in the control of vertebrate translesion synthesis.     

BRCT domain-containing protein TopBP1 functions in DNA replication and damage response

Topoisomerase IIbeta-binding protein (TopBP1), a human protein with eight BRCT domains, is similar to Saccharomyces cerevisiae Dpb11 and Schizosaccharomyces pombe Cut5 checkpoint proteins and closely related to Drosophila Mus101. We show that human TopBP1 is required for DNA replication and that it interacts with DNA polymerase epsilon. In S phase TopBP1 colocalizes with Brca1 to foci that do not represent sites of ongoing DNA replication. Inhibition of DNA synthesis leads to relocalization of TopBP1 together with Brca1 to replication forks, suggesting a role in rescue of stalled forks. DNA damage induces formation of distinct TopBP1 foci that colocalize with Brca1 in S phase, but not in G(1) phase. We also show that TopBP1 interacts with the checkpoint protein hRad9. Thus, these results implicate TopBP1 in replication and checkpoint functions.     

Replication protein A phosphorylation and the cellular response to DNA damage

Defects in cellular DNA metabolism have a direct role in many human disease processes. Impaired responses to DNA damage and basal DNA repair have been implicated as causal factors in diseases with DNA instability like cancer, Fragile X and Huntington's. Replication protein A (RPA) is essential for multiple processes in DNA metabolism including DNA replication, recombination and DNA repair pathways (including nucleotide excision, base excision and double-strand break repair). RPA is a single-stranded DNA-binding protein composed of subunits of 70-, 32- and 14-kDa. RPA binds ssDNA with high affinity and interacts specifically with multiple proteins. Cellular DNA damage causes the N-terminus of the 32-kDa subunit of human RPA to become hyper-phosphorylated. Current data indicates that hyper-phosphorylation causes a change in RPA conformation that down-regulates activity in DNA replication but does not affect DNA repair processes. This suggests that the role of RPA phosphorylation in the cellular response to DNA damage is to help regulate DNA metabolism and promote DNA repair.     

Poly(ADP-ribose) in the cellular response to DNA damage

Poly(ADP-ribose) polymerase is a chromatin-bound enzyme which, on activation by DNA strand breaks, catalyzes the successive transfer of ADP-ribose units from NAD to nuclear proteins. Poly(ADP-ribose) synthesis is stimulated by DNA strand breaks, and the polymer may alter the structure and/or function of chromosomal proteins to facilitate the DNA repair process. Electronmicroscopic studies show that poly(ADP-ribose) unwinds the tightly packed nucleosomal structure of isolated chromatin. Recent studies also show that the presence of poly(ADP-ribose) enhances the activity of DNA ligase. This may increase the capacity of the cell to complete DNA repair. Inhibitors of poly(ADP-ribose) polymerase or deficiencies of the substrate, NAD, lead to retardation of the DNA repair process. When DNA strand breaks are extensive or when breaks fail to be repaired, the stimulus for activation of poly(ADP-ribose) persists and the activated enzyme is capable of totally consuming cellular pools of NAD. Depletion of NAD and consequent lowering of cellular ATP pools, due to activation of poly(ADP-ribose) polymerase, may account for rapid cell death before DNA repair takes place and before the genetic effects of DNA damage become manifest.     

Acylpeptide hydrolase is a component of the cellular response to DNA damage

Acylpeptide hydrolase (APEH) deacetylates N-alpha-acetylated peptides and selectively degrades oxidised proteins, but the biochemical pathways that are regulated by this protease are unknown. Here, we identify APEH as a component of the cellular response to DNA damage. Although APEH is primarily localised in the cytoplasm, we show that a sub-fraction of this enzyme is sequestered at sites of nuclear damage following UVA irradiation or following oxidative stress. We show that localization of APEH at sites of nuclear damage is mediated by direct interaction with XRCC1, a scaffold protein that accelerates the repair of DNA single-strand breaks. We show that APEH interacts with the amino-terminal domain of XRCC1, and that APEH facilitates both single-strand break repair and cell survival following exposure to H₂O₂ in human cells. These data identify APEH as a novel proteolytic component of the DNA damage response.     

Activation of the DNA damage response by telomere attrition: a passage to cellular senescence

Critical telomere shortening induces senescence in many normal human cell types grown in culture. Recent data have revealed that dysfunctional telomeres can resemble certain forms of DNA damage, and point to a role for DNA damage signaling in the establishment and maintenance of telomere-initiated senescence. Here, we review these new observations and highlight potential avenues of future research. We consider the identities of the key DNA damage response factors involved in senescence and discuss a model for the molecular events occurring in pre-senescent cells that ultimately lead to a permanent cell cycle arrest phenotype.     

Ubiquitin-binding domains and their role in the DNA damage response

The modification of eukaryotic proteins by covalent attachment of ubiquitin is a versatile signaling event with a wide range of possible consequences. Canonical poly-ubiquitination by Lys-48 linked chains usually destines a protein for degradation by the proteasome. By contrast, attachment of a single ubiquitin or ubiquitin chains linked through Lys-63 or Lys-6 serves a non-proteolytic role. Over the last years, evidence has accumulated that several nuclear proteins become ubiquitinated in response to DNA damage. Typically, these proteins carry mono-ubiquitin or non-classical ubiquitin chains and are localized close to the site of DNA damage. Of particular interest are PCNA and the variant histone H2AX, two key proteins whose ubiquitination serves to recruit factors needed by the cell to cope with the damage. A prerequisite for docking effector proteins to the site of the lesion is the detection of a specific ubiquitin modification, a process that can be mediated by a range of dedicated ubiquitin-binding domains (UBDs). As the same types of ubiquitin modification are involved in entirely different processes, the recognition of the ubiquitin mark has to go along with the recognition of the modified protein. Thus, ubiquitin-binding domains gain their specificity through combination with other recognition domains and motifs. This review discusses ubiquitin-binding domains relevant to the DNA damage response, including their binding mode, their specificity, and their interdependence with other factors. For several repair pathways, current knowledge of the events downstream of the ubiquitin mark is sketchy. A closer look at orphan UBD proteins might lead to the identification of missing pieces in the DNA response puzzle.     

Phosphopeptide binding specificities of BRCA1 COOH-terminal (BRCT) domains

Protein phosphorylation by protein kinases may generate docking sites for other proteins. It thus allows the assembly of signaling complexes in response to kinase activation. Several protein domains that bind phosphoserine or phosphothreonine residues have been identified, including the 14-3-3, PIN1, FHA, KIX, WD-40 domain, and polo box (Yaffe, M. B., and Elia, A. E. (2001) Curr. Opin. Cell Biol. 13, 131-138; Elia, A. E., Cantley, L. C., and Yaffe, M. B. (2003) Science 299, 1228-1231). The BRCA1 COOH-terminal (BRCT) domains are protein modules found in many proteins that regulate DNA damage responses (Koonin, E. V., Altschul, S. F., and Bork, P. (1996) Nat. Genet. 13, 266-268). Whether BRCT domains can mediate phosphorylation-dependent interactions has not been systematically investigated. We report here that the BRCT domains also recognize phosphopeptides. Oriented peptide library analysis indicated that the BRCT domains from BRCA1, MDC1, BARD1, and DNA Ligase IV preferred distinct phosphoserine-containing peptides. In addition, the interaction between BRCA1 and the BRCT binding motif of BACH1 was required for BACH1 checkpoint activity. Furthermore, BRCT domains of the yeast DNA repair protein Rad9 could bind phosphopeptides, suggesting that the BRCT domains represent a class of ancient phosphopeptide-binding modules. Potential targets of BRCT domains were identified through data base search. Structural analysis of BRCA1 BRCT repeats also predicted conserved residues that may form the phosphopeptide-binding pocket. Thus, the BRCT repeats are a new family of phosphopeptide-binding domains in DNA damage responses.