The linker histone H1-BRCA1 axis is a crucial mediator of replication fork stability.

The maintenance of genome integrity relies on replication fork stabilization upon encountering endogenous and exogenous sources of DNA damage. How this process is coordinated with the local chromatin environment remains poorly defined. Here, we show that the replication-dependent histone H1 variants interact with the tumour suppressor BRCA1 in a replication stress-dependent manner. Transient loss of the replication-dependent histones H1 does not affect fork progression in unchallenged conditions but leads to the accumulation of stalled replication intermediates. Upon challenge with hydroxyurea, cells deficient for histone H1 variants fail to recruit BRCA1 to stalled replication forks and undergo MRE11-dependent fork resection and collapse, which ultimately leads to genomic instability and cell death. In summary, our work defines an essential role of the replication-dependent histone H1 variants in mediating BRCA1-dependent fork protection and genome stability.

Microtubule-associated proteins MAP7 and MAP7D1 promote DNA double-strand break repair in the G1 cell cycle phase.

The DNA-damage response is a complex signaling network that guards genomic integrity. The microtubule cytoskeleton is involved in the repair of DNA double-strand breaks; however, little is known about which cytoskeleton-related proteins are involved in DNA repair and how. Using quantitative proteomics, we discovered that microtubule associated proteins MAP7 and MAP7D1 interact with several DNA repair proteins including DNA double-strand break repair proteins RAD50, BRCA1 and 53BP1. We observed that downregulation of MAP7 and MAP7D1 leads to increased phosphorylation of p53 after γ-irradiation. Moreover, we determined that the downregulation of MAP7D1 leads to a strong G1 arrest and that the downregulation of MAP7 and MAP7D1 in G1 arrested cells negatively affects DNA repair, recruitment of RAD50 to chromatin and localization of 53BP1 to the sites of damage. These findings describe for the first time a novel function of MAP7 and MAP7D1 in cell cycle regulation and repair of DNA double-strand breaks.

Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations.

BACKGROUND: Pathological forms of TAR DNA-binding protein 43 (TDP-43) are present in motor neurons of almost all amyotrophic lateral sclerosis (ALS) patients, and mutations in TDP-43 are also present in ALS. Loss and gain of TDP-43 functions are implicated in pathogenesis, but the mechanisms are unclear. While the RNA functions of TDP-43 have been widely investigated, its DNA binding roles remain unclear. However, recent studies have implicated a role for TDP-43 in the DNA damage response. METHODS: We used NSC-34 motor neuron-like cells and primary cortical neurons expressing wildtype TDP-43 or TDP-43 ALS associated mutants (A315T, Q331K), in which DNA damage was induced by etoposide or H2O2 treatment. We investigated the consequences of depletion of TDP-43 on DNA repair using small interfering RNAs. Specific non homologous end joining (NHEJ) reporters (EJ5GFP and EJ2GFP) and cells lacking DNA-dependent serine/threonine protein kinase (DNA-PK) were used to investigate the role of TDP-43 in DNA repair. To investigate the recruitment of TDP-43 to sites of DNA damage we used single molecule super-resolution microscopy and a co-immunoprecipitation assay. We also investigated DNA damage in an ALS transgenic mouse model, in which TDP-43 accumulates pathologically in the cytoplasm. We also examined fibroblasts derived from ALS patients bearing the TDP-43 M337V mutation for evidence of DNA damage. RESULTS: We demonstrate that wildtype TDP-43 is recruited to sites of DNA damage where it participates in classical NHEJ DNA repair. However, ALS-associated TDP-43 mutants lose this activity, which induces DNA damage. Furthermore, DNA damage is present in mice displaying TDP-43 pathology, implying an active role in neurodegeneration. Additionally, DNA damage triggers features typical of TDP-43 pathology; cytoplasmic mis-localisation and stress granule formation. Similarly, inhibition of NHEJ induces TDP-43 mis-localisation to the cytoplasm. CONCLUSIONS: This study reveals that TDP-43 functions in DNA repair, but loss of this function triggers DNA damage and is associated with key pathological features of ALS.

Nuclear localisation of 53BP1 is regulated by phosphorylation of the nuclear localisation signal.

BACKGROUND INFORMATION: Repair of damaged DNA is essential for maintaining genomic stability. TP53-binding protein 1 (53BP1) plays an important role in repair of the DNA double-strand breaks. Nuclear localisation of 53BP1 depends on importin ß and nucleoporin 153, but the type and location of 53BP1 nuclear localisation signal (NLS) have yet to be determined. RESULTS: Here, we show that nuclear import of 53BP1 depends on two basic regions, namely 1667-KRK-1669 and 1681-KRGRK-1685, which are both needed for importin binding. Lysine 1667 is essential for interaction with importin and its substitution to arginine reduced nuclear localisation of 53BP1. Furthermore, we have found that CDK1-dependent phosphorylation of 53BP1 at S1678 impairs importin binding during mitosis. Phosphorylation-mimicking mutant S1678D showed reduced nuclear localisation, suggesting that phosphorylation of the NLS interferes with nuclear import of the 53BP1 CONCLUSIONS: We show that 53BP1 contains a classical bipartite NLS 1666-GKRKLITSEEERSPAKRGRKS-1686, which enables the importin-mediated nuclear transport of 53BP1. Additionally, we found that posttranslational modification within the NLS region can regulate 53BP1 nuclear import. SIGNIFICANCE: Our results indicate that integrity of the NLS is important for 53BP1 nuclear localisation. Precise mapping of the NLS will facilitate further studies on the effect of posttranslational modifications and somatic mutations on the nuclear localisation 53BP1 and DNA repair.

Assembly of the U5 snRNP component PRPF8 is controlled by the HSP90/R2TP chaperones.

Splicing is catalyzed by the spliceosome, a complex of five major small nuclear ribonucleoprotein particles (snRNPs). The pre-mRNA splicing factor PRPF8 is a crucial component of the U5 snRNP, and together with EFTUD2 and SNRNP200, it forms a central module of the spliceosome. Using quantitative proteomics, we identified assembly intermediates containing PRPF8, EFTUD2, and SNRNP200 in association with the HSP90/R2TP complex, its ZNHIT2 cofactor, and additional proteins. HSP90 and R2TP bind unassembled U5 proteins in the cytoplasm, stabilize them, and promote the formation of the U5 snRNP. We further found that PRPF8 mutants causing Retinitis pigmentosa assemble less efficiently with the U5 snRNP and bind more strongly to R2TP, with one mutant retained in the cytoplasm in an R2TP-dependent manner. We propose that the HSP90/R2TP chaperone system promotes the assembly of a key module of U5 snRNP while assuring the quality control of PRPF8. The proteomics data further reveal new interactions between R2TP and the tuberous sclerosis complex (TSC), pointing to a potential link between growth signals and the assembly of key cellular machines.

Human RIF1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation.

The human RIF1 protein controls DNA replication, but the molecular mechanism is largely unknown. Here, we demonstrate that human RIF1 negatively regulates DNA replication by forming a complex with protein phosphatase 1 (PP1) that limits phosphorylation-mediated activation of the MCM replicative helicase. We identify specific residues on four MCM helicase subunits that show hyperphosphorylation upon RIF1 depletion, with the regulatory N-terminal domain of MCM4 being particularly strongly affected. In addition to this role in limiting origin activation, we discover an unexpected new role for human RIF1-PP1 in mediating efficient origin licensing. Specifically, during the G1 phase of the cell cycle, RIF1-PP1 protects the origin-binding ORC1 protein from untimely phosphorylation and consequent degradation by the proteasome. Depletion of RIF1 or inhibition of PP1 destabilizes ORC1, thereby reducing origin licensing. Consistent with reduced origin licensing, RIF1-depleted cells exhibit increased spacing between active origins. Human RIF1 therefore acts as a PP1-targeting subunit that regulates DNA replication positively by stimulating the origin licensing step, and then negatively by counteracting replication origin activation.

Substrate recognition and function of the R2TP complex in response to cellular stress.

The R2TP complex is a HSP90 co-chaperone, which consists of four subunits: PIH1D1, RPAP3, RUVBL1, and RUVBL2. It is involved in the assembly of large protein or protein-RNA complexes such as RNA polymerase, small nucleolar ribonucleoproteins (snoRNPs), phosphatidylinositol 3 kinase-related kinases (PIKKs), and their complexes. While RPAP3 has a HSP90 binding domain and the RUVBLs comprise ATPase activities important for R2TP functions, PIH1D1 contains a PIH-N domain that specifically recognizes phosphorylated substrates of the R2TP complex. In this review we provide an overview of the current knowledge of the R2TP complex with the focus on the recently identified structural and mechanistic features of the R2TP complex functions. We also discuss the way R2TP regulates cellular response to stress caused by low levels of nutrients or by DNA damage and its possible exploitation as a target for anti-cancer therapy.

Phosphorylation-dependent PIH1D1 interactions define substrate specificity of the R2TP cochaperone complex.

The R2TP cochaperone complex plays a critical role in the assembly of multisubunit machines, including small nucleolar ribonucleoproteins (snoRNPs), RNA polymerase II, and the mTORC1 and SMG1 kinase complexes, but the molecular basis of substrate recognition remains unclear. Here, we describe a phosphopeptide binding domain (PIH-N) in the PIH1D1 subunit of the R2TP complex that preferentially binds to highly acidic phosphorylated proteins. A cocrystal structure of a PIH-N domain/TEL2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which Lys57 and 64 in PIH1D1, along with a conserved DpSDD phosphopeptide motif within TEL2, are essential and sufficient for binding. Proteomic analysis of PIH1D1 interactors identified R2TP complex substrates that are recruited by the PIH-N domain in a sequence-specific and phosphorylation-dependent manner suggestive of a common mechanism of substrate recognition. We propose that protein complexes assembled by the R2TP complex are defined by phosphorylation of a specific motif and recognition by the PIH1D1 subunit.

HELQ promotes RAD51 paralogue-dependent repair to avert germ cell loss and tumorigenesis.

Repair of interstrand crosslinks (ICLs) requires the coordinated action of the intra-S-phase checkpoint and the Fanconi anaemia pathway, which promote ICL incision, translesion synthesis and homologous recombination (reviewed in refs 1, 2). Previous studies have implicated the 3'-5' superfamily 2 helicase HELQ in ICL repair in Drosophila melanogaster (MUS301 (ref. 3)) and Caenorhabditis elegans (HELQ-1 (ref. 4)). Although in vitro analysis suggests that HELQ preferentially unwinds synthetic replication fork substrates with 3' single-stranded DNA overhangs and also disrupts protein-DNA interactions while translocating along DNA, little is known regarding its functions in mammalian organisms. Here we report that HELQ helicase-deficient mice exhibit subfertility, germ cell attrition, ICL sensitivity and tumour predisposition, with Helq heterozygous mice exhibiting a similar, albeit less severe, phenotype than the null, indicative of haploinsufficiency. We establish that HELQ interacts directly with the RAD51 paralogue complex BCDX2 and functions in parallel to the Fanconi anaemia pathway to promote efficient homologous recombination at damaged replication forks. Thus, our results reveal a critical role for HELQ in replication-coupled DNA repair, germ cell maintenance and tumour suppression in mammals.

Tripartite Motif-containing 33 (TRIM33) protein functions in the poly(ADP-ribose) polymerase (PARP)-dependent DNA damage response through interaction with Amplified in Liver Cancer 1 (ALC1) protein.

Activation of poly(ADP-ribose) polymerase (PARP) near sites of DNA breaks facilitates recruitment of DNA repair proteins and promotes chromatin relaxation in part through the action of chromatin-remodeling enzyme Amplified in Liver Cancer 1 (ALC1). Through proteomic analysis we find that ALC1 interacts after DNA damage with Tripartite Motif-containing 33 (TRIM33), a multifunctional protein implicated in transcriptional regulation, TGF-ß signaling, and tumorigenesis. We demonstrate that TRIM33 is dynamically recruited to DNA damage sites in a PARP1- and ALC1-dependent manner. TRIM33-deficient cells show enhanced sensitivity to DNA damage and prolonged retention of ALC1 at sites of DNA breaks. Conversely, overexpression of TRIM33 alleviates the DNA repair defects conferred by ALC1 overexpression. Thus, TRIM33 plays a role in PARP-dependent DNA damage response and regulates ALC1 activity by promoting its timely removal from sites of DNA damage.

CK2 phospho-dependent binding of R2TP complex to TEL2 is essential for mTOR and SMG1 stability.

TEL2 interacts with and is essential for the stability of all phosphatidylinositol 3-kinase-related kinases (PIKKs), but its mechanism of action remains unclear. Here, we show that TEL2 is constitutively phosphorylated on conserved serines 487 and 491 by casein kinase 2 (CK2). Proteomic analyses establish that the CK2 phosphosite of TEL2 confers binding to the R2TP/prefoldin-like complex, which possesses chaperon/prefoldin activities required during protein complex assembly. The PIH1D1 subunit of the R2TP complex binds directly to the CK2 phosphosite of TEL2 in vitro and is required for the TEL2-R2TP/prefoldin-like complex interaction in vivo. Although the CK2 phosphosite mutant of TEL2 retains association with the PIKKs and HSP90 in cells, failure to interact with the R2TP/prefoldin-like complex results in instability of the PIKKs, principally mTOR and SMG1. We propose that TEL2 acts as a scaffold to coordinate the activities of R2TP/prefoldin-like and HSP90 chaperone complexes during the assembly of the PIKKs.

Preventing nonhomologous end joining suppresses DNA repair defects of Fanconi anemia.

Fanconi anemia (FA) is a complex cancer susceptibility disorder associated with DNA repair defects and infertility, yet the precise function of the FA proteins in genome maintenance remains unclear. Here we report that C. elegans FANCD2 (fcd-2) is dispensable for normal meiotic recombination but is required in crossover defective mutants to prevent illegitimate repair of meiotic breaks by nonhomologous end joining (NHEJ). In mitotic cells, we show that DNA repair defects of C. elegans fcd-2 mutants and FA-deficient human cells are significantly suppressed by eliminating NHEJ. Moreover, NHEJ factors are inappropriately recruited to sites of replication stress in the absence of FANCD2. Our findings are consistent with the interpretation that FA results from the promiscuous action of NHEJ during DNA repair. We propose that a critical function of the FA pathway is to channel lesions into accurate, as opposed to error-prone, repair pathways.

Poly(ADP-ribose)-dependent regulation of DNA repair by the chromatin remodeling enzyme ALC1.

Posttranslational modifications play key roles in regulating chromatin plasticity. Although various chromatin-remodeling enzymes have been described that respond to specific histone modifications, little is known about the role of poly[adenosine 5'-diphosphate (ADP)-ribose] in chromatin remodeling. Here, we identify a chromatin-remodeling enzyme, ALC1 (Amplified in Liver Cancer 1, also known as CHD1L), that interacts with poly(ADP-ribose) and catalyzes PARP1-stimulated nucleosome sliding. Our results define ALC1 as a DNA damage-response protein whose role in this process is sustained by its association with known DNA repair factors and its rapid poly(ADP-ribose)-dependent recruitment to DNA damage sites. Furthermore, we show that depletion or overexpression of ALC1 results in sensitivity to DNA-damaging agents. Collectively, these results provide new insights into the mechanisms by which poly(ADP-ribose) regulates DNA repair.

FANCM-FAAP24 and HCLK2: roles in ATR signalling and the Fanconi anemia pathway.

The ATR signalling pathway coordinates the cellular response to replication stress, which is essential for the maintenance of genome integrity. HCLK2/Tel2 is a highly conserved orphan protein that binds directly to ATR and other PI3-kinase related kinases and plays a central role in checkpoint signalling responses.(1) Proteomic analyses of HCLK2 complexes confirmed ATR, ATRIP and DNA-PKcs as HCLK2 interacting factors and also uncovered two surprising interacting proteins, the heterodimeric Fanconi Anemia (FA) proteins FANCM and FAAP24. Our subsequent findings that ATR signalling is attenuated in FANCM and FAAP24-depleted cells, together with recent biochemical studies, suggested that remodelling of stalled replication forks by FANCM-FAAP24 is required to facilitate efficient activation of ATR signalling in response to replication stress.(2) Furthermore, our study revealed that the DNA translocase activity of FANCM is essential for efficient activation of the ATR signalling, a function that is separate and distinct from its role in targeting the FA core complex to sites of DNA damage. In this review we discuss the importance of these findings in the context of recent data and raise questions regarding the role of HCLK2 and FANCM-FAAP24 in human disease.

FANCM and FAAP24 function in ATR-mediated checkpoint signaling independently of the Fanconi anemia core complex.

The Fanconi anemia (FA) pathway is implicated in DNA repair and cancer predisposition. Central to this pathway is the FA core complex, which is targeted to chromatin by FANCM and FAAP24 following replication stress. Here we show that FANCM and FAAP24 interact with the checkpoint protein HCLK2 independently of the FA core complex. In addition to defects in FA pathway activation, downregulation of FANCM or FAAP24 also compromises ATR/Chk1-mediated checkpoint signaling, leading to defective Chk1, p53, and FANCE phosphorylation; 53BP1 focus formation; and Cdc25A degradation. As a result, FANCM and FAAP24 deficiency results in increased endogenous DNA damage and a failure to efficiently invoke cell-cycle checkpoint responses. Moreover, we find that the DNA translocase activity of FANCM, which is dispensable for FA pathway activation, is required for its role in ATR/Chk1 signaling. Our data suggest that DNA damage recognition and remodeling activities of FANCM and FAAP24 cooperate with ATR/Chk1 to promote efficient activation of DNA damage checkpoints.

DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis.

During the evolution of cancer, the incipient tumour experiences 'oncogenic stress', which evokes a counter-response to eliminate such hazardous cells. However, the nature of this stress remains elusive, as does the inducible anti-cancer barrier that elicits growth arrest or cell death. Here we show that in clinical specimens from different stages of human tumours of the urinary bladder, breast, lung and colon, the early precursor lesions (but not normal tissues) commonly express markers of an activated DNA damage response. These include phosphorylated kinases ATM and Chk2, and phosphorylated histone H2AX and p53. Similar checkpoint responses were induced in cultured cells upon expression of different oncogenes that deregulate DNA replication. Together with genetic analyses, including a genome-wide assessment of allelic imbalances, our data indicate that early in tumorigenesis (before genomic instability and malignant conversion), human cells activate an ATR/ATM-regulated DNA damage response network that delays or prevents cancer. Mutations compromising this checkpoint, including defects in the ATM-Chk2-p53 pathway, might allow cell proliferation, survival, increased genomic instability and tumour progression.

Distinct functional domains of Nbs1 modulate the timing and magnitude of ATM activation after low doses of ionizing radiation.

The ATM kinase is a tumour suppressor and a key activator of genome integrity checkpoints in mammalian cells exposed to ionizing radiation (IR) and other insults that elicit DNA double-strand breaks (DSBs). In response to IR, autophosphorylation on serine 1981 causes dissociation of ATM dimers and initiates cellular ATM kinase activity. Here, we show that the kinetics and magnitude of ATM Ser1981 phosphorylation after exposure of human fibroblasts to low doses (2 Gy) of IR are altered in cells deficient in Nbs1, a substrate of ATM and a component of the MRN (Mre11-Rad50-Nbs1) complex involved in processing/repair of DSBs and ATM-dependent cell cycle checkpoints. Timely phosphorylation of both ATM Ser1981 and the ATM substrate Smc1 after IR were rescued via retrovirally mediated reconstitution of Nbs1-deficient cells by wild-type Nbs1 or mutants of Nbs1 defective in the FHA domain or nonphosphorylatable by ATM, but not by Nbs1 lacking the Mre11-interaction domain. Our data indicate that apart from its role downstream of ATM in the DNA damage checkpoint network, the MRN complex serves also as a modulator/amplifier of ATM activity. Although not absolutely required for ATM activation, the MRN nuclease complex may help reach the threshold activity of ATM necessary for optimal genome maintenance and prevention of cancer.

The twist gene is a common target of retroviral integration and transcriptional deregulation in experimental nephroblastoma.

The genes involved in the transformation of kidney blastema cells were searched for in avian nephroblastomas induced by the MAV2 retrovirus. The twist gene was identified as a common site of provirus integration in tumor cells. Twist was rearranged by the MAV2 provirus in three out of 76 independent nephroblastoma samples. The MAV2 integration sites were localized within 40 nucleotides of the twist 5'UTR region, right upstream from the ATG initiation codon. The integrated proviruses were deleted at their 5'ends. As a consequence, twist transcription became controlled by the retroviral 3'LTR promoter and was strongly upregulated, more than 200 times. In addition, 2-100 times elevated twist transcription was also detected in the majority of other nephroblastoma samples not containing MAV2 in the twist locus. We propose that chicken nephroblastoma originates from a single blastemic cell in which the MAV retrovirus, through its integration, has deregulated specific combinations of genes controlling proliferation and differentiation. The activation of the twist gene expression appears to contribute to tumorigenesis, as there is an in vivo positive selection of tumor cell clones containing the twist gene hyperactivated by MAV2 sequences inserted within the twist promoter.