David S. Yu

Insights into DNA recombination from the structure of a RAD51-BRCA2 complex

The breast cancer susceptibility protein BRCA2 controls the function of RAD51, a recombinase enzyme, in pathways for DNA repair by homologous recombination. We report here the structure of a complex between an evolutionarily conserved sequence in BRCA2 (the BRC repeat) and the RecA-homology domain of RAD51. The BRC repeat mimics a motif in RAD51 that serves as an interface for oligomerization between individual RAD51 monomers, thus enabling BRCA2 to control the assembly of the RAD51 nucleoprotein filament, which is essential for strand-pairing reactions during DNA recombination. The RAD51 oligomerization motif is highly conserved among RecA-like recombinases, highlighting a common evolutionary origin for the mechanism of nucleoprotein filament formation, mirrored in the BRC repeat. Cancer-associated mutations that affect the BRC repeat disrupt its predicted interaction with RAD51, yielding structural insight into mechanisms for cancer susceptibility.

EMSY Links the BRCA2 Pathway to Sporadic Breast and Ovarian Cancer

The BRCA2 gene is mutated in familial breast and ovarian cancer, and its product is implicated in DNA repair and transcriptional regulation. Here we identify a protein, EMSY, which binds BRCA2 within a region (exon 3) deleted in cancer. EMSY is capable of silencing the activation potential of BRCA2 exon 3, associates with chromatin regulators HP1beta and BS69, and localizes to sites of repair following DNA damage. EMSY maps to chromosome 11q13.5, a region known to be involved in breast and ovarian cancer. We show that the EMSY gene is amplified almost exclusively in sporadic breast cancer (13%) and higher-grade ovarian cancer (17%). In addition, EMSY amplification is associated with worse survival, particularly in node-negative breast cancer, suggesting that it may be of prognostic value. The remarkable clinical overlap between sporadic EMSY amplification and familial BRCA2 deletion implicates a BRCA2 pathway in sporadic breast and ovarian cancer.

Full-length archaeal Rad51 structure and mutants: Mechanisms for RAD51 assembly and control by BRCA2

To clarify RAD51 interactions controlling homologous recombination, we report here the crystal structure of the full‐length RAD51 homolog from Pyrococcus furiosus. The structure reveals how RAD51 proteins assemble into inactive heptameric rings and active DNA‐bound filaments matching three‐dimensional electron microscopy reconstructions. A polymerization motif (RAD51‐PM) tethers individual subunits together to form assemblies. Subunit interactions support an allosteric ‘switch’ promoting ATPase activity and DNA binding roles for the N‐terminal domain helix–hairpin–helix (HhH) motif. Structural and mutational results characterize RAD51 interactions with the breast cancer susceptibility protein BRCA2 in higher eukaryotes. A designed P.furiosus RAD51 mutant binds BRC repeats and forms BRCA2‐dependent nuclear foci in human cells in response to γ‐irradiation‐induced DNA damage, similar to human RAD51. These results show that BRCA2 repeats mimic the RAD51‐PM and imply analogous RAD51 interactions with RAD52 and RAD54. Both BRCA2 and RAD54 may act as antagonists and chaperones for RAD51 filament assembly by coupling RAD51 interface exchanges with DNA binding. Together, these structural and mutational results support an interface exchange hypothesis for coordinated protein interactions in homologous recombination.

Dynamic Control of Rad51 Recombinase by Self-Association and Interaction with BRCA2

Here, we visualize GFP-Rad51 fusion proteins in the nucleus of living cells to demonstrate the dynamic compartmentalization of Rad51 by self-association or by binding to BRCA2. Mutants of Rad51 that fail to oligomerize and/or to bind BRCA2 distinguish three fractions of Rad51 within the nucleoplasm: a relatively mobile fraction, an immobile oligomerized fraction, and an immobile BRCA2-bound fraction. Strikingly, inhibition of replication by hydroxyurea reduces the immobile fraction of nucleoplasmic Rad51. This effect is specific to Rad51 mutants that retain the capacity to bind BRCA2, indicating that the BRCA2-bound fraction is selectively mobilized. We propose that arrested replication triggers a switch between dual functions of BRCA2 in sequestering or mobilizing a small fraction of nucleoplasmic Rad51 and suggest a mechanism for the dynamic control of protein complexes that participate in homologous recombination.

Cyclin‐dependent kinase 9–cyclin K functions in the replication stress response

Cyclin‐dependent kinase 9 (CDK9) is a well‐characterized subunit of the positive transcription elongation factor b complex in which it regulates transcription elongation in cooperation with cyclin T. However, CDK9 also forms a complex with cyclin K, the function of which is less clear. Using a synthetic lethal RNA interference screen in human cells, we identified CDK9 as a component of the replication stress response. Loss of CDK9 activity causes an increase in spontaneous levels of DNA damage signalling in replicating cells and a decreased ability to recover from a transient replication arrest. This activity is restricted to CDK9–cyclin K complexes and is independent of CDK9–cyclin T complex. CDK9 accumulates on chromatin in response to replication stress and limits the amount of single‐stranded DNA in cells under stress. Furthermore, we show that CDK9 and cyclin K interact with ataxia telangiectasia and Rad3‐related protein and other checkpoint signalling proteins. These results reveal an unexpectedly direct role for CDK9–cyclin K in checkpoint pathways that maintain genome integrity in response to replication stress.

Ultrasound GLCM texture analysis of radiation-induced parotid-gland injury in head-and-neck cancer radiotherapy: an in vivo study of late toxicity.

PURPOSE Xerostomia (dry mouth), secondary to irradiation of the parotid glands, is one of the most common side effects of head-and-neck cancer radiotherapy. Diagnostic tools able to accurately and efficiently measure parotid gland injury have yet to be introduced into the clinic. This studys purpose is to investigate sonographic textural features as potential imaging signatures for quantitative assessment of parotid-gland injury after head-and-neck radiotherapy. METHODS The authors have investigated a series of sonographic features obtained from the gray level co-occurrence matrix (GLCM) - a second order statistical method of texture analysis. These GLCM textural features were selected based on empirical observations that the normal parotid gland exhibits homogeneous echotexture, whereas the postradiotherapy parotid gland often exhibits heterogeneous echotexture. We employed eight sonographic features: (1) angular second moment (ASM), (2) inverse differential moment (IDM), (3) contrast, (4) variance, (5) correlation, (6) entropy, (7) cluster shade, and (8) cluster prominence. Altogether, sonographic properties of the parotid glands were quantified by their degrees of homogeneity (ASM and IDM), heterogeneity (contrast and variance), smoothness (correlation), randomness (entropy), and symmetry (cluster shade and prominence). The sonographic features were tested in a pilot study of 12 postradiotherapy patients and 7 healthy volunteers. The mean follow-up time for the postradiotherapy patients was 17.2 months (range: 12.1-23.9 months) and the mean radiation dose to the parotid glands was 32.3 Gy (range: 11.0-63.4 Gy). Each participant underwent one ultrasound study in which longitudinal (vertical) ultrasound scans were performed on the bilateral parotids - a total of 24 postirradiation and 14 normal parotid glands were examined. The 14 normal parotid glands served as the control group. A radiologist contoured the parotid glands on the B-mode images and the sonographic features were computed from the contoured region-of-interest. RESULTS The authors observed significant differences (p < 0.05) in all sonographic features between the normal and postradiotherapy parotid glands. The sonographic findings were consistent with the clinical observations of the ultrasound images: normal parotid glands exhibited homogeneous texture, while the postradiotherapy parotid glands exhibited heterogeneous echotexture (e.g., hyperechoic lines and spots), which likely represents fibrosis. CONCLUSIONS The authors have demonstrated the feasibility of ultrasonic texture evaluation of parotid glands; and the sonographic features may serve as imaging signatures to assess radiation-induced parotid injury.

Activation of Chromosomal DNA Replication in Saccharomyces cerevisiae by Acidic Transcriptional Activation Domains

ABSTRACT A large body of evidence from viral systems has established that transcription factors play an important and direct role in activating viral DNA replication. Among the transcriptional activation domains that can stimulate viral DNA replication are acidic domains such as those derived from herpes simplex virus VP16 and the tumor suppressor p53. Here we show that acidic activation domains can also activate a cellular origin of replication in a chromosomal context. When tethered to the yeast ARS1 (autonomously replicating sequence 1) origin of replication, both VP16 and p53 activation domains can enhance origin function. In addition, the C-terminal acidic region of the yeast transcription factor ABF1, which normally activates the ARS1 origin, is sufficient for activating ARS1 function when tethered to the origin. Mutations at residues Trp-53 and Phe-54 of a 20-residue (41 to 60) activation region of p53 abolish the activation of both replication and transcription, suggesting that the same structural determinants may be employed to activate both processes in yeast. Furthermore, using a two-dimensional gel electrophoresis method, we demonstrate that the GAL4-p53 chimeric activator can activate initiation of chromosomal replication from an origin inserted at the native ARS1 locus. These findings strongly suggest functional conservation of the mechanisms used by the acidic activation domains to activate viral DNA replication in mammalian cells and chromosomal replication in yeast.

Comparison of manual and automatic segmentation methods for brain structures in the presence of space-occupying lesions: a multi-expert study

The purpose of this work was to characterize expert variation in segmentation of intracranial structures pertinent to radiation therapy, and to assess a registration-driven atlas-based segmentation algorithm in that context. Eight experts were recruited to segment the brainstem, optic chiasm, optic nerves, and eyes, of 20 patients who underwent therapy for large space-occupying tumors. Performance variability was assessed through three geometric measures: volume, Dice similarity coefficient, and Euclidean distance. In addition, two simulated ground truth segmentations were calculated via the simultaneous truth and performance level estimation algorithm and a novel application of probability maps. The experts and automatic system were found to generate structures of similar volume, though the experts exhibited higher variation with respect to tubular structures. No difference was found between the mean Dice similarity coefficient (DSC) of the automatic and expert delineations as a group at a 5% significance level over all cases and organs. The larger structures of the brainstem and eyes exhibited mean DSC of approximately 0.8-0.9, whereas the tubular chiasm and nerves were lower, approximately 0.4-0.5. Similarly low DSCs have been reported previously without the context of several experts and patient volumes. This study, however, provides evidence that experts are similarly challenged. The average maximum distances (maximum inside, maximum outside) from a simulated ground truth ranged from (-4.3, +5.4) mm for the automatic system to (-3.9, +7.5) mm for the experts considered as a group. Over all the structures in a rank of true positive rates at a 2 mm threshold from the simulated ground truth, the automatic system ranked second of the nine raters. This work underscores the need for large scale studies utilizing statistically robust numbers of patients and experts in evaluating quality of automatic algorithms.

Ipsilateral cortical fMRI responses after peripheral nerve damage in rats reflect increased interneuron activity

In the weeks following unilateral peripheral nerve injury, the deprived primary somatosensory cortex (SI) responds to stimulation of the ipsilateral intact limb as demonstrated by functional magnetic resonance imaging (fMRI) responses. The neuronal basis of these responses was studied by using high-resolution fMRI, in vivo electrophysiological recordings, and juxtacellular neuronal labeling in rats that underwent an excision of the forepaw radial, median, and ulnar nerves. These nerves were exposed but not severed in control rats. Significant bilateral increases of fMRI responses in SI were observed in denervated rats. In the healthy SI of the denervated rats, increases in fMRI responses were concordant with increases in local field potential (LFP) amplitude and an increased incidence of single units responding compared with control rats. In contrast, in the deprived SI, increases in fMRI responses were associated with a minimal change in LFP amplitude but with increased incidence of single units responding. Based on action potential duration, juxtacellular labeling, and immunostaining results, neurons responding to intact forepaw stimulation in the deprived cortex were identified as interneurons. These results suggest that the increases in fMRI responses in the deprived cortex reflect increased interneuron activity.

Pronecrotic mixed lineage kinase domain-like protein expression is a prognostic biomarker in patients with early-stage resected pancreatic adenocarcinoma

Mixed lineage kinase domain‐like protein (MLKL) is a necrosome component mediating programmed necrosis that may be an important determinant of cancer cell death. The goal of the current study was to evaluate the prognostic value of MLKL expression in patients with pancreatic adenocarcinoma (PAC).