Analysis of mutations that dissociate G(2) and essential S phase functions of human ataxia telangiectasia-mutated and Rad3-related (ATR) protein kinase

ATR (ataxia telangiectasia-mutated and Rad3-related) contains 16 conserved candidate autophosphorylation sites that match its preferred S/TQ consensus. To determine whether any is functionally important, we mutated the 16 candidate residues to alanine in a single cDNA to create a 16A-ATR mutant. The 16A-ATR mutant maintains kinase and G(2) checkpoint activities. However, it fails to rescue the essential function of ATR in maintaining cell viability and fails to promote replication recovery from a transient exposure to replication stress. Further analysis identified T1566A/T1578A/T1589A (3A-ATR) as critical mutations causing this separation of function activity. Secondary structure predictions indicate that these residues occur in a region between ATR HEAT repeats 31R and 32R that aligns with regions of ATM and DNA-PK containing regulatory autophosphorylation sites. Although this region is important for ATR function, the 3A-ATR residues do not appear to be sites of autophosphorylation. Nevertheless, our analysis identifies an important regulatory region of ATR that is shared among the PI3K-related protein kinase family. Furthermore, our data indicate that the essential function of ATR for cell viability is linked to its function in promoting proper replication in the context of replication stress and is independent of G(2) checkpoint activity.     

Tethering DNA damage checkpoint mediator proteins topoisomerase IIbeta-binding protein 1 (TopBP1) and Claspin to DNA activates ataxia-telangiectasia mutated and RAD3-related (ATR) phosphorylation of checkpoint kinase 1 (Chk1)

The ataxia-telangiectasia mutated and RAD3-related (ATR) kinase initiates DNA damage signaling pathways in human cells after DNA damage such as that induced upon exposure to ultraviolet light by phosphorylating many effector proteins including the checkpoint kinase Chk1. The conventional view of ATR activation involves a universal signal consisting of genomic regions of replication protein A-covered single-stranded DNA. However, there are some indications that the ATR-mediated checkpoint can be activated by other mechanisms. Here, using the well defined Escherichia coli lac repressor/operator system, we have found that directly tethering the ATR activator topoisomerase IIβ-binding protein 1 (TopBP1) to DNA is sufficient to induce ATR phosphorylation of Chk1 in an in vitro system as well as in vivo in mammalian cells. In addition, we find synergistic activation of ATR phosphorylation of Chk1 when the mediator protein Claspin is also tethered to the DNA with TopBP1. Together, these findings indicate that crowding of checkpoint mediator proteins on DNA is sufficient to activate the ATR kinase.     

Thr-1989 phosphorylation is a marker of active ataxia telangiectasia-mutated and Rad3-related (ATR) kinase

The DNA damage response kinases ataxia telangiectasia-mutated (ATM), DNA-dependent protein kinase (DNA-PK), and ataxia telangiectasia-mutated and Rad3-related (ATR) signal through multiple pathways to promote genome maintenance. These related kinases share similar methods of regulation, including recruitment to specific nucleic acid structures and association with protein activators. ATM and DNA-PK also are regulated via phosphorylation, which provides a convenient biomarker for their activity. Whether phosphorylation regulates ATR is unknown. Here we identify ATR Thr-1989 as a DNA damage-regulated phosphorylation site. Selective inhibition of ATR prevents Thr-1989 phosphorylation, and phosphorylation requires ATR activation. Cells engineered to express only a non-phosphorylatable T1989A mutant exhibit a modest ATR functional defect. Our results suggest that, like ATM and DNA-PK, phosphorylation regulates ATR, and phospho-peptide specific antibodies to Thr-1989 provide a proximal marker of ATR activation.     

A Dbf4 mutant contributes to bypassing the Rad53-mediated block of origins of replication in response to genotoxic stress

An intra-S phase checkpoint slows the rate of DNA replication in response to DNA damage and replication fork blocks in eukaryotic cells. In the budding yeast Saccharomyces cerevisiae, such down-regulation is achieved through the Rad53 kinase-dependent block of origins of replication. We have identified the Rad53 phosphorylation sites on Dbf4, the activator subunit of the essential S phase Dbf4-dependent kinase, and generated a non-phosphorylatable Dbf4 mutant (dbf4(7A)). We show here that dbf4(7A) is a bona fide intra-S phase checkpoint bypass allele that contributes to abrogating the Rad53 block of origin firing in response to genotoxic stress.     

The unstructured C-terminal tail of yeast Dpb11 (human TopBP1) protein is dispensable for DNA replication and the S phase checkpoint but required for the G2/M checkpoint

Budding yeast Dpb11 (human TopBP1, fission yeast Cut5) is an essential protein required for replisome assembly and for the DNA damage checkpoint. Previous studies with the temperature-sensitive dpb11-1 allele, truncated at amino acid 583 of the 764-amino acid protein, have suggested the model that Dpb11 couples DNA replication to the replication checkpoint. However, the dpb11-1 allele shows distinct replication defects even at permissive temperatures. Here, we determine that the 1-600-amino acid domain of DPB11 is both required and sufficient for full replication function of Dpb11 but that this domain is defective for activation of the principal checkpoint kinase Mec1 (human ataxia telangiectasia and Rad3-related) in vitro and in vivo. Remarkably, mutants of DPB11 that leave its replication function intact but abrogate its ability to activate Mec1 are proficient for the replication checkpoint, but they are compromised for the G(2)/M DNA damage checkpoint. These data suggest that replication checkpoint defects may result indirectly from defects in replisome assembly. Two conserved aromatic amino acids in the C terminus of Dpb11 are critical for Mec1 activation in vitro and for the G(2)/M checkpoint in yeast. Together with aromatic motifs identified previously in the Ddc1 subunit of 9-1-1, another activator of Mec1 kinase, they define a consensus structure for Mec1 activation.     

Fanconi anemia complementation group A (FANCA) protein has intrinsic affinity for nucleic acids with preference for single-stranded forms

The Fanconi anemia complementation group A (FANCA) gene is one of 15 disease-causing genes and has been found to be mutated in ～60% of Fanconi anemia patients. Using purified protein, we report that human FANCA has intrinsic affinity for nucleic acids. FANCA binds to both single-stranded (ssDNA) and double-stranded (dsDNA) DNAs; however, its affinity for ssDNA is significantly higher than for dsDNA in an electrophoretic mobility shift assay. FANCA also binds to RNA with an intriguingly higher affinity than its DNA counterpart. FANCA requires a certain length of nucleic acids for optimal binding. Using DNA and RNA ladders, we determined that the minimum number of nucleotides required for FANCA recognition is ～30 for both DNA and RNA. By testing the affinity between FANCA and a variety of DNA structures, we found that a 5'-flap or 5'-tail on DNA facilitates its interaction with FANCA. A patient-derived FANCA truncation mutant (Q772X) has diminished affinity for both DNA and RNA. In contrast, the complementing C-terminal fragment of Q772X, C772-1455, retains the differentiated nucleic acid-binding activity (RNA > ssDNA > dsDNA), indicating that the nucleic acid-binding domain of FANCA is located primarily at its C terminus, where most disease-causing mutations are found.     

Degradation of p12 Subunit by CRL4Cdt2 E3 Ligase Inhibits Fork Progression after DNA Damage

After acute DNA damage, the cell arrests S-phase progression by inhibiting origin initiation and fork progression to repair damaged DNA. The intra-S-phase checkpoint kinase Chk1 phosphorylates Cdc25A to target the latter for degradation by CRL1^β-TrCP and so inhibit origin firing. The mechanism for inhibiting fork progression, however, has not been identified. Here, we show that degradation of p12, the fourth subunit of DNA polymerase δ, is critical for inhibiting fork progression. CRL4^Cdt2 is an E3 ligase that ubiquitinates and degrades p12 after UV treatment. Cells expressing a stable form of p12 exhibit UV-resistant DNA synthesis. DNA fiber assay and alkaline-sucrose gradient assay demonstrate that the impairment of fork progression after DNA damage requires p12 degradation. These results suggest that ubiquitination of p12 through CRL4^Cdt2 and subsequent degradation form one mechanism by which a cell responds to DNA damage to inhibit fork progression.     

Structural analysis of Shu proteins reveals a DNA binding role essential for resisting damage

The yeast Shu complex, consisting of the proteins Shu1, Shu2, Psy3, and Csm2, maintains genomic stability by coupling post-replication repair to homologous recombination. However, a lack of biochemical and structural information on the Shu proteins precludes revealing their precise roles within the pathway. Here, we report on the 1.9-Å crystal structure of the Psy3-Csm2 complex. The crystal structure shows that Psy3 forms a heterodimer with Csm2 mainly through a hydrophobic core. Unexpectedly, Psy3 and Csm2 share a similar architecture that closely resembles the ATPase core domain of Rad51. The L2 loop present in Psy3 and Csm2 is similar to that of Rad51 and confers the DNA binding activity of the Shu complex. As with Rad51, the Shu complex appears to form a nucleoprotein filament by binding nonspecifically to DNA. Structure-based mutagenesis studies have demonstrated that the DNA binding activity of the Shu complex is essential for repair of the methyl methanesulfonate-induced DNA damage. Our findings provide good foundations for the understanding of the Srs2 regulation by the Shu complex.     

Human DNA helicase B (HDHB) binds to replication protein A and facilitates cellular recovery from replication stress

Maintenance of genomic stability in proliferating cells depends on a network of proteins that coordinate chromosomal replication with DNA damage responses. Human DNA helicase B (HELB or HDHB) has been implicated in chromosomal replication, but its role in this coordinated network remains undefined. Here we report that cellular exposure to UV irradiation, camptothecin, or hydroxyurea induces accumulation of HDHB on chromatin in a dose- and time-dependent manner, preferentially in S phase cells. Replication stress-induced recruitment of HDHB to chromatin is independent of checkpoint signaling but correlates with the level of replication protein A (RPA) recruited to chromatin. We show using purified proteins that HDHB physically interacts with the N-terminal domain of the RPA 70-kDa subunit (RPA70N). NMR spectroscopy and site-directed mutagenesis reveal that HDHB docks on the same RPA70N surface that recruits S phase checkpoint signaling proteins to chromatin. Consistent with this pattern of recruitment, cells depleted of HDHB display reduced recovery from replication stress.     

RING finger and WD repeat domain 3 (RFWD3) associates with replication protein A (RPA) and facilitates RPA-mediated DNA damage response

DNA damage response is crucial for maintaining genomic integrity and preventing cancer by coordinating the activation of checkpoints and the repair of damaged DNA. Central to DNA damage response are the two checkpoint kinases ATM and ATR that phosphorylate a wide range of substrates. RING finger and WD repeat domain 3 (RFWD3) was initially identified as a substrate of ATM/ATR from a proteomic screen. Subsequent studies showed that RFWD3 is an E3 ubiquitin ligase that ubiquitinates p53 in vitro and positively regulates p53 levels in response to DNA damage. We report here that RFWD3 associates with replication protein A (RPA), a single-stranded DNA-binding protein that plays essential roles in DNA replication, recombination, and repair. Binding of RPA to single-stranded DNA (ssDNA), which is generated by DNA damage and repair, is essential for the recruitment of DNA repair factors to damaged sites and the activation of checkpoint signaling. We show that RFWD3 is physically associated with RPA and rapidly localizes to sites of DNA damage in a RPA-dependent manner. In vitro experiments suggest that the C terminus of RFWD3, which encompass the coiled-coil domain and the WD40 domain, is necessary for binding to RPA. Furthermore, DNA damage-induced phosphorylation of RPA and RFWD3 is dependent upon each other. Consequently, loss of RFWD3 results in the persistent foci of DNA damage marker γH2AX and the repair protein Rad51 in damaged cells. These findings suggest that RFWD3 is recruited to sites of DNA damage and facilitates RPA-mediated DNA damage signaling and repair.     

The PSO4 Protein Complex Associates with Replication Protein A (RPA) and Modulates the Activation of Ataxia Telangiectasia-mutated and Rad3-related (ATR)

The PSO4 core complex is composed of PSO4/PRP19/SNEV, CDC5L, PLRG1, and BCAS2/SPF27. Besides its well defined functions in pre-mRNA splicing, the PSO4 complex has been shown recently to participate in the DNA damage response. However, the specific role for the PSO4 complex in the DNA damage response pathways is still not clear. Here we show that both the BCAS2 and PSO4 subunits of the PSO4 complex directly interact and colocalize with replication protein A (RPA). Depletion of BCAS2 or PSO4 impairs the recruitment of ATR-interacting protein (ATRIP) to DNA damage sites and compromises CHK1 activation and RPA2 phosphorylation. Moreover, we demonstrate that both the RPA1-binding ability of BCAS2 and the E3 ligase activity of PSO4 are required for efficient accumulation of ATRIP at DNA damage sites and the subsequent CHK1 activation and RPA2 phosphorylation. Our results suggest that the PSO4 complex functionally interacts with RPA and plays an important role in the DNA damage response.     

Activation of the Prereplication Complex Is Blocked by Mimosine through Reactive Oxygen Species-activated Ataxia Telangiectasia Mutated (ATM) Protein without DNA Damage

Mimosine is an effective cell synchronization reagent used for arresting cells in late G₁ phase. However, the mechanism underlying mimosine-induced G₁ cell cycle arrest remains unclear. Using highly synchronous cell populations, we show here that mimosine blocks S phase entry through ATM activation. HeLa S3 cells are exposed to thymidine for 15 h, released for 9 h by washing out the thymidine, and subsequently treated with 1 mm mimosine for a further 15 h (thymidine → mimosine). In contrast to thymidine-induced S phase arrest, mimosine treatment synchronizes >90% of cells at the G₁-S phase boundary by inhibiting the transition of the prereplication complex to the preinitiation complex. Mimosine treatment activates ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint signaling without inducing DNA damage. Inhibition of ATM activity is found to induce mimosine-arrested cells to enter S phase. In addition, ATM activation by mimosine treatment is mediated by reactive oxygen species (ROS). These results suggest that, upon mimosine treatment, ATM blocks S phase entry in response to ROS, which prevents replication fork stalling-induced DNA damage.     

Alpha Thalassemia/Mental Retardation Syndrome X-linked Gene Product ATRX Is Required for Proper Replication Restart and Cellular Resistance to Replication Stress

Alpha thalassemia/mental retardation syndrome X-linked (ATRX) is a member of the SWI/SNF protein family of DNA-dependent ATPases. It functions as a chromatin remodeler and is classified as an SNF2-like helicase. Here, we showed somatic knock-out of ATRX displayed perturbed S-phase progression as well as hypersensitivity to replication stress. ATRX is recruited to sites of DNA damage, required for efficient checkpoint activation and faithful replication restart. In addition, we identified ATRX as a binding partner of MRE11-RAD50-NBS1 (MRN) complex. Together, these results suggest a non-canonical function of ATRX in guarding genomic stability.     

Molecular cooperation between the Werner syndrome protein and replication protein A in relation to replication fork blockage

The premature aging and cancer-prone disease Werner syndrome is caused by loss of function of the RecQ helicase family member Werner syndrome protein (WRN). At the cellular level, loss of WRN results in replication abnormalities and chromosomal aberrations, indicating that WRN plays a role in maintenance of genome stability. Consistent with this notion, WRN possesses annealing, exonuclease, and ATPase-dependent helicase activity on DNA substrates, with particularly high affinity for and activity on replication and recombination structures. After certain DNA-damaging treatments, WRN is recruited to sites of blocked replication and co-localizes with the human single-stranded DNA-binding protein replication protein A (RPA). In this study we examined the physical and functional interaction between WRN and RPA specifically in relation to replication fork blockage. Co-immunoprecipitation experiments demonstrated that damaging treatments that block DNA replication substantially increased association between WRN and RPA in vivo, and a direct interaction between purified WRN and RPA was confirmed. Furthermore, we examined the combined action of RPA (unmodified and hyperphosphorylation mimetic) and WRN on model replication fork and gapped duplex substrates designed to bind RPA. Even with RPA bound stoichiometrically to this gap, WRN efficiently catalyzed regression of the fork substrate. Further analysis showed that RPA could be displaced from both substrates by WRN. RPA displacement by WRN was independent of its ATPase- and helicase-dependent remodeling of the fork. Taken together, our results suggest that, upon replication blockage, WRN and RPA functionally interact and cooperate to help properly resolve replication forks and maintain genome stability.     

Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System

DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5′ to 3′ exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5′ to 3′ exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.     

RECQL4, the protein mutated in Rothmund-Thomson syndrome, functions in telomere maintenance

Telomeres are structures at the ends of chromosomes and are composed of long tracks of short tandem repeat DNA sequences bound by a unique set of proteins (shelterin). Telomeric DNA is believed to form G-quadruplex and D-loop structures, which presents a challenge to the DNA replication and repair machinery. Although the RecQ helicases WRN and BLM are implicated in the resolution of telomeric secondary structures, very little is known about RECQL4, the RecQ helicase mutated in Rothmund-Thomson syndrome (RTS). Here, we report that RTS patient cells have elevated levels of fragile telomeric ends and that RECQL4-depleted human cells accumulate fragile sites, sister chromosome exchanges, and double strand breaks at telomeric sites. Further, RECQL4 localizes to telomeres and associates with shelterin proteins TRF1 and TRF2. Using recombinant proteins we showed that RECQL4 resolves telomeric D-loop structures with the help of shelterin proteins TRF1, TRF2, and POT1. We also found a novel functional synergistic interaction of this protein with WRN during D-loop unwinding. These data implicate RECQL4 in telomere maintenance.     

Role of human DNA glycosylase Nei-like 2 (NEIL2) and single strand break repair protein polynucleotide kinase 3'-phosphatase in maintenance of mitochondrial genome

The repair of reactive oxygen species-induced base lesions and single strand breaks (SSBs) in the nuclear genome via the base excision (BER) and SSB repair (SSBR) pathways, respectively, is well characterize, and important for maintaining genomic integrity. However, the role of mitochondrial (mt) BER and SSBR proteins in mt genome maintenance is not completely clear. Here we show the presence of the oxidized base-specific DNA glycosylase Nei-like 2 (NEIL2) and the DNA end-processing enzyme polynucleotide kinase 3'-phosphatase (PNKP) in purified human mitochondrial extracts (MEs). Confocal microscopy revealed co-localization of PNKP and NEIL2 with the mitochondrion-specific protein cytochrome c oxidase subunit 2 (MT-CO2). Further, chromatin immunoprecipitation analysis showed association of NEIL2 and PNKP with the mitochondrial genes MT-CO2 and MT-CO3 (cytochrome c oxidase subunit 3); importantly, both enzymes also associated with the mitochondrion-specific DNA polymerase γ. In cell association of NEIL2 and PNKP with polymerase γ was further confirmed by proximity ligation assays. PNKP-depleted ME showed a significant decrease in both BER and SSBR activities, and PNKP was found to be the major 3'-phosphatase in human ME. Furthermore, individual depletion of NEIL2 and PNKP in human HEK293 cells caused increased levels of oxidized bases and SSBs in the mt genome, respectively. Taken together, these studies demonstrate the critical role of NEIL2 and PNKP in maintenance of the mammalian mitochondrial genome.     

Oligomerization of MDC1 protein is important for proper DNA damage response

Mediator of DNA damage checkpoint 1 (MDC1) plays an important role in the DNA damage response (DDR). MDC1 functions as a mediator protein and binds multiple proteins involved in different aspects of the DDR. However, little is know about the organization of MDC1 complexes. Here we show that ataxia telangiectasia, mutated (ATM) phosphorylates MDC1 at Thr-98 following DNA damage, which promotes its oligomerization. Oligomerization of MDC1 is important for the accumulation of MDC1 complex at the sites of DNA damage. Mutation of Thr-98 (T98A) would abolish its oligomerization and result in a defect in DNA damage checkpoint activation and increased sensitivity to irradiation. Taken together, these results suggest that the oligomerization of MDC1 plays an important role in DDR and help understand the formation of proteins complexes at the sites of DNA damage.     

Formation of a stable RuvA protein double tetramer is required for efficient branch migration in vitro and for replication fork reversal in vivo

In bacteria, RuvABC is required for the resolution of Holliday junctions (HJ) made during homologous recombination. The RuvAB complex catalyzes HJ branch migration and replication fork reversal (RFR). During RFR, a stalled fork is reversed to form a HJ adjacent to a DNA double strand end, a reaction that requires RuvAB in certain Escherichia coli replication mutants. The exact structure of active RuvAB complexes remains elusive as it is still unknown whether one or two tetramers of RuvA support RuvB during branch migration and during RFR. We designed an E. coli RuvA mutant, RuvA2(KaP), specifically impaired for RuvA tetramer-tetramer interactions. As expected, the mutant protein is impaired for complex II (two tetramers) formation on HJs, although the binding efficiency of complex I (a single tetramer) is as wild type. We show that although RuvA complex II formation is required for efficient HJ branch migration in vitro, RuvA2(KaP) is fully active for homologous recombination in vivo. RuvA2(KaP) is also deficient at forming complex II on synthetic replication forks, and the binding affinity of RuvA2(KaP) for forks is decreased compared with wild type. Accordingly, RuvA2(KaP) is inefficient at processing forks in vitro and in vivo. These data indicate that RuvA2(KaP) is a separation-of-function mutant, capable of homologous recombination but impaired for RFR. RuvA2(KaP) is defective for stimulation of RuvB activity and stability of HJ·RuvA·RuvB tripartite complexes. This work demonstrates that the need for RuvA tetramer-tetramer interactions for full RuvAB activity in vitro causes specifically an RFR defect in vivo.