Structural basis for intra- and intermolecular interactions on RAD9 subunit of 9-1-1 checkpoint clamp implies functional 9-1-1 regulation by RHINO.

Eukaryotic DNA clamp is a trimeric protein featuring a toroidal ring structure that binds DNA on the inside of the ring and multiple proteins involved in DNA transactions on the outside. Eukaryotes have two types of DNA clamps: the replication clamp PCNA and the checkpoint clamp RAD9-RAD1-HUS1 (9-1-1). 9-1-1 activates the ATR-CHK1 pathway in DNA damage checkpoint, regulating cell cycle progression. Structure of 9-1-1 consists of two moieties: a hetero-trimeric ring formed by PCNA-like domains of three subunits and an intrinsically disordered C-terminal region of the RAD9 subunit, called RAD9 C-tail. The RAD9 C-tail interacts with the 9-1-1 ring and disrupts the interaction between 9-1-1 and DNA, suggesting a negative regulatory role for this intramolecular interaction. In contrast, RHINO, a 9-1-1 binding protein, interacts with both RAD1 and RAD9 subunits, positively regulating checkpoint activation by 9-1-1. This study presents a biochemical and structural analysis of intra- and inter-molecular interactions on the 9-1-1 ring. Biochemical analysis indicates that RAD9 C-tail binds to the hydrophobic pocket on the PCNA-like domain of RAD9, implying that the pocket is involved in multiple protein-protein interactions. The crystal structure of the 9-1-1 ring in complex with a RHINO peptide reveals that RHINO binds to the hydrophobic pocket of RAD9, shedding light on the RAD9-binding motif. Additionally, the study proposes a structural model of the 9-1-1-RHINO quaternary complex. Together, these findings provide functional insights into the intra- and inter-molecular interactions on the front side of RAD9, elucidating the roles of RAD9 C-tail and RHINO in checkpoint activation.

The 9-1-1 DNA clamp subunit RAD1 forms specific interactions with clamp loader RAD17, revealing functional implications for binding-protein RHINO.

The RAD9-RAD1-HUS1 complex (9-1-1) is a eukaryotic DNA clamp with a crucial role at checkpoints for DNA damage. The ring-like structure of 9-1-1 is opened for loading onto 5' recessed DNA by the clamp loader RAD17 RFC-like complex (RAD17-RLC), in which the RAD17 subunit is responsible for specificity to 9-1-1. Loading of 9-1-1 is required for activation of the ATR-CHK1 checkpoint pathway and the activation is stimulated by a 9-1-1 interacting protein, RHINO, which interacts with 9-1-1 via a recently identified RAD1-binding motif. This discovery led to the hypothesis that other interacting proteins may contain a RAD1-binding motif as well. Here, we show that vertebrate RAD17 proteins also have a putative RAD1-binding motif in their N-terminal regions, and we report the crystal structure of human 9-1-1 bound to a human RAD17 peptide incorporating the motif at 2.1 Å resolution. Our structure confirms that the N-terminal region of RAD17 binds to the RAD1 subunit of 9-1-1 via specific interactions. Furthermore, we show that the RAD1-binding motif of RHINO disturbs the interaction of the N-terminal region of RAD17 with 9-1-1. Our results provide deeper understanding of how RAD17-RLC specifically recognizes 9-1-1 and imply that RHINO has a functional role in 9-1-1 loading/unloading and checkpoint activation.

Structure of the RAD9-RAD1-HUS1 checkpoint clamp bound to RHINO sheds light on the other side of the DNA clamp.

DNA clamp, a highly conserved ring-shaped protein, binds dsDNA within its central pore. Also, DNA clamp interacts with various nuclear proteins on its front, thereby stimulating their enzymatic activities and biological functions. It has been assumed that the DNA clamp is a functionally single-faced ring from bacteria to humans. Here, we report the crystal structure of the heterotrimeric RAD9-RAD1-HUS1 (9-1-1) checkpoint clamp bound to a peptide of RHINO, a recently identified cancer-related protein that interacts with 9-1-1 and promotes activation of the DNA damage checkpoint. This is the first structure of 9-1-1 bound to its partner. The structure reveals that RHINO is unexpectedly bound to the edge and around the back of the 9-1-1 ring through specific interactions with the RAD1 subunit of 9-1-1. Our finding indicates that 9-1-1 is a functionally double-faced DNA clamp.

Colonic distention at CT colonography: randomized evaluation of both IV hyoscine butylbromide and automated carbon dioxide insufflation.

OBJECTIVE: The purpose of this article is to evaluate the efficacy of IV hyoscine butylbromide as a bowel relaxant and automated carbon dioxide insufflation in CT colonography in terms of colonic distention and perceived burden. SUBJECTS AND METHODS; Two hundred twenty-four participants were randomly allocated to one of four groups: control (no bowel relaxant and IV saline placebo before CT colonography with manual carbon dioxide insufflation), hyoscine butylbromide (IV hyoscine butylbromide before examination with manual carbon dioxide insufflation), automated (no bowel relaxant before examination with automated carbon dioxide insufflation), and combined (hyoscine butylbromide before examination with automated carbon dioxide insufflation). The degree of colonic distention on a 4-point scale, examination time, and participants' satisfaction, as measured by their responses to a questionnaire, were assessed. RESULTS: The mean distention grades of all the colonic segments and both positions were 3.22 in the control group, 3.28 in the hyoscine butylbromide group, 3.77 in the automated group, and 3.74 in the combined group. Compared with manual carbon dioxide insufflation, automated carbon dioxide insufflation significantly improved the clinical adequacy of colonic distention and shortened examination time. No statistically significant difference was seen in the clinical adequacy of distention between participants who received hyoscine butylbromide and those who did not, or in examination time. Overall, the participants' experiences were not different. CONCLUSION: Colonic distention was statistically significantly improved by automated carbon dioxide insufflation, but not by the administration of hyoscine butylbromide. The participants' tolerance was similar in each group.

Evaluation of dose reduction and image quality in CT colonography: comparison of low-dose CT with iterative reconstruction and routine-dose CT with filtered back projection.

OBJECTIVE: To prospectively evaluate the radiation dose and image quality comparing low-dose CT colonography (CTC) reconstructed using different levels of iterative reconstruction techniques with routine-dose CTC reconstructed with filtered back projection. METHODS: Following institutional ethics clearance and informed consent procedures, 210 patients underwent screening CTC using automatic tube current modulation for dual positions. Examinations were performed in the supine position with a routine-dose protocol and in the prone position, randomly applying four different low-dose protocols. Supine images were reconstructed with filtered back projection and prone images with iterative reconstruction. Two blinded observers assessed the image quality of endoluminal images. Image noise was quantitatively assessed by region-of-interest measurements. RESULTS: The mean effective dose in the supine series was 1.88 mSv using routine-dose CTC, compared to 0.92, 0.69, 0.57, and 0.46 mSv at four different low doses in the prone series (p < 0.01). Overall image quality and noise of low-dose CTC with iterative reconstruction were significantly improved compared to routine-dose CTC using filtered back projection. The lowest dose group had image quality comparable to routine-dose images. CONCLUSIONS: Low-dose CTC with iterative reconstruction reduces the radiation dose by 48.5 to 75.1% without image quality degradation compared to routine-dose CTC with filtered back projection. KEY POINTS: • Low-dose CTC reduces radiation dose ≥ 48.5% compared to routine-dose CTC. • Iterative reconstruction improves overall CTC image quality compared with FBP. • Iterative reconstruction reduces overall CTC image noise compared with FBP. • Automated exposure control with iterative reconstruction is useful for low-dose CTC.

Effect of listening to music and essential oil inhalation on patients undergoing screening CT colonography: a randomized controlled trial.

OBJECTIVE: To prospectively evaluate the effect of listening to music and inhaling aroma oil on patients undergoing screening computed tomography colonography. MATERIALS AND METHODS: Two hundred and twenty four participants were randomly allocated to one of the four groups including: (1) combined music and aroma, (2) music alone, (3) aroma alone, and (4)control. The visual analog scale for pain and a questionnaire were used for subjective outcomes. We also used a pre-test­post-test design to compare the differences in blood pressure and heart rate as objective outcomes. RESULTS: There were no statistical differences between the control group and other groups in the visual analog scale or changes in heart rate. Changes in blood pressure were similar. Participants reported good overall experiences. There were no differences in terms of overall satisfaction, pain rating, willingness to repeat the computed tomography colonography procedure in the future, or preference between colonoscopy and computed tomography colonography. More participants using music and/or aroma requested music and/or aroma during the next computed tomography colonography (P < 0.0001). CONCLUSION: Although audio and olfactory intervention had little effect on perceived pain or discomfort and vital signs, participants who listened to music and inhaled aroma during the computed tomography colonography preferred music and aroma during the next computed tomography colonography.