Shan Yan

TopBP1 and DNA polymerase-α directly recruit the 9-1-1 complex to stalled DNA replication forks

TopBP1 and the Rad9–Rad1–Hus1 (9-1-1) complex activate the ataxia telangiectasia mutated and Rad3-related (ATR) protein kinase at stalled replication forks. ATR is recruited to stalled forks through its binding partner, ATR-interacting protein (ATRIP); however, it is unclear how TopBP1 and 9-1-1 are recruited so that they may join ATR–ATRIP and initiate signaling. In this study, we use Xenopus laevis egg extracts to determine the requirements for 9-1-1 loading. We show that TopBP1 is required for the recruitment of both 9-1-1 and DNA polymerase (pol)-α to sites of replication stress. Furthermore, we show that pol-α is also directly required for Rad9 loading. Our study identifies an assembly pathway, which is controlled by TopBP1 and includes pol-α, that mediates the loading of the 9-1-1 complex onto stalled replication forks. These findings clarify early events in the assembly of checkpoint signaling complexes on DNA and identify TopBP1 as a critical sensor of replication stress.

Functional interplay between ATM/ATR-mediated DNA damage response and DNA repair pathways in oxidative stress

To maintain genome stability, cells have evolved various DNA repair pathways to deal with oxidative DNA damage. DNA damage response (DDR) pathways, including ATM-Chk2 and ATR-Chk1 checkpoints, are also activated in oxidative stress to coordinate DNA repair, cell cycle progression, transcription, apoptosis, and senescence. Several studies demonstrate that DDR pathways can regulate DNA repair pathways. On the other hand, accumulating evidence suggests that DNA repair pathways may modulate DDR pathway activation as well. In this review, we summarize our current understanding of how various DNA repair and DDR pathways are activated in response to oxidative DNA damage primarily from studies in eukaryotes. In particular, we analyze the functional interplay between DNA repair and DDR pathways in oxidative stress. A better understanding of cellular response to oxidative stress may provide novel avenues of treating human diseases, such as cancer and neurodegenerative disorders.

Continued primer synthesis at stalled replication forks contributes to checkpoint activation.

An increased number of primer–template junctions generated by PCNA, Pol-δ, and Pol-ε at stalled replication forks activates Chk1.

Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling

TopBP1-like proteins, which include Xenopus laevis Xmus101, are required for DNA replication and have been linked to replication checkpoint control. A direct role for TopBP1/Mus101 in checkpoint control has been difficult to prove, however, because of the requirement for replication in generating the DNA structures that activate the checkpoint. Checkpoint activation occurs in X. laevis egg extracts upon addition of an oligonucleotide duplex (AT70). We show that AT70 bypasses the requirement for replication in checkpoint activation. We take advantage of this replication-independent checkpoint system to determine the role of Xmus101 in the checkpoint. We find that Xmus101 is essential for AT70-mediated checkpoint signaling and that it functions to promote phosphorylation of Claspin bound Chk1 by the ataxia-telangiectasia and Rad-3–related (ATR) protein kinase. We also identify a separation-of-function mutant of Xmus101. In extracts expressing this mutant, replication of sperm chromatin occurs normally; however, the checkpoint response to stalled replication forks fails. These data demonstrate that Xmus101 functions directly during signal relay from ATR to Chk1.

APE2 is required for ATR-Chk1 checkpoint activation in response to oxidative stress

The base excision repair pathway is largely responsible for the repair of oxidative stress-induced DNA damage. However, it remains unclear how the DNA damage checkpoint is activated by oxidative stress at the molecular level. Here, we provide evidence showing that hydrogen peroxide (H2O2) triggers checkpoint kinase 1 (Chk1) phosphorylation in an ATR [ataxia-telangiectasia mutated (ATM) and Rad3-related]-dependent but ATM-independent manner in Xenopus egg extracts. A base excision repair protein, Apurinic/apyrimidinic (AP) endonuclease 2 (APE2, APN2, or APEX2), is required for the generation of replication protein A (RPA)-bound single-stranded DNA, the recruitment of a checkpoint protein complex [ATR, ATR-interacting protein (ATRIP), and Rad9] to damage sites, and H2O2-induced Chk1 phosphorylation. A conserved proliferating cell nuclear antigen interaction protein box of APE2 is important for the recruitment of APE2 to H2O2-damaged chromatin. APE2 3′-phosphodiesterase and 3′-5′ exonuclease activity is essential for single-stranded DNA generation in the 3′–5′ direction from single-stranded breaks, referred to as single-stranded break end resection. In addition, APE2 associates with Chk1, and a serine residue (S86) in the Chk1-binding motif of APE2 is essential for Chk1 phosphorylation, indicating a Claspin-like but distinct role for APE2 in ATR-Chk1 signaling. Our data indicate that APE2 plays a vital and previously unexpected role in ATR-Chk1 checkpoint signaling in response to oxidative stress. Thus, our findings shed light on a distinct mechanism of how an ATR-Chk1–dependent DNA damage checkpoint is mediated by APE2 in the oxidative stress response.

TopBP1 and DNA polymerase alpha-mediated recruitment of the 9-1-1 complex to stalled replication forks: Implications for a replication restart-based mechanism for ATR checkpoint activation

Upon sensing DNA damage or replication stress, cells trigger checkpoint response pathways that control cell cycle progression and maintain genomic stability. A variety of DNA lesions can activate the ATR (ATM and Rad3-related) protein kinase, which phosphorylates its critical substrate Chk1 to relay the checkpoint signal. ATR activation requires several factors, including the BRCT repeat-containing TopBP1 protein and the 9-1-1 clamp protein. Here, we summarize recent advances in understanding the multiple roles played by TopBP1 in ATR activation at stalled replication forks. We review recent studies showing that TopBP1 controls the loading of 9-1-1 onto stalled replication forks via a pathway that also requires DNA polymerase alpha (pol α). Based on these recent studies, we present a revised model for ATR activation, and speculate that TopBP1-mediated recruitment of pol α and 9-1-1 may couple checkpoint activation to replication restart, when DNA synthesis is blocked on the leading strand of a replication fork. Lastly, we present a new experiment that examines how TopBP1 binds to stalled replication forks, and we identify important new questions that our recent studies have raised regarding how stalled replication forks are sensed by the ATR checkpoint pathway.

Oxidative Stress, Bone Marrow Failure, and Genome Instability in Hematopoietic Stem Cells

Reactive oxygen species (ROS) can be generated by defective endogenous reduction of oxygen by cellular enzymes or in the mitochondrial respiratory pathway, as well as by exogenous exposure to UV or environmental damaging agents. Regulation of intracellular ROS levels is critical since increases above normal concentrations lead to oxidative stress and DNA damage. A growing body of evidence indicates that the inability to regulate high levels of ROS leading to alteration of cellular homeostasis or defective repair of ROS-induced damage lies at the root of diseases characterized by both neurodegeneration and bone marrow failure as well as cancer. That these diseases may be reflective of the dynamic ability of cells to respond to ROS through developmental stages and aging lies in the similarities between phenotypes at the cellular level. This review summarizes work linking the ability to regulate intracellular ROS to the hematopoietic stem cell phenotype, aging, and disease.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop. Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle. The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures. DNA damage can be induced by ultraviolet (UV) irradiation, γ-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphys laboratory and is widely used to induce ATR/Chk1 checkpoint signaling. Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Importin β-dependent nuclear import of TopBP1 in ATR–Chk1 checkpoint in Xenopus egg extracts

TopBP1, a multiple-BRCT-containing protein, plays diverse functions in DNA metabolism including DNA replication, DNA damage response and transcriptional regulation. The cytoplasmic localization of TopBP1 has been found to be associated with breast cancer susceptibility in clinical studies, suggesting the biological significance of TopBP1s sub-cellular localization. However, it remains elusive how TopBP1 is shuttled into nucleus and recruited to chromatin under normal or stressful conditions. Taking advantage of Xenopus egg extract, we identified Importin β as a new interacting protein of the TopBP1 C-terminus. We verified the TopBP1-Importin β association via GST pulldown and coimmunoprecipitation assays. We then demonstrated that TopBP1s C-terminal motif (designated as CTM, 23 amino acids) containing a putative NLS (nuclear localization signal) was required for Importin β interaction and that CT100 of Importin β (100 amino acids of extreme C-terminus of Importin β) was required for TopBP1 interaction. Further structure-function analysis reveals that the CTM of TopBP1 is essential for TopBP1s nuclear import and subsequent chromatin recruitment, thereby playing important roles in DNA replication and mitomycin C (MMC)-induced Chk1 phosphorylation. In addition, Importin β-specific inhibitor importazole inhibits TopBP1s nuclear import and the MMC-induced Chk1 phosphorylation. With ongoing DNA replication, the Importin β-dependent nuclear import of TopBP1 was indeed required for the MMC-induced Chk1 phosphorylation. Our data also suggest that checkpoint activation requires more TopBP1 than DNA replication does. The requirement of TopBP1s CTM motif for ATR-Chk1 checkpoint can be bypassed in a nucleus-free AT70 system. Taken together, our findings suggest that the CTM motif-mediated TopBP1 shuttling into nucleus via Importin β plays an important role in the ATR-Chk1 checkpoint signaling in Xenopus egg extracts.

Direct Binding to Replication Protein A (RPA)-coated Single-stranded DNA Allows Recruitment of the ATR Activator TopBP1 to Sites of DNA Damage.

A critical event for the ability of cells to tolerate DNA damage and replication stress is activation of the ATR kinase. ATR activation is dependent on the BRCT (BRCA1 C terminus) repeat-containing protein TopBP1. Previous work has shown that recruitment of TopBP1 to sites of DNA damage and stalled replication forks is necessary for downstream events in ATR activation; however, the mechanism for this recruitment was not known. Here, we use protein binding assays and functional studies in Xenopus egg extracts to show that TopBP1 makes a direct interaction, via its BRCT2 domain, with RPA-coated single-stranded DNA. We identify a point mutant that abrogates this interaction and show that this mutant fails to accumulate at sites of DNA damage and that the mutant cannot activate ATR. These data thus supply a mechanism for how the critical ATR activator, TopBP1, senses DNA damage and stalled replication forks to initiate assembly of checkpoint signaling complexes.