PTIP Regulates 53BP1 and SMC1 at the DNA Damage Sites

Although PTIP is implicated in the DNA damage response, through interactions with 53BP1, the function of PTIP in the DNA damage response remain elusive. Here, we show that RNF8 controls DNA damage-induced nuclear foci formation of PTIP, which in turn regulates 53BP1 localization to the DNA damage sites. In addition, SMC1, a substrate of ATM, could not be phosphorylated at the DNA damage sites in the absence of PTIP. The PTIP-dependent pathway is important for DNA double strand breaks repair and DNA damage-induced intra-S phase checkpoint activation. Taken together, these results suggest that the role of PTIP in the DNA damage response is downstream of RNF8 and upstream of 53BP1. Thus, PTIP regulates 53BP1-dependent signaling pathway following DNA damage.The DNA damage response pathways are signal transduction pathways with DNA damage sensors, mediators, and effectors, which are essential for maintaining genomic stability (1–3). Following DNA double strand breaks, histone H2AX at the DNA damage sites is rapidly phosphorylated by ATM/ATR/DNAPK (4–10), a family homologous to phosphoinositide 3-kinases (11, 12). Subsequently, phospho-H2AX (γH2AX) provides the platform for accumulation of a larger group of DNA damage response factors, such as MDC1, BRCA1, 53BP1, and the MRE11·RAD50·NBS1 complex (13, 14), at the DNA damage sites. Translocalization of these proteins to the DNA double strand breaks (DSBs)³ facilitates DNA damage checkpoint activation and enhances the efficiency of DNA damage repair (14, 15).Recently, PTIP (Pax2 transactivation domain-interacting protein, or Paxip) has been identified as a DNA damage response protein and is required for cell survival when exposed to ionizing radiation (IR) (1, 16–18). PTIP is a 1069-amino acid nuclear protein and has been originally identified in a yeast two-hybrid screening as a partner of Pax2 (19). Genetic deletion of the PTIP gene in mice leads to early embryonic lethality at embryonic day 8.5, suggesting that PTIP is essential for early embryonic development (20). Structurally, PTIP contains six tandem BRCT (BRCA1 carboxyl-terminal) domains (16–18, 21). The BRCT domain is a phospho-group binding domain that mediates protein-protein interactions (17, 22, 23). Interestingly, the BRCT domain has been found in a large number of proteins involved in the cellular response to DNA damages, such as BRCA1, MDC1, and 53BP1 (7, 24–29). Like other BRCT domain-containing proteins, upon exposure to IR, PTIP forms nuclear foci at the DSBs, which is dependent on its BRCT domains (16–18). By protein affinity purification, PTIP has been found in two large complexes. One includes the histone H3K4 methyltransferase ALR and its associated cofactors, the other contains DNA damage response proteins, including 53BP1 and SMC1 (30, 31). Further experiments have revealed that DNA damage enhances the interaction between PTIP and 53BP1 (18, 31).To elucidate the DNA damage response pathways, we have examined the upstream and downstream partners of PTIP. Here, we report that PTIP is downstream of RNF8 and upstream of 53BP1 in response to DNA damage. Moreover, PTIP and 53BP1 are required for the phospho-ATM association with the chromatin, which phosphorylates SMC1 at the DSBs. This PTIP-dependent pathway is involved in DSBs repair.     

Dynamic assembly and sustained retention of 53BP1 at the sites of DNA damage are controlled by Mdc1/NFBD1

53BP1 is a key component of the genome surveillance network activated by DNA double strand breaks (DSBs). Despite its known accumulation at the DSB sites, the spatiotemporal aspects of 53BP1 interaction with DSBs and the role of other DSB regulators in this process remain unclear. Here, we used real-time microscopy to study the DSB-induced redistribution of 53BP1 in living cells. We show that within minutes after DNA damage, 53BP1 becomes progressively, yet transiently, immobilized around the DSB-flanking chromatin. Quantitative imaging of single cells revealed that the assembly of 53BP1 at DSBs significantly lagged behind Mdc1/NFBD1, another DSB-interacting checkpoint mediator. Furthermore, short interfering RNA-mediated ablation of Mdc1/NFBD1 drastically impaired 53BP1 redistribution to DSBs and triggered premature dissociation of 53BP1 from these regions. Collectively, these in vivo measurements identify Mdc1/NFBD1 as a key upstream determinant of 53BP1's interaction with DSBs from its dynamic assembly at the DSB sites through sustained retention within the DSB-flanking chromatin up to the recovery from the checkpoint.     

Saccharomyces Cerevisiae Rad52 Alleles Temperature-Sensitive for the Repair of DNA Double-Strand Breaks

We have screened for mutations of the Saccharomyces cerevisiae RAD52 gene which confer a temperature-sensitive (ts) phenotype with respect to either the repair of DNA lesions caused by methyl methanesulfonate (MMS) or the recombination of an intrachromosomal recombination reporter. We were readily able to isolate alleles ts for the repair of lesions caused by MMS but were unable to find alleles with a severe ts deficiency in intrachromosomal recombination. We extensively characterized four strains conferring ts growth on MMS agar. These strains also exhibit ts survival when exposed to γ-radiation or when the HO endonuclease is constitutively expressed. Although none of the four alleles confers a severe ts defect in intrachromosomal recombination, two confer significant defects in tests of mitotic, interchromosomal recombination carried out in diploid strains. The mutant diploids sporulate, but the two strains with defects in interchromosomal recombination have reduced spore viability. Meiotic recombination is not depressed in the two diploids with reduced spore viability. Thus, in the two strains with reduced spore viability, defects in mitotic and meiotic recombination do not correlate. Sequence analysis revealed that in three of the four ts alleles the causative mutations are in the first one-third of the open reading frame while the fourth is in the C-terminal third.     

Homologous Recombination, but Not DNA Repair, Is Reduced in Vertebrate Cells Deficient in RAD52

Rad52 plays a pivotal role in double-strand break (DSB) repair and genetic recombination in Saccharomyces cerevisiae, where mutation of this gene leads to extreme X-ray sensitivity and defective recombination. Yeast Rad51 and Rad52 interact, as do their human homologues, which stimulates Rad51-mediated DNA strand exchange in vitro, suggesting that Rad51 and Rad52 act cooperatively. To define the role of Rad52 in vertebrates, we generated RAD52^−/− mutants of the chicken B-cell line DT40. Surprisingly, RAD52^−/− cells were not hypersensitive to DNA damages induced by γ-irradiation, methyl methanesulfonate, or cis-platinum(II)diammine dichloride (cisplatin). Intrachromosomal recombination, measured by immunoglobulin gene conversion, and radiation-induced Rad51 nuclear focus formation, which is a putative intermediate step during recombinational repair, occurred as frequently in RAD52^−/− cells as in wild-type cells. Targeted integration frequencies, however, were consistently reduced in RAD52^−/− cells, showing a clear role for Rad52 in genetic recombination. These findings reveal striking differences between S. cerevisiae and vertebrates in the functions of RAD51 and RAD52.     

Coordinated response of mammalian Rad51 and Rad52 to DNA damage

Biochemical analysis has shown that mammalian Rad51 and Rad52 interact and synergize in DNA recombination reactions in vitro, but these proteins have not been shown to function together in response to DNA damage in vivo. By analysis of murine cells expressing murine Rad52 tagged with green fluorescent protein (GFP)–Rad52, we now show that DNA damage causes Rad51 and GFP–Rad52 to colocalize in distinct nuclear foci. Cells expressing GFP–Rad52 show both increased survival and an increased number of Rad51 foci, raising the possibility that Rad52 is limiting for repair. These observations provide evidence of coordinated function of Rad51 and Rad52 in vivo and support the hypothesis that Rad52 plays an important role in the DNA damage response in mammalian cells.     

The Srs2 Suppressor of Rad6 Mutations of Saccharomyces Cerevisiae Acts by Channeling DNA Lesions into the Rad52 DNA Repair Pathway

rad6 mutants of Saccharomyces cerevisiae are defective in the repair of damaged DNA, DNA damage induced mutagenesis, and sporulation. In order to identify genes that can substitute for RAD6 function, we have isolated genomic suppressors of the UV sensitivity of rad6 deletion (rad6 delta) mutations and show that they also suppress the gamma-ray sensitivity but not the UV mutagenesis or sporulation defects of rad6. The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of gamma-ray sensitivity. The six suppressor mutations we isolated are all alleles of the same locus and are also allelic to a previously described suppressor of the rad6-1 nonsense mutation, SRS2. We show that suppression of rad6 delta is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6 delta SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes. Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into the RAD52 DNA repair pathway are discussed.     

Ionizing radiation-induced foci formation of mammalian Rad51 and Rad54 depends on the Rad51 paralogs, but not on Rad52

Homologous recombination is of major importance for the prevention of genomic instability during chromosome duplication and repair of DNA damage, especially double-strand breaks. Biochemical experiments have revealed that during the process of homologous recombination the RAD52 group proteins, including Rad51, Rad52 and Rad54, are involved in an essential step: formation of a joint molecule between the broken DNA and the intact repair template. Accessory proteins for this reaction include the Rad51 paralogs and BRCA2. The significance of homologous recombination for the cell is underscored by the evolutionary conservation of the Rad51, Rad52 and Rad54 proteins from yeast to humans. Upon treatment of cells with ionizing radiation, the RAD52 group proteins accumulate at the sites of DNA damage into so-called foci. For the yeast Saccharomyces cerevisiae, foci formation of Rad51 and Rad54 is abrogated in the absence of Rad52, while Rad51 foci formation does occur in the absence of the Rad51 paralog Rad55. By contrast, we show here that in mammalian cells, Rad52 is not required for foci formation of Rad51 and Rad54. Furthermore, radiation-induced foci formation of Rad51 and Rad54 is impaired in all Rad51 paralog and BRCA2 mutant cell lines tested, while Rad52 foci formation is not influenced by a mutation in any of these recombination proteins. Despite their evolutionary conservation and biochemical similarities, S. cerevisiae and mammalian Rad52 appear to differentially contribute to the DNA-damage response.     

The Tumor Suppressor PML Specifically Accumulates at RPA/Rad51-Containing DNA Damage Repair Foci but Is Nonessential for DNA Damage-Induced Fibroblast Senescence

The PML tumor suppressor has been functionally implicated in DNA damage response and cellular senescence. Direct evidence for such a role based on PML knockdown or knockout approaches is still lacking. We have therefore analyzed the irradiation-induced DNA damage response and cellular senescence in human and mouse fibroblasts lacking PML. Our data show that PML nuclear bodies (NBs) nonrandomly associate with persistent DNA damage foci in unperturbed human skin and in high-dose-irradiated cell culture systems. PML bodies do not associate with transient γH2AX foci after low-dose gamma irradiation. Superresolution microscopy reveals that all PML bodies within a nucleus are engaged at Rad51- and RPA-containing repair foci during ongoing DNA repair. The lack of PML (i) does not majorly affect the DNA damage response, (ii) does not alter the efficiency of senescence induction after DNA damage, and (iii) does not affect the proliferative potential of primary mouse embryonic fibroblasts during serial passaging. Thus, while PML NBs specifically accumulate at Rad51/RPA-containing lesions and senescence-derived persistent DNA damage foci, they are not essential for DNA damage-induced and replicative senescence of human and murine fibroblasts.     

Rad22Rad52-dependent repair of ribosomal DNA repeats cleaved by Slx1-Slx4 endonuclease

Slx1 and Slx4 are subunits of a structure-specific DNA endonuclease that is found in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and other eukaryotic species. It is thought to initiate recombination events or process recombination structures that occur during the replication of the tandem repeats of the ribosomal DNA (rDNA) locus. Here, we present evidence that fission yeast Slx1-Slx4 initiates homologous recombination events in the rDNA repeats that are processed by a mechanism that requires Rad22 (Rad52 homologue) but not Rhp51 (Rad51 homologue). Slx1 is required to generate approximately 50% of the spontaneous Rad22 DNA repair foci that occur in cycling cells. Most of these foci colocalize with the nucleolus, which contains the rDNA repeats. The increased fork pausing at the replication fork barriers in the rDNA repeats in a strain that lacks Rqh1 DNA helicase is further increased by expression of a dominant negative form of Slx1. These data suggest that Slx1-Slx4 cleaves paused replication forks in the rDNA, leading to Rad22-dependent homologous recombination that is used to maintain rDNA copy number.     

A Cell Cycle-Dependent Regulatory Circuit Composed of 53BP1-RIF1 and BRCA1-CtIP Controls DNA Repair Pathway Choice

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Highlights? 53BP1 inhibits BRCA1 recruitment to DSB sites in G1 ? RIF1 is the effector of 53BP1 during DSB repair ? Class-switch recombination requires RIF1 ? RIF1 recruitment to DSB sites in S/G2 is inhibited by BRCA1-CtIP     

Functional Interplay between the 53BP1-Ortholog Rad9 and the Mre11 Complex Regulates Resection,End-Tethering and Repair of a Double-Strand Break

The Mre11-Rad50-Xrs2 nuclease complex, together with Sae2, initiates the 5′-to-3′ resection of Double-Strand DNA Breaks (DSBs). Extended 3′ single stranded DNA filaments can be exposed from a DSB through the redundant activities of the Exo1 nuclease and the Dna2 nuclease with the Sgs1 helicase. In the absence of Sae2, Mre11 binding to a DSB is prolonged, the two DNA ends cannot be kept tethered, and the DSB is not efficiently repaired. Here we show that deletion of the yeast 53BP1-ortholog RAD9 reduces Mre11 binding to a DSB, leading to Rad52 recruitment and efficient DSB end-tethering, through an Sgs1-dependent mechanism. As a consequence, deletion of RAD9 restores DSB repair either in absence of Sae2 or in presence of a nuclease defective MRX complex. We propose that, in cells lacking Sae2, Rad9/53BP1 contributes to keep Mre11 bound to a persistent DSB, protecting it from extensive DNA end resection, which may lead to potentially deleterious DNA deletions and genome rearrangements.     

Role of ATM and the Damage Response Mediator Proteins 53BP1 and MDC1 in the Maintenance of G2/M Checkpoint Arrest

ATM-dependent initiation of the radiation-induced G₂/M checkpoint arrest is well established. Recent results have shown that the majority of DNA double-strand breaks (DSBs) in G₂ phase are repaired by DNA nonhomologous end joining (NHEJ), while ∼15% of DSBs are slowly repaired by homologous recombination. Here, we evaluate how the G₂/M checkpoint is maintained in irradiated G₂ cells, in light of our current understanding of G₂ phase DSB repair. We show that ATM-dependent resection at a subset of DSBs leads to ATR-dependent Chk1 activation. ATR-Seckel syndrome cells, which fail to efficiently activate Chk1, and small interfering RNA (siRNA) Chk1-treated cells show premature mitotic entry. Thus, Chk1 significantly contributes to maintaining checkpoint arrest. Second, sustained ATM signaling to Chk2 contributes, particularly when NHEJ is impaired by XLF deficiency. We also show that cells lacking the mediator proteins 53BP1 and MDC1 initially arrest following radiation doses greater than 3 Gy but are subsequently released prematurely. Thus, 53BP1^−/− and MDC1^−/− cells manifest a checkpoint defect at high doses. This failure to maintain arrest is due to diminished Chk1 activation and a decreased ability to sustain ATM-Chk2 signaling. The combined repair and checkpoint defects conferred by 53BP1 and MDC1 deficiency act synergistically to enhance chromosome breakage.DNA double-strand breaks (DSBs) activate the DNA damage response (DDR), a coordinated process that functions to enhance survival and maintain genomic stability. The DDR includes pathways of DSB repair and a signal transduction response that activates apoptosis and cell cycle checkpoint arrest and influences DSB repair (15). DNA nonhomologous end joining (NHEJ) and homologous recombination (HR) represent the major DSB repair mechanisms, NHEJ being the major mechanism in G₀/G₁, while both processes function in G₂ (9, 32). Ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) are related phosphoinositol 3-kinase-like kinases (PIKKs) that regulate the DNA damage signaling response. ATM is activated by DSBs, while ATR is activated at single-strand (ss) regions of DNA via a process that involves ATRIP-replication protein A (RPA)-ssDNA association. Ionizing radiation (IR) induces DSBs, base damage, and ss nicks. Since neither base damage nor ss nicks activate ATR, IR-induced signaling in the G₁ and G₂ phases is predominantly ATM dependent (3, 29). In S phase, ATR can be activated by both endogenous and exogenously induced lesions following replication fork stalling/collapse (8).Recent work has shown that in G₂ phase, DSBs can undergo resection via an ATM-dependent process generating ssDNA regions that can activate ATR following RPA association (11). ATR activation at resected DSBs is coupled to loss of ATM activation (11). Although ATM and ATR share overlapping substrates, there is specificity in their signaling to the transducer kinases; ATM uniquely phosphorylates Chk2, while ATR phosphorylates Chk1. Phosphorylation of either Chk1 or Chk2 causes their activation. Critical targets of Chk1/Chk2 are the Cdc25 phosphatases, which regulate the cyclin-dependent kinases (Cdks), including Cdk1, the regulator of mitotic entry (18). Collectively, these studies suggest that two components of ATM-dependent signaling to the G₂/M checkpoint machinery can occur: ATM-Chk2 signaling at unresected DSBs and ATM-ATR-Chk1 signaling at resected DSBs.Although much is known about the mechanism leading to G₂/M checkpoint activation, few studies have addressed how arrest is maintained and how release coordinates with the status of DSB repair. We examine here the maintenance of checkpoint arrest during the immediate phase of DSB repair. We do not address the issue of checkpoint adaptation, a distinct phenomenon which occurs after prolonged checkpoint arrest (22). Further, we focus on the process maintaining arrest in irradiated G₂-phase cells and do not consider how arrest is maintained in irradiated S-phase cells that progress into G₂ phase. (Previous studies have shown that while G₂/M arrest is ATM dependent at early times post-IR, at later times it becomes ATR dependent as S-phase cells progress into G₂ phase [2, 33].) To focus on mechanisms maintaining ATM-dependent signaling in G₂-phase cells, we use aphidicolin (APH) to prevent S-phase cells from progressing into G₂ during analysis. We, thus, examine checkpoint maintenance in cells irradiated in G₂ phase and do not evaluate arrest regulated by ATR following replication fork stalling. The basis for our work stems from two recent advances. First, we evaluate the impact of ATM-mediated ATR activation in the light of recent findings that resection occurs in G₂ phase (11). Second, we consider the finding that NHEJ represents the major DSB repair mechanism in G₂ and that a 15 to 20% subset of DSBs, representing those that are rejoined with slow kinetics in an ATM-dependent manner, undergo resection and repair by HR (3, 25). Thus, contrary to the notion that HR represents the major DSB repair pathway in G₂ phase, it repairs only 15 to 20% of X- or gamma-ray-induced DSBs and represents the slow component of DSB repair in G₂ phase. Given these findings, several potential models for how checkpoint arrest is maintained in G₂ can be envisaged. A simple model is that the initial signal generated by IR is maintained for a defined time to allow for DSB repair. Such a model appears to explain the kinetics of checkpoint signaling in fission yeast after moderate IR (17). In mammalian cells, the duration of arrest depends on dose and DSB repair capacity (6). Thus, it is possible that the status of ongoing repair is communicated to the checkpoint machinery to coordinate timely release with the process of DSB repair. Here, we consider the impact of resection leading to ATM-ATR-Chk1 signaling versus ATM-Chk2 signaling from nonresected DSBs and how they interplay to maintain rather than initiate checkpoint arrest.Mediator proteins, including 53BP1 and MDC1, assemble at DSBs in an ATM-dependent manner, but their roles in the DDR are unclear. Cells lacking 53BP1 or MDC1 are proficient in checkpoint initiation after moderate IR doses, leading to the suggestion that these proteins are required for amplification of the ATM signal after exposure to low doses but are dispensable after high doses, when a robust signal is generated, even in their absence (7, 16, 28, 31). Despite their apparent subtle role in ATM signaling, cells lacking these mediator proteins display significant genomic instability (19). We thus also examine whether the mediator proteins contribute to the maintenance of checkpoint arrest.We identify two ATM-dependent processes that contribute to the maintenance of checkpoint arrest in G₂-phase cells: (i) ATR-Chk1 activation at resected DSBs and (ii) a process that involves sustained signaling from ATM to Chk2 at unrepaired DSBs. Further, we show that 53BP1 and MDC1 are required for maintaining checkpoint arrest, even following exposure to high radiation doses due to roles in ATR-Chk1 activation and sustained ATM-Chk2 signaling, and that this contributes to their elevated genomic instability.     

The GAR Motif of 53BP1 is Arginine Methylated by PRMT1 and is Necessary for 53BP1 DNA Binding Activity

The p53-binding protein 1 (53BP1) is rapidly recruited to sites of DNA double-strand breaks and forms characteristics nuclear foci, demonstrating its role in the early events of detection, signaling and repair of damaged DNA. 53BP1 contains a glycine arginine rich (GAR) motif of unknown function within its kinetochore binding domain. Herein, we show that the GAR motif of 53BP1 is arginine methylated by protein arginine methyltransferase 1 (PRMT1), the same methyltransferase that methylates MRE11. 53BP1 contains asymmetric dimethylarginines (aDMA) within cells, as detected with methylarginine-specific antibodies. Amino acid substitution of the arginines within the GAR motif of 53BP1 abrogated binding to single and double-stranded DNA, demonstrating that the GAR motif is required for DNA binding activity of 53BP1. Fibroblast cells treated with methylase inhibitors failed to relocalize 53BP1 to sites of DNA damage and formed few ?-H2AX foci, consistent with our previous data that MRE11 fails to relocalize to DNA damage sites in cells treated with methylase inhibitors. Our findings identify the GAR motif as a region required for 53BP1 DNA binding activity and is the site of methylation by PRMT1.     

Photoenzymatic Repair of Ultraviolet Damage in DNA : I. Kinetics of the reaction

As previously reported, ultraviolet-inactivated bacterial transforming DNA can be restored to activity by an enzyme-like agent from bakers' yeast which requires light for its activity. Kinetics of this reaction, in the presence and absence of inhibitors, are found consistent with the Michaelis-Menten reaction scheme, with the sites of ultraviolet damage on the DNA serving as substrate and the repaired structure as product. Kinetic studies with different light intensities suggest that the necessary illumination causes photolysis of the enzyme-substrate complex with concurrent repair of the DNA. Competitive inhibition of irradiated transforming DNA repair, which occurs when irradiated non-transforming DNA is present in the same reaction mixture, permits ultraviolet damage (of the kind capable of being photoreactivated) to be detected in any type of DNA.     

Rad51 and Rad52 Are Involved in Homologous Recombination of Replicating Herpes Simplex Virus DNA

Replication of herpes simplex virus 1 is coupled to recombination, but the molecular mechanisms underlying this process are poorly characterized. The role of Rad51 and Rad52 recombinases in viral recombination was examined in human fibroblast cells 1BR.3.N (wild type) and in GM16097 with replication defects caused by mutations in DNA ligase I. Intermolecular recombination between viruses, tsS and tsK, harboring genetic markers gave rise to ∼17% recombinants in both cell lines. Knock-down of Rad51 and Rad52 by siRNA reduced production of recombinants to 11% and 5%, respectively, in wild type cells and to 3% and 5%, respectively, in GM16097 cells. The results indicate a specific role for Rad51 and Rad52 in recombination of replicating herpes simplex virus 1 DNA. Mixed infections using clinical isolates with restriction enzyme polymorphisms in the US4 and US7 genes revealed recombination frequencies of 0.7%/kbp in wild type cells and 4%/kbp in GM16097 cells. Finally, tandem repeats in the US7 gene remained stable upon serial passage, indicating a high fidelity of recombination in infected cells.