Biomedical subjects

Junjie Chen

Publications and source records attributed to Junjie Chen.

At least 55 records · Page 3Linked to original sources

PubMed2004

Structural basis of BACH1 phosphopeptide recognition by BRCA1 tandem BRCT domains.

BRCT tandem domains, found in many proteins involved in DNA damage checkpoint and DNA repair pathways, were recently shown to be phosphopeptide binding motifs. Using solution nuclear magnetic resonance (NMR) spectroscopy and mutational analysis, we have characterized the interaction of BRCA1-BRCT domains with a phosphoserine-containing peptide derived from the DNA repair helicase BACH1. We show that a phenylalanine in the +3 position from the phosphoserine of BACH1 is bound to a conserved hydrophobic pocket formed between the two BRCT domains and that recognition of the phosphate group is mediated by lysine and serine side chains from the amino-terminal BRCT domain. Mutations that prevent phosphopeptide binding abolish BRCA1 function in DNA damage-induced checkpoint control. Our NMR data also reveal a dynamic interaction between BRCA1-BRCT and BACH1, where the bound phosphopeptide exists as an equilibrium of two conformations and where BRCA1-BRCT undergoes a transition to a more rigid conformation upon peptide binding.

BRCA1 Protein↗

PubMed2004

Early events in the DNA damage response.

The ability to sense DNA damage and activate response pathways that coordinate cell cycle progression and DNA repair is essential for the maintenance of genomic integrity and the viability of organisms. During the last couple of years, several proteins have been identified that participate very early in the DNA damage response. Here we review the current understanding of the mechanisms by which mammalian cells detect DNA lesions, especially double-strand breaks, and mediate the signal to downstream transducers.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Human immunodeficiency virus type 1 Vpr-mediated G2 arrest requires Rad17 and Hus1 and induces nuclear BRCA1 and gamma-H2AX focus formation.

Eukaryotic cells have evolved a complex mechanism for sensing DNA damage during genome replication. Activation of this pathway prevents entry into mitosis to allow for either DNA repair or, in the event of irreparable damage, commitment to apoptosis. Under conditions of replication stress, the damage signal is initiated by the ataxia-telangiectasia-mutated and Rad3-related kinase ATR. We recently demonstrated that the human immunodeficiency virus type 1 (HIV-1) gene product viral protein R (Vpr) arrests infected cells in the G(2) phase via the activation of ATR. In the present study, we show that the activation of ATR by Vpr is analogous to activation by certain genotoxic agents, both mechanistically and in its downstream consequences. Specifically, we show a requirement for Rad17 and Hus1 to induce G(2) arrest as well as Vpr-induced phosphorylation of histone 2A variant X (H2AX) and formation of nuclear foci containing H2AX and breast cancer susceptibility protein 1. These results demonstrate that G(2) arrest mediated by the HIV-1 gene product Vpr utilizes the cellular signaling pathway whose physiological function is to recognize replication stress. These findings should contribute to a greater understanding of how HIV-1 manipulates the CD4(+)-lymphocyte cell cycle and apoptosis induction in the progressive CD4(+)-lymphocyte depletion characteristic of HIV-1 pathogenesis.

Animals↗

PubMed2004

DNA damage-induced cell cycle checkpoint control requires CtIP, a phosphorylation-dependent binding partner of BRCA1 C-terminal domains.

The BRCA1 C-terminal (BRCT) domain has recently been implicated as a phospho-protein binding domain. We demonstrate here that a CTBP-interacting protein CtIP interacts with BRCA1 BRCT domains in a phosphorylation-dependent manner. The CtIP/BRCA1 complex only exists in G(2) phase and is required for DNA damage-induced Chk1 phosphorylation and the G(2)/M transition checkpoint. However, the CtIP/BRCA1 complex is not required for the damage-induced G(2) accumulation checkpoint, which is controlled by a separate BRCA1/BACH1 complex. Taken together, these data not only implicate CtIP as a critical player in cell cycle checkpoint control but also provide molecular mechanisms by which BRCA1 controls multiple cell cycle transitions after DNA damage.

Animals↗

PubMed2004

Use of siRNA to study the function of MDC1 in DNA damage responses.

Small interfering RNA (siRNA) technology has emerged as a powerful genetic tool to investigate gene function in mammalian cells. Here we use siRNA to study a mediator of DNA damage-checkpoint protein 1 (MDC1), previously known as Kiaa0170 or NFBD1, in DNA damage responses. We show that MDC1 siRNA specifically and efficiently down-regulates MDC1 expression, resulting in defective radiation-induced apoptosis in A549 cells. Transfection with siRNA-resistant MDC1 restores radiation-induced apoptosis. These findings suggest that siRNA can be a very useful tool for the exploration of gene function in mammalian checkpoint responses.

Adaptor Proteins, Signal Transducing↗

PubMed2003

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.

Adaptor Proteins, Signal Transducing↗

PubMed2003

The BRCT domain is a phospho-protein binding domain.

The carboxyl-terminal domain (BRCT) of the Breast Cancer Gene 1 (BRCA1) protein is an evolutionarily conserved module that exists in a large number of proteins from prokaryotes to eukaryotes. Although most BRCT domain-containing proteins participate in DNA-damage checkpoint or DNA-repair pathways, or both, the function of the BRCT domain is not fully understood. We show that the BRCA1 BRCT domain directly interacts with phosphorylated BRCA1-Associated Carboxyl-terminal Helicase (BACH1). This specific interaction between BRCA1 and phosphorylated BACH1 is cell cycle regulated and is required for DNA damage-induced checkpoint control during the transition from G2 to M phase of the cell cycle. Further, we show that two other BRCT domains interact with their respective physiological partners in a phosphorylation-dependent manner. Thirteen additional BRCT domains also preferentially bind phospho-peptides rather than nonphosphorylated control peptides. These data imply that the BRCT domain is a phospho-protein binding domain involved in cell cycle control.

Amino Acid Motifs↗

PubMed2003

Autophosphorylation of checkpoint kinase 2 at serine 516 is required for radiation-induced apoptosis.

In response to ionizing radiation, checkpoint kinase 2 (Chk2) is activated in an ataxia telangiectasia mutation-dependent manner and induces either cell cycle arrest or apoptosis. Chk2 is also autophosphorylated following DNA damage. It is proposed that autophosphorylation of Chk2 may contribute to Chk2 activation. To fully understand the regulation of Chk2, we mapped an in vitro Chk2 autophosphorylation site at C-terminal serine 516 site (Ser-516). Ser-516 of Chk2 is phosphorylated following radiation in vivo, and this phosphorylation depends on the kinase activity of Chk2. Mutation of this autophosphorylation site (S516A) results in reduced Chk2 kinase activity, suggesting that Chk2 autophosphorylation is required for full kinase activation following DNA damage. Moreover, the S516A mutant of Chk2 is defective in ionizing radiation-induced apoptosis, suggesting that Chk2 autophosphorylation is critical for Chk2 function following DNA damage.

Amino Acid Sequence↗

PubMed2003

Both DNA topoisomerase II-binding protein 1 and BRCA1 regulate the G2-M cell cycle checkpoint.

Cell cycle checkpoints play a central role in genomic stability. The human DNA topoisomerase II-binding protein 1 (TopBP1) protein contains eight BRCA1 COOH terminus motifs and shares similarities with Cut5, a yeast checkpoint Rad protein. TopBP1 also shares many features with BRCA1. We report that, when expression of TopBP1 protein is inhibited in BRCA1 mutant cells, mimicking a TopBP1, BRCA1 double-negative condition, the G(2)-M checkpoint is strongly abrogated and apoptosis is increased after ionizing radiation. However, a BRCA1-negative or a TopBP1-negative background resulted in only partial abrogation of the G(2)-M checkpoint. The BRCA1 mutant and TopBP1-reduced condition specifically destroys regulation of the Chk1 kinase but not the Chk2 kinase, suggesting involvement in the ataxia telangiectasia-related pathway. These results indicate that both TopBP1 and BRCA1 specifically regulate the G(2)-M checkpoint, partially compensating each function.

Apoptosis↗

PubMed2003

Human claspin is required for replication checkpoint control.

Claspin is a newly identified protein that regulates Chk1 activation in Xenopus. In the present study we investigated the role of human Claspin in the DNA damage/replication checkpoint in mammalian cells. We observed that human Claspin is a cell cycle regulated protein that peaks at S/G2 phase. Claspin localizes in the nuclei, but it only associates with Chk1 following replication stress or other types of DNA damage. In addition, Claspin is phosphorylated in response to replication stress, and this phosphorylation appears to be required for its association with Chk1. Moreover, Claspin interacts with the checkpoint proteins ATR and Rad9. Given that both the ATR and Rad9-Rad1-Hus1 complexes are involved in Chk1 activation, it is possible that Claspin works as an adaptor molecule bringing these molecules together. Using small interfering RNA technology, we have shown that down-regulation of Claspin expression inhibits Chk1 activation in response to replication stress. More importantly, down-regulation of Claspin augments the premature chromatin condensation induced by hydroxyurea, inhibits the UV-induced reduction of DNA synthesis, and decreases cell survival. Taken together, these data imply a potentially critical role for Claspin in replication checkpoint control in mammalian cells.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Cloning and characterization of hCTF18, hCTF8, and hDCC1. Human homologs of a Saccharomyces cerevisiae complex involved in sister chromatid cohesion establishment.

A growing body of evidence suggests that establishment of sister chromatid cohesion is dependent on replication fork passage over a precohesion area. In Saccharomyces cerevisiae, this process involves an alternative replication factor C (RFC) complex that contains the four small RFC subunits as well as CTF18, CTF8, and DCC1. Here, we show that an evolutionarily conserved homologous complex exists in the nucleus of human cells. We demonstrate that hCTF18, hCTF8, and hDCC1 interact with each other as well as with the p38 subunit of RFC. This alternative RFC-containing complex interacts with proliferating cell nuclear antigen but not with the Rad9/Rad1/Hus1 complex, a proliferating cell nuclear antigen-like clamp involved in the DNA damage response. hCTF18 preferentially binds chromatin during S phase, suggesting a role during replication. Our data provide evidence for the existence of an alternative RFC complex with a probable role in mammalian sister chromatid cohesion establishment.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2003

Remodeling of cardiac fiber structure after infarction in rats quantified with diffusion tensor MRI.

Structural remodeling of myocardium after infarction plays a critical role in functional adaptation. Diffusion tensor magnetic resonance imaging (DTMRI) provides a means for rapid and nondestructive characterization of the three-dimensional fiber architecture of cardiac tissues. In this study, microscopic structural changes caused by MI were evaluated in Fischer 344 rats 4 wk after infarct surgery. DTMRI studies were performed on 15 excised, formalin-fixed rat hearts of both infarct (left anterior descending coronary artery occlusion, n = 8) and control (sham, n = 7) rats. Infarct myocardium exhibited increased water diffusivity (41% increase in trace values) and decreased diffusion anisotropy (37% decrease in relative anisotropy index). The reduced diffusion anisotropy correlated negatively with microscopic fiber disarray determined by histological analysis (R = 0.81). Transmural courses of fiber orientation angles in infarct zones were similar to those of normal myocardium. However, regional angular deviation of the diffusion tensor increased significantly in the infarct myocardium and correlated strongly with microscopic fiber disarray (R = 0.86). These results suggest that DTMRI may provide a valuable tool for defining structural remodeling in diseased myocardium at the cellular and tissue level.

Animals↗

PubMed2003

BRCA proteins and DNA damage checkpoints.

BRCA1 and BRCA2 are frequently mutated in breast cancer patients, responsible for the majority cases of familial breast cancer. Through genetic and biochemical analysis, the function of BRCA1 and BRCA2 is emerging. This review examines recent progresses in understanding the roles of BRCA proteins in DNA damage checkpoint control and how they are regulated in response to DNA damage

Animals↗

PubMed2003

Accumulation of checkpoint protein 53BP1 at DNA breaks involves its binding to phosphorylated histone H2AX.

53BP1 participates in the cellular response to DNA damage. Like many proteins involved in the DNA damage response, 53BP1 becomes hyperphosphorylated after radiation and colocalizes with phosphorylated H2AX in megabase regions surrounding the sites of DNA strand breaks. However, it is not yet clear whether the phosphorylation status of 53BP1 determines its localization or vice versa. In this study we mapped a region upstream of the 53BP1 C terminus that is required and sufficient for the recruitment of 53BP1 to these DNA break areas. In vitro assays revealed that this region binds to phosphorylated but not unphosphorylated H2AX. Moreover, using H2AX-deficient cells reconstituted with wild-type or a phosphorylation-deficient mutant of H2AX, we have shown that phosphorylation of H2AX at serine 140 is critical for efficient 53BP1 foci formation, implying that a direct interaction between 53BP1 and phosphorylated H2AX is required for the accumulation of 53BP1 at DNA break sites. On the other hand, radiation-induced phosphorylation of the 53BP1 N terminus by the ATM (ataxia-telangiectasia mutated) kinase is not essential for 53BP1 foci formation and takes place independently of 53BP1 redistribution. Thus, these two damage-induced events, hyperphosphorylation and relocalization of 53BP1, occur independently in the cell.

Carrier Proteins↗

PubMed2003

MDC1 is coupled to activated CHK2 in mammalian DNA damage response pathways.

Forkhead-homology-associated (FHA) domains function as protein-protein modules that recognize phosphorylated serine/threonine motifs. Interactions between FHA domains and phosphorylated proteins are thought to have essential roles in the transduction of DNA damage signals; however, it is unclear how FHA-domain-containing proteins participate in mammalian DNA damage responses. Here we report that a FHA-domain-containing protein-mediator of DNA damage checkpoint protein 1 (MDC1; previously known as KIAA0170)--is involved in DNA damage responses. MDC1 localizes to sites of DNA breaks and associates with CHK2 after DNA damage. This association is mediated by the MDC1 FHA domain and the phosphorylated Thr 68 of CHK2. Furthermore, MDC1 is phosphorylated in an ATM/CHK2-dependent manner after DNA damage, suggesting that MDC1 may function in the ATM-CHK2 pathway. Consistent with this hypothesis, suppression of MDC1 expression results in defective S-phase checkpoint and reduced apoptosis in response to DNA damage, which can be restored by the expression of wild-type MDC1 but not MDC1 with a deleted FHA domain. Suppression of MDC1 expression results in decreased p53 stabilization in response to DNA damage. These results suggest that MDC1 is recruited through its FHA domain to the activated CHK2, and has a critical role in CHK2-mediated DNA damage responses.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Mediator of DNA damage checkpoint protein 1 regulates BRCA1 localization and phosphorylation in DNA damage checkpoint control.

BRCA1 is a tumor suppressor involved in DNA repair and damage-induced checkpoint controls. In response to DNA damage, BRCA1 relocalizes to nuclear foci at the sites of DNA lesions. However, little is known about the regulation of BRCA1 relocalization following DNA damage. Here we show that mediator of DNA damage checkpoint protein 1 (MDC1), previously named NFBD1 or Kiaa0170, is a proximate mediator of DNA damage responses that regulates BRCA1 function. MDC1 regulates ataxia-telangiectasia-mutated (ATM)-dependent phosphorylation events at the site of DNA damage. Importantly down-regulation of MDC1 abolishes the relocalization and hyperphosphorylation of BRCA1 following DNA damage, which coincides with defective G(2)/M checkpoint control in response to DNA damage. Taken together these data suggest that MDC1 regulates BRCA1 function in DNA damage checkpoint control.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Improved molecular imaging contrast agent for detection of human thrombus.

Molecular imaging of microthrombus within fissures of unstable atherosclerotic plaques requires sensitive detection with a thrombus-specific agent. Effective molecular imaging has been previously demonstrated with fibrin-targeted Gd-DTPA-bis-oleate (BOA) nanoparticles. In this study, the relaxivity of an improved fibrin-targeted paramagnetic formulation, Gd-DTPA-phosphatidylethanolamine (PE), was compared with Gd-DTPA-BOA at 0.05-4.7 T. Ion- and particle-based r(1) relaxivities (1.5 T) for Gd-DTPA-PE (33.7 (s*mM)(-1) and 2.48 x 10(6) (s*mM)(-1), respectively) were about twofold higher than for Gd-DTPA-BOA, perhaps due to faster water exchange with surface gadolinium. Gd-DTPA-PE nanoparticles bound to thrombus surfaces via anti-fibrin antibodies (1H10) induced 72% +/- 5% higher change in R(1) values at 1.5 T (deltaR(1) = 0.77 +/- 0.02 1/s) relative to Gd-DTPA-BOA (deltaR(1) = 0.45 +/- 0.02 1/s). These studies demonstrate marked improvement in a fibrin-specific molecular imaging agent that might allow sensitive, early detection of vascular microthrombi, the antecedent to stroke and heart attack.

Contrast Media↗

PubMed2003

p53 Binding protein 53BP1 is required for DNA damage responses and tumor suppression in mice.

53BP1 is a p53 binding protein of unknown function that binds to the central DNA-binding domain of p53. It relocates to the sites of DNA strand breaks in response to DNA damage and is a putative substrate of the ataxia telangiectasia-mutated (ATM) kinase. To study the biological role of 53BP1, we disrupted the 53BP1 gene in the mouse. We show that, similar to ATM(-/-) mice, 53BP1-deficient mice were growth retarded, immune deficient, radiation sensitive, and cancer prone. 53BP1(-/-) cells show a slight S-phase checkpoint defect and prolonged G(2)/M arrest after treatment with ionizing radiation. Moreover, 53BP1(-/-) cells feature a defective DNA damage response with impaired Chk2 activation. These data indicate that 53BP1 acts downstream of ATM and upstream of Chk2 in the DNA damage response pathway and is involved in tumor suppression.

Animals↗