RNA polymerase III is required for the repair of DNA double-strand breaks by homologous recombination.

End resection in homologous recombination (HR) and HR-mediated repair of DNA double-strand breaks (DSBs) removes several kilobases from 5' strands of DSBs, but 3' strands are exempted from degradation. The mechanism by which the 3' overhangs are protected has not been determined. Here, we established that the protection of 3' overhangs is achieved through the transient formation of RNA-DNA hybrids. The DNA strand in the hybrids is the 3' ssDNA overhang, while the RNA strand is newly synthesized. RNA polymerase III (RNAPIII) is responsible for synthesizing the RNA strand. Furthermore, RNAPIII is actively recruited to DSBs by the MRN complex. CtIP and MRN nuclease activity is required for initiating the RNAPIII-mediated RNA synthesis at DSBs. A reduced level of RNAPIII suppressed HR, and genetic loss > 30 bp increased at DSBs. Thus, RNAPIII is an essential HR factor, and the RNA-DNA hybrid is an essential repair intermediate for protecting the 3' overhangs in DSB repair.

Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells.

During DNA replication in eukaryotic cells, short single-stranded DNA segments known as Okazaki fragments are first synthesized on the lagging strand. The Okazaki fragments originate from ∼35-nucleotide-long RNA-DNA primers. After Okazaki fragment synthesis, these primers must be removed to allow fragment joining into a continuous lagging strand. To date, the models of enzymatic machinery that removes the RNA-DNA primers have come almost exclusively from biochemical reconstitution studies and some genetic interaction assays, and there is little direct evidence to confirm these models. One obstacle to elucidating Okazaki fragment processing has been the lack of methods that can directly examine primer removal in vivo In this study, we developed an electron microscopy assay that can visualize nucleotide flap structures on DNA replication forks in fission yeast (Schizosaccharomyces pombe). With this assay, we first demonstrated the generation of flap structures during Okazaki fragment processing in vivo The mean and median lengths of the flaps in wild-type cells were ∼51 and ∼41 nucleotides, respectively. We also used yeast mutants to investigate the impact of deleting key DNA replication nucleases on these flap structures. Our results provided direct in vivo evidence for a previously proposed flap cleavage pathway and the critical function of Dna2 and Fen1 in cleaving these flaps. In addition, we found evidence for another previously proposed exonucleolytic pathway involving RNA-DNA primer digestion by exonucleases RNase H2 and Exo1. Taken together, our observations suggest a dual mechanism for Okazaki fragment maturation in lagging strand synthesis and establish a new strategy for interrogation of this fascinating process.

Conformational Change of Human Checkpoint Kinase 1 (Chk1) Induced by DNA Damage.

Phosphorylation of Chk1 by ataxia telangiectasia-mutated and Rad3-related (ATR) is critical for checkpoint activation upon DNA damage. However, how phosphorylation activates Chk1 remains unclear. Many studies suggest a conformational change model of Chk1 activation in which phosphorylation shifts Chk1 from a closed inactive conformation to an open active conformation during the DNA damage response. However, no structural study has been reported to support this Chk1 activation model. Here we used FRET and bimolecular fluorescence complementary techniques to show that Chk1 indeed maintains a closed conformation in the absence of DNA damage through an intramolecular interaction between a region (residues 31-87) at the N-terminal kinase domain and the distal C terminus. A highly conserved Leu-449 at the C terminus is important for this intramolecular interaction. We further showed that abolishing the intramolecular interaction by a Leu-449 to Arg mutation or inducing ATR-dependent Chk1 phosphorylation by DNA damage disrupts the closed conformation, leading to an open and activated conformation of Chk1. These data provide significant insight into the mechanisms of Chk1 activation during the DNA damage response.

[Effectiveness of transverse double "8"-shaped tension band technique for Lawrence zone â fracture of the fifth metatarsal base].

Objective: To explore the effectiveness of transverse double "8"-shaped tension band technique in the treatment of Lawrence zoneⅠfracture of the 5th metatarsal base. Methods: Between February 2019 and October 2021, 15 patients with Lawrence zoneⅠfracture of the 5th metatarsal base were treated with transverse double "8"-shaped tension band technique. There were 8 males and 7 females, with a median age of 40 years (range, 23-59 years). The fractures were caused by sprains. The time from injury to operation was 3-7 days (mean, 4.1 days). X-ray films were taken to observe the fracture healing and the anchor looseness and detachment. The foot function was evaluated by American Orthopaedic Foot and Ankle Society (AOFAS) score, visual analogue scale (VAS) score, and the eversion angle of the calcaneal talus joint. Results: The incisions healed by first intention after operation in 14 cases and the incision healed poorly in 1 case. All patients were followed up 8-12 months (median, 10 months). The imaging examination showed that all fractures healed well, with a healing time of 10-14 weeks (mean, 11.7 weeks). At last follow-up, AOFAS score was 82-100 (median, 98); 13 cases were excellent and 2 cases were good, with an excellent and good rate of 100%. VAS score was 0-3 (median, 1). Three cases had mild limited ankle joint range of motion, while 12 cases had normal range of motion. The eversion angle of the calcaneal talus joint was 25°-32° (median, 30°). Conclusion: The application of transverse double "8"-shaped tension band technique for Lawrence zone Ⅰ fracture of the 5th metatarsal base has advantages such as simple operation, avoidance of secondary operation, and reduction of foreign body sensation, with definite effectiveness.

Coupling cellular localization and function of checkpoint kinase 1 (Chk1) in checkpoints and cell viability.

Chk1 plays a key role in regulating the replication checkpoint and DNA damage response. Recent evidence suggests that mammalian Chk1 regulates both the nuclear and cytoplasmic checkpoint events. However, mechanisms regulating cellular mobilization of Chk1 were not well understood. Here, we report the identification of regions of human Chk1 that regulate its protein cellular localization and checkpoint function. We demonstrate that the two highly conserved motifs (CM1 and CM2) at the C terminus of Chk1 function as a nuclear export signal and nuclear localization signal, respectively. Mutating five highly conserved residues within these two motifs of Chk1 resulted in its accumulation mainly in the cytoplasm. These cytoplasmic Chk1 mutants were less stable and exhibited significantly reduced phosphorylation by DNA damage treatment, yet they retained, at least partially, checkpoint function. Using an adenovirus-mediated gene targeting technique, we attempted to create an HCT116 cell line in which endogenous Chk1 is mutated so that it is expressed exclusively in the cytoplasm. However, we failed to obtain homozygous mutant cell lines. We found that even the heterozygous mutant cell lines showed cell survival defects accompanied by spontaneous cell death. Together, these results reveal novel regulatory mechanisms that couple protein cellular localization with the checkpoint response and cell viability of Chk1.

[Cloning, eukaryotic expression and spatial expression patterns of porcine MNSFbeta].

MNSFbeta (Monoclonal nonspecific suppressor factor beta) is a natural immunosuppressive factor which has been reported to be involved in various biological processes, such as immune responses, cell division, stress response, cell apoptosis, and nuclear transport. However, study on porcine MNSFbeta has been rarely reported. In this study, the full-length sequence of porcine MNSFbeta (GenBank accession number: KF77642500) was predicted in silicon and its cDNA sequence was obtained through RT-PCR from porcine spleen. The nucleic acid and protein sequences were analyzed. Then, the gene was subcloned into pEGFP-C1 to construct a recombinant plasmid pEGFP-MNSFbeta which was transfected into swine umbilical vein endothelial cells (SUVECs) using Lipofectamine 2000. The expression of GFP was detected by fluorescence microscopy, Western blot, and laser confocal fluorescence microscopy. The spatial expression patterns of porcine MNSFbeta were detected by real-time qPCR. Results showed that the full length of porcine MNSFbeta was 402 bp encoding 133 amino acids with only one exon. Bioinformatics analysis showed that porcine MNSFbeta protein was a stable protein consisting of a ubiquitin-like domain fused to the ribosomal protein S30 with no signal peptide. The analyses of homology and phylogenetic tree of porcine MNSFbeta and its homologs in other 18 species showed that the identities of MNSFbeta protein sequence were higher than 91% among different species and the evolutionary distance was less than 0.05. It indicates that MNSFbeta is highly conserved in the process of evolution. Fluorescence signal showed that the fusion protein GFP-MNSFbeta was successfully expressed in SUVECs which was then confirmed by Western blot. Laser confocal fluorescence microscopy showed that MNSFbeta was expressed in both nucleus and cytoplasm. Analysis of spatial expression patterns showed that procine MNSFbeta was widely expressed in immune tissues, but not in lung, suggesting that MNSFbeta may play an important role in immune response.

How destination brand experience influences tourist citizenship behavior: Testing mediation of brand relationship quality and moderation effects on commitment.

This study examines the potential predictors of tourist citizenship behavior based on the Stimulus-Organism-Response framework. The studies were conducted in China. Data were collected via questionnaire surveys. Structural equation path modeling and mediation as well as moderation role were used for data analyses. This model was used to test the hypotheses using a sample of 325 individuals with tourism experience in Guangzhou city. The results reveal that tourism destination brand experience and brand relationship quality significantly affect tourist citizenship behavior. Furthermore, the results show that brand relationship quality significantly mediates the relationship between tourism destination brand experience and tourist citizenship behavior and demonstrate that commitment plays a significant moderating role between brand relationship quality and tourist citizenship behavior. This study clearly shows the relationship between tourism destination brand experience, brand relationship quality, and tourist citizenship behavior. Thus, this study contributes to existing tourism studies by identifying gaps and proposing a holistic view to understand tourist citizenship behavior in the tourism industry.

RNA polymerase III directly participates in DNA homologous recombination.

A recent study showed that RNA transcription is directly involved in DNA homologous recombination (HR). The first step in HR is end resection, which degrades a few kilobases or more from the 5'-end strand at DNA breaks, but the 3'-end strand remains strictly intact. Such protection of the 3'-end strand is achieved by the transient formation of an RNA-DNA hybrid structure. The RNA strand in the hybrid is newly synthesized by RNA polymerase III. The revelation of the existence of an RNA-DNA hybrid intermediate should further help resolve several long-standing questions of HR. In this article, we also put forward our views on some controversial issues related to RNA-DNA hybrids, RNA polymerases, and the protection of 3'-end strands.

Factors associated with the depression status of Chinese parents who have lost their only child.

Aim: This study aimed to assess the risk factors for depression among parents who have lost their only child (PLOCs). Methods: We used a cross-sectional survey to reveal the risk factors of depression among PLOCs. Multi-stage, stratified, cluster sampling was used to recruit the participants. The cluster sampling method was used to select PLOCs in Hangzhou, Zhejiang Province, and Wuhu, Anhui Province, while the stratified cluster sampling method was used in Anshun, Guizhou Province. A total of 651 PLOCs were recruited in this study. Participants completed the Social Support Rating Scale (SSRS) and the Geriatric Depression Scale-15 (GDS-15). Socio-demographics were also collected, including age, sex, monthly income, education level, marital status, self-reported health, and a number of diseases were collected as well. Chi-square tests and binary logistic regression were conducted to analyze the influence of these factors on PLOCs' mental status. Results: Two hundred and fifty-eight PLOCs (39.56%) reported depression. Compared to PLOCs living in Wuhu, those living in Hangzhou (OR = 3.374, CI = 2.337-4.870) had a higher risk of depression. Being single (OR = 1.449, CI = 1.019-2.061) and the presence/absence of grandchildren (OR = 0.430, CI = 0.274-0.676)were significantly associated with the depression status of PLOCs. Conclusion: The sampled Chinese PLOCs reported a high prevalence of depression that was influenced by their place of residence, marital status, and presence/absence of grandchildren. This may highlight the need for routine assessment and help of this group by the relevant stakeholders (including government, non-profit social organizations, and professional psychologists) with more attention paid to single and low-income PLOCs that have no grandchildren. It is imperative to build a comprehensive care system of "extended family-community-society-government" for this vulnerable group.

Down-regulation of PKCzeta expression inhibits chemotaxis signal transduction in human lung cancer cells.

Metastasis is the major cause of mortality in lung cancer. Chemotaxis plays a vital role in cancer cell metastasis. In the current study, we reported that epidermal growth factor (EGF) induced a robust chemotaxis of A549 and H1299 cells, two representative human non-small cell lung cancer (NSCLC) cells. Chelerythrine chloride, an inhibitor of all protein kinase C (PKC) isozymes, significantly reduced the chemotactic capacity of NSCLC cells while inhibitors of classical or novel PKC isozymes, such as Gö6976, calphostin C, or Gö6850, showed no effect, which suggested that atypical PKC might be involved in the chemotactic process of NSCLC cells. EGF-elicited translocation and phosphorylation of atypical PKCzeta, indicating that EGF could activate PKCzeta. Treatment with a PKCzeta specific inhibitor, a myristoylated pseudosubstrate, blocked the chemotaxis in a dose-dependent manner, further confirming that atypical PKCzeta was required for NSCLC chemotaxis. Mechanistic studies suggested that PKCzeta was regulated by phosphatidylinositol 3 kinase (PI3K)/Akt. Furthermore, PKCzeta-mediated chemotaxis by regulating actin polymerization and cell adhesion. Taken together, our study suggested that PKCzeta was required in NSCLC cell chemotaxis, thus could be used as a target to develop anti-lung cancer metastasis therapies.

Reduction of Akt2 expression inhibits chemotaxis signal transduction in human breast cancer cells.

Protein kinase Czeta PKCzeta mediates cancer cell chemotaxis by regulating cytoskeleton rearrangement and cell adhesion. In the research for its upstream regulator, we investigated the role of Akt2 in chemotaxis and metastasis of human breast cancer cells. Reduction of Akt2 expression by siRNA inhibited chemotaxis of MDA-MB-231, T47D, and MCF7 cells, three representative human breast cancer cells. Expression of a wild type Akt2 in siRNA transfected cells rescued the phenotype. EGF-induced integrin beta1 phosphorylation was dampened, consistent with defects in adhesion. Phosphorylation of LIMK and cofilin, a critical step of cofilin recycle and actin polymerization, was also impaired. Thus, Akt2 regulates both cell adhesion and cytoskeleton rearrangement during chemotaxis. Depletion of Akt2 by siRNA impaired the activation of PKCzeta while inhibition of PKCzeta did not interfere with EGF induced phosphorylation of Akt. Furthermore, EGF induced co-immunoprecipitation between PKCzeta and Akt2, but not Akt1, suggesting that a direct interaction between PKCzeta and Akt2 in chemotaxis. Protein levels of integrin beta1, LIMK, cofilin, and PKCzeta didn't alter, suggesting that Akt2 does not regulate the expression of these signaling molecules. In a Severe Combine Immunodeficiency mouse model, Akt2 depleted MDA-MB-231 cells showed a marked reduction in metastasis to mouse lungs, demonstrating the biological relevancy of Akt2 in cancer metastasis in vivo. Taken together, our results suggest that Akt2 directly mediates EGF-induced chemotactic signaling pathways through PKCzeta and its expression is critical during the extravasation of circulating cancer cells.

Investigate the role of PTEN in chemotaxis of human breast cancer cells.

Chemotaxis plays an important role in metastasis of cancer cells. In the current study, we investigated the role of PTEN, a tumor suppressor, in chemotaxis of human breast cancer cells. Over-expression of PTEN inhibited EGF-induced chemotaxis, probably due to an overall reduction of PIP(3) levels. Disruption of PTEN by siRNA caused a marked decrease in chemokinesis, cell adhesion, and membrane spreading, resulting in a severe defect in chemotaxis. In PTEN disrupted cells, PDK1, AKT, and PKCzeta exhibited elevated basal activities, which prevented EGF-induced further activation of these molecules. In the absence of EGF, active PDK1 was detected on multiple directions of the plasma membranes of PTEN disrupted cells, which competed against EGF-induced gradient sensing. To confirm the biological relevance of in vitro studies, both PTEN disrupted cells and its parental human breast cancer cells were injected into tail veins of SCID mice. Mice injected with PTEN disrupted cancer cells showed a marked decrease in lung metastasis. Taken together, our data show that PTEN plays a non-redundant role in EGF-induced chemotaxis of human breast cancer cells, and an optimal level of PTEN is required in these responses.

Application of Monte Carlo cross-validation to identify pathway cross-talk in neonatal sepsis.

To explore genetic pathway cross-talk in neonates with sepsis, an integrated approach was used in this paper. To explore the potential relationships between differently expressed genes between normal uninfected neonates and neonates with sepsis and pathways, genetic profiling and biologic signaling pathway were first integrated. For different pathways, the score was obtained based upon the genetic expression by quantitatively analyzing the pathway cross-talk. The paired pathways with high cross-talk were identified by random forest classification. The purpose of the work was to find the best pairs of pathways able to discriminate sepsis samples versus normal samples. The results found 10 pairs of pathways, which were probably able to discriminate neonates with sepsis versus normal uninfected neonates. Among them, the best two paired pathways were identified according to analysis of extensive literature. Impact statement To find the best pairs of pathways able to discriminate sepsis samples versus normal samples, an RF classifier, the DS obtained by DEGs of paired pathways significantly associated, and Monte Carlo cross-validation were applied in this paper. Ten pairs of pathways were probably able to discriminate neonates with sepsis versus normal uninfected neonates. Among them, the best two paired pathways ((7) IL-6 Signaling and Phospholipase C Signaling (PLC); (8) Glucocorticoid Receptor (GR) Signaling and Dendritic Cell Maturation) were identified according to analysis of extensive literature.

Hospital-acquired lower respiratory tract infections among high risk hospitalized patients in a tertiary care teaching hospital in China: An economic burden analysis.

BACKGROUND: Data on the economic burden of hospital-acquired lower respiratory tract infection (LRTI) among high risk hospitalized patients are lacking in China. This study aims to fill this knowledge gap. METHODS: We used a prospective matched cohort design, comparing patients with LRTIs and 1:1 matched patients without LRTIs. Study period was from January 2013 to December 2015 analyzing inpatients from high risk wards - intensive care unit (ICU), dialysis, hematology, etc. - in a tertiary hospital. Hospital information system and hospital infection surveillance system were applied to extract necessary information. The primary outcome was incidence of hospital-acquired LRTIs, and the secondary was economic burden outcomes, including incremental medical costs and prolonged length of stay (LOS). Wilcoxon's signed rank test was used to explore the differences in the economic burden. RESULTS: Among 5990 hospital visitors over the period of time, 895 (14.94%) had hospital-acquired LRTIs. We analyzed 340 patients with LRTIs and 340 respective controls without infections. The median hospital costs for patients with ICU-acquired LRTIs were significantly higher than those without LRTIs in other wards ($12,301.17 vs. $4674.64, P<0.01). The average attributable cost per patient was $2853.93 ($6916.48 vs. $4062.55, P<0.01). Patients from hematology department had the longest LOS, at 15days (25days vs. 10 days, P<0.01). An LRTI led to an attributable increase in LOS by 8days on average (P<0.01). Western medicine, treatment and laboratory test were the dominant contributors to the growth in overall medical costs in hospital-acquired LRTIs. CONCLUSIONS: Hospital-acquired LRTI imposed considerable economic burden on patients hospitalized in high risk wards in China. This study provides the first data for economic evaluation of LRTI, highlighting the urgent need to establish targeted preventive strategies to minimize the occurrence of this complication to reduce economic burden.

Nosocomial infections among acute leukemia patients in China: An economic burden analysis.

BACKGROUND: The economic burden associated nosocomial infections (NIs) in patients with acute leukemia (AL) in China was unclear. A prospective study was conducted to quantify the medical cost burden of NIs among AL patients. METHODS: Nine hundred ninety-four patients diagnosed with AL between January 2011 and December 2013 were included. Relevant necessary information was extracted from the hospital information system and hospital infection surveillance system. The primary outcome was incidence of NIs and the secondary was economic burden results, including extra medical costs and prolonged length of stay (LOS). We estimated the total incremental cost of NIs by comparing all-cause health care costs in patients with versus without infections. Prolonged duration of stay was compared in patients with different infections. RESULTS: Of 994 patients with AL, 277 (27.9%) experienced NIs. NI was associated with a total incremental cost of $3,092 per patient ($5,227 vs $2,135; P < .01) and infected patients experienced a longer LOS (21 vs 10 days; P < .01). Patients with multisite infection had the highest total medical cost ($8,474.90 vs $2,209.90; P < .01) and the longest LOS (25 vs 15 days; P < .01). Western medicine was the main contributor to the rise of total cost in all kinds of infections. CONCLUSIONS: NI was associated with higher medical costs, which imposed an economic burden on patients with AL. The study highlights the influence of NIs on LOS and health care costs and appeal to the establishment of prophylactic measures for NIs to reduce the unnecessary waste of medical resources in the long run.

The prevalence and predictive factors of urinary tract infection in patients undergoing renal transplantation: A meta-analysis.

BACKGROUND: Urinary tract infections (UTIs) are the main cause of infectious complications in renal transplant (RTx) recipients and are considered as a potential risk factor for poorer graft outcomes. However, the risk factors of UTIs are controversial. We estimated the incidence and predisposing factors of UTIs in patients undergoing RTx. METHODS: Seventeen studies (6,671 patients) evaluated the prevalence and the risk factors of UTIs in patients with RTx published January 2000-October 2014 were included. The data were pooled using the fixed effect model or DerSimonian-Laird random effect model according to I2. RESULTS: Thirteen eligible articles with a total of 3,364 patients were evaluated and the pooled prevalence of UTIs was 38.0% (95% confidence interval [CI], 29%-47%; P < .01). The estimated risk factors for UTI include female sex (odds ratio [OR], 3.11; 95% CI, 2.10-4.13), older age (OR, 1.032; 95% CI, 1.01-1.04), duration of catheter (OR, 1.52; 95% CI, 1.03-2.03), acute rejection episodes (OR, 1.64; 95% CI, 1.11-2.41), and receiving a kidney from a deceased donor (OR, 1.28; 95% CI, 1.09-1.52). CONCLUSIONS: More than one-third of RTx patients had at least 1 UTI after surgery. Female sex, older age of the recipient, long duration of catheter, acute rejection episodes, and cadaveric donor were associated with higher risk of UTI.

A potential molecular model for studying apoptosis enhanced by the interaction of BCL-G with JAB1 in swine.

BCL-G, an apoptotic factor in Bcl-2 family, is involved in several kinds of diseases by interacting with several proteins. Although many studies on mouse and human BCL-G have been reported, porcine BCL-G (pBCL-G) has been little investigated. In this study, our results showed that pBCL-G was universally expressed in porcine tissues. The BH2 domain affected the subcellular distribution of pBCL-G protein. pBCL-G could interact with porcine JAB1 (pJAB1), by which its subcellular distribution was affected. pBCL-G promoted staurosporine-induced apoptosis that was significantly enhanced by interaction of pBCL-G with pJAB1. The apoptosis at least partially depended on the activated caspase-8, -9 and -3. Owing to the close phylogenetic distance between pigs and humans and their many physiological similarities, our findings may provide a potential molecular model to study human BCL-G and also may have implications in the treatment of diseases relevant with BCL-G.

Autoregulatory mechanisms of phosphorylation of checkpoint kinase 1.

Checkpoint kinase 1 (Chk1), a serine/threonine protein kinase, is centrally involved in cell-cycle checkpoints and cellular response to DNA damage. Phosphorylation of Chk1 at 2 Ser/Gln (SQ) sites, Ser-317 and Ser-345, by the upstream kinase ATR is critical for checkpoint activation. However, the precise molecular mechanisms controlling Chk1 phosphorylation and subsequent checkpoint activation are not well understood. Here, we report unique autoregulatory mechanisms that control protein phosphorylation of human Chk1, as well as checkpoint activation and cell viability. Phosphorylation of Ser-317 is required, but not sufficient, for maximal phosphorylation at Ser-345. The N-terminal kinase domain of Chk1 prevents Chk1 phosphorylation at the C-terminus by ATR in the absence of DNA damage. Loss of the inhibitory effect imposed by the N-terminus causes constitutive phosphorylation of Chk1 by ATR under normal growth conditions, which in turn triggers artificial checkpoints that suppress the S-phase progression. Furthermore, two point mutations were identified that rendered Chk1 constitutively active, and expression of the constitutively active mutant form of Chk1 inhibited cancer cell proliferation. Our findings therefore reveal unique regulatory mechanisms of Chk1 phosphorylation and suggest that expression of constitutively active Chk1 may represent a novel strategy to suppress tumor growth. Cancer Res; 72(15); 3786-94. ©2012 AACR.

A new in vitro system for activating the cell cycle checkpoint.

In response to DNA damage, cells launch elegant networks of genome surveillance mechanisms, called cell cycle checkpoints, to detect and repair damaged DNA to maintain the genome stability. Key components of cell cycle checkpoints are two PI3K-related protein kinases (PIKK), ATR and ATM, which participate in both sensing the DNA damage and transducing the damage signal through phosphorylating two target protein kinases, Chk1 and Chk2, respectively. However, how exactly cell cycle checkpoints are activated, maintained, and terminated are not completely understood. Given the complexity of the cell cycle checkpoint signaling and the cellular environment, systems that can faithfully mimic the cell cycle checkpoint activation in vitro, such as the Xenopus egg extracts, are of extreme value in dissecting the precise molecular mechanisms underlying DNA damage response. Here we describe that the well-established in vitro transcription and translation (IVTNT) system has the capability to induce protein phosphorylation of substrates for ATR or ATM, including Chk1, Rad17, and ATM itself. These phosphorylation events highly mimic those occurring in cells when treated with DNA damaging agents. Our results demonstrate that the IVTNT system could be developed into a novel in vitro system to facilitating the dissecting of mechanisms leading to cell cycle checkpoint activation in vivo.

Targeting the checkpoint kinase Chk1 in cancer therapy.

A paramount objective of the eukaryotic cell division cycle is to overcome numerous internal and external insults to faithfully duplicate the genetic information once per every cycle. This is carried out by elaborate networks of genome surveillance signaling pathways, termed replication checkpoints. Central to replication checkpoints are two protein kinases, the upstream kinase ATR, and its downstream target kinase, Chk1. When the DNA replication process is interrupted, the ATR-Chk1 pathway transmits signals to delay cell cycle progression, and to maintain fork viability so that DNA duplication can resume after the initial damage is corrected. Previous studies showed that replicative stress not only activated Chk1, but also triggered the ubiquitin-dependent destruction of Chk1 in cultured human cells. In a recent study, we identified the F-box protein, Fbx6, as the mediator that regulates Chk1 ubiquitination and degradation in both normally cycling cells and during replication stress. We further showed that expression levels of Chk1 and Fbx6 exhibited an overall inverse correlation in both cultured cancer cell lines and in breast tumor tissues, and that defects in Chk1 degradation, for instance, due to reduced expression of Fbx6, rendered tumor cells resistant to anticancer treatment. Here we highlight those findings and their implications in the replication checkpoint and cellular sensitivity to cancer therapies.