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IntroductionIn goal-directed tasks, visual prompts before the appearance of goals can make people ready in advance, which helps them to complete the movement better, and the presentation type of the visual prompt is very important. In previous studies, it has not been clear how different types of visual prompts guide attention in goal-directed tasks.MethodsAccording to the characteristics of goal-directed tasks, our research designed three different prompts: the cue prompt (featuring static arrow), the preparation prompt (involving dynamic countdown), and the combination prompt of cue and preparation information (simultaneously incorporating arrow and countdown). We used event-related potential components (CNV and P300) and graph theory indicators (clustering coefficient and characteristic path length) under the brain function connection to analyze the attention state of the brain.ResultsThe results showed that the combination prompts better guided the participants’ sustained attention during the prompt stage, making them well prepared for the movement. Thus, after the target appeared, the participants had better executive control and achieved a faster response to the target. However, under the combination prompt, the participants consumed more attention resources during the prompt stage.DiscussionWe believe that for the participants with impaired cognitive function, cue prompts or preparation prompts can be considered, which also play a role in guiding the participants’ attention and helping them make motor preparations when less attention resources are consumed. This study provides a neurophysiological and behavioral foundation for the design of visual prompts in goal-directed tasks.

Abstract Background Cardiac implantable electronic devices (CIEDs) has proven to be an invaluable tool in the practice of cardiology. Patients who have undergone CIED surgery with local anesthesia may result in fear, insecurity and suffering. Some studies have put efforts on ways to improve intraoperative experience of patients with local anesthesia, but researches concerning experiences of CIED patients during surgery is in its infancy. Methods Based on semi-structured and in-depth interviews, a qualitative design was conducted in a tertiary general hospital in China from May 2022 to July 2023.Purposeful sampling of 17 patients received CIED surgery and 20 medical staff were interviewed. Thematic analysis with an inductive approach was used to identify dominant themes. Results Four themes emerged from the data: (1) Safety and success is priority; (2) Humanistic Caring is a must yet be lacking; (3) Paradox of surgery information given; (4) Ways to improve surgery experiences in the operation. Conclusions Intraoperative care is significant for CIED surgery. To improve care experience during surgery, healthcare professionals should pay attention to patients’ safety and the factors that affecting humanistic caring in clinical practice. In addition, information support should consider information-seeking styles and personal needs. Besides, the four approaches presented in this study are effective to improve the intraoperative care experience.

Abstract Although some existing sparse representation (SR) methods are robust for target detection in passive bistatic radar (PBR), they still face the challenges of high computational complexity and poor detection performance for extremely low-signal-to-clutter ratio (SCR) target. So, an average effective subcarrier (AES)-domain sparse representation approach is investigated in this paper. Firstly, the AES-based SR model is proposed to solve the problem of high computational complexity, which is established by utilizing the sparseness of the orthogonal frequency-division multiplexing (OFDM) with cyclic prefix (CP) signals in each effective subcarrier domain. Then, considering the difficulty of detecting extremely low-SCR targets, clutter cancellation is implemented by the SR-based optimization model. Two AES-S algorithms, namely AES-S-based clutter cancellation in the time domain (AES-S-T) and AES-S-based clutter cancellation in the subcarrier domain (AES-S-C), are proposed, and the computational complexity is further reduced. Finally, extensive simulation and experimental results illustrate that the proposed algorithms have good detection performance and low computational complexity in PBR detection scene.

Abstract A novel 2‐D beam steering technology using a Fabry–Pérot antenna with a liquid‐based reconfigurable metasurface is presented. The antenna employs a reconfigurable partially reflecting surface to regulate phase distribution and adopts a microstrip antenna as feed to realise 2‐D beam steering. The antenna beam can be tilted in four different directions by injecting liquid metal into the specific area of the microfluidic channels embedded in the metasurface. Moreover, the antenna has a simple and compact structure with a low profile. A prototype is manufactured, and good agreement between simulated and experimental results verifies the correctness of the design. The measured results of the manufactured antenna prototype demonstrate that the main beam tilts to maximum values of ±15° and ±28° in the yoz and xoz planes, respectively, between 9.5 and 9.7 GHz.

IntroductionIn studies on consciousness detection for patients with disorders of consciousness, difference comparison of EEG responses based on active and passive task modes is difficult to sensitively detect patients’ consciousness, while a single potential analysis of EEG responses cannot comprehensively and accurately determine patients’ consciousness status. Therefore, in this paper, we designed a new consciousness detection paradigm based on a multi-stage cognitive task that could induce a series of event-related potentials and ERD/ERS phenomena reflecting different consciousness contents. A simple and direct task of paying attention to breathing was designed, and a comprehensive evaluation of consciousness level was conducted using multi-feature joint analysis.MethodsWe recorded the EEG responses of 20 healthy subjects in three modes and reported the consciousness-related mean event-related potential amplitude, ERD/ERS phenomena, and the classification accuracy, sensitivity, and specificity of the EEG responses under different conditions.ResultsThe results showed that the EEG responses of the subjects under different conditions were significantly different in the time domain and time-frequency domain. Compared with the passive mode, the amplitudes of the event-related potentials in the breathing mode were further reduced, and the theta-ERS and alpha-ERD phenomena in the frontal region were further weakened. The breathing mode showed greater distinguishability from the active mode in machine learning-based classification.DiscussionBy analyzing multiple features of EEG responses in different modes and stimuli, it is expected to achieve more sensitive and accurate consciousness detection. This study can provide a new idea for the design of consciousness detection methods.

IntroductionRhythmic visual cues (RVCs) may influence gait initiation by modulating cognition resources. However, it is unknown how RVCs modulate cognitive resources allocation during gait movements. This study focused on investigating the effects of RVCs on cortical hemodynamic response features during stepping to evaluate the changes of cognitive resources.MethodsWe recorded cerebral hemoglobin concentration changes of 14 channels in 17 healthy subjects using functional near-infrared spectroscopy (fNIRS) during stepping tasks under exposure to RVCs and non-rhythmic visual cues (NRVCs). We reported mean oxygenated hemoglobin (HbO) concentration changes, β-values, and functional connectivity (FC) between channels.ResultsThe results showed that, the RVC conditions revealed lower HbO responses compared to the NRVC conditions during the preparation and early stepping. Correspondingly, the β-values reflected that RVCs elicited lower hemodynamic responses than NRVCs, and there was a decreasing trend in stimulus-evoked cortical activation as the task progressed. However, the FC between channels were stronger under RVCs than under NRVCs during the stepping progress, and there were more significant differences in FC during the early stepping.DiscussionIn conclusion, there were lower cognitive demand and stronger FC under RVC conditions than NRVC conditions, which indicated higher efficiency of cognitive resources allocation during stepping tasks. This study may provide a new insight for further understanding the mechanism on how RVCs alleviate freezing of gait.

Cilia project from cells to serve sensory functions, and ciliary disruption can result in multiple disorders known as ciliopathies. Here the authors show that the ciliopathy gene TCTN1 functions to regulate the ciliary transition zone and ectosome formation.

Rhythmic visual cues can affect the allocation of cognitive resources during gait initiation (GI) and motor preparation. However, it is unclear how the input of rhythmic visual information modulates the allocation of cognitive resources and affects GI. The purpose of this study was to explore the effect of rhythmic visual cues on the dynamic allocation of cognitive resources by recording electroencephalographic (EEG) activity during exposure to visual stimuli. This study assessed event-related potentials (ERPs), event-related synchronization/desynchronization (ERS/ERD), and EEG microstates at 32 electrodes during presentation of non-rhythmic and rhythmic visual stimuli in 20 healthy participants. The ERP results showed that the amplitude of the C1 component was positive under exposure to rhythmic visual stimuli, while the amplitude of the N1 component was higher under exposure to rhythmic visual stimuli compared to their non-rhythmic counterparts. Within the first 200 ms of the onset of rhythmic visual stimuli, ERS in the theta band was highly pronounced in all brain regions analyzed. The results of microstate analysis showed that rhythmic visual stimuli were associated with an increase in cognitive processing over time, while non-rhythmic visual stimuli were associated with a decrease. Overall, these findings indicated that, under exposure to rhythmic visual stimuli, consumption of cognitive resources is lower during the first 200 ms of visual cognitive processing, but the consumption of cognitive resources gradually increases over time. After approximately 300 ms, cognitive processing of rhythmic visual stimuli consumes more cognitive resources than processing of stimuli in the non-rhythmic condition. This indicates that the former is more conducive to the completion of gait-related motor preparation activities, based on processing of rhythmic visual information during the later stages. This finding indicates that the dynamic allocation of cognitive resources is the key to improving gait-related movement based on rhythmic visual cues.

Timely completion of eukaryotic genome duplication requires coordinated DNA replication initiation at multiple origins. Replication begins with the loading of the Mini-Chromosome Maintenance (MCM) complex, proceeds by the activation of the Cdc45-MCM-GINS (CMG) helicase, and ends with CMG removal after chromosomes are fully replicated. Post-translational modifications on the MCM and associated factors ensure an orderly transit of these steps. Although the mechanisms of CMG activation and removal are partially understood, regulated MCM loading is not, leaving an incomplete understanding of how DNA replication begins. Here we describe a site-specific modification of Mcm3 by the Small Ubiquitin-like MOdifier (SUMO). Mutations that prevent this modification reduce the MCM loaded at replication origins and lower CMG levels, resulting in impaired cell growth, delayed chromosomal replication, and the accumulation of gross chromosomal rearrangements (GCRs). These findings demonstrate the existence of a SUMO-dependent regulation of origin-bound MCM and show that this pathway is needed to prevent genome rearrangements.

Abstract The existence of walls surrounding targets leads to multi-path return at the radar receiver, which provides additional information about the target, and thus can be exploited to strengthen the quality of through-wall radar imaging (TWRI). Based on this, a multi-path exploitation method is proposed to identify the location of the multi-path ghost. An algorithm that combining the modified Green’s function with back projection algorithm is presented to associate and map the multi-path ghosts to the location of real targets. The theoretical analysis is verified according to the simulation results obtained using gprMAX software as well as practical radar measured data, and our proposed method is shown to outperform that in conventional multi-path exploitation method (Setlur et al., IEEE Trans. Geosci. Remote Sens. 49:4021–4034, 2011).

In motor imagery (MI) brain–computer interface (BCI) research, some researchers have designed MI paradigms of force under a unilateral upper-limb static state. It is difficult to apply these paradigms to the dynamic force interaction process between the robot and the patient in a brain-controlled rehabilitation robot system, which needs to induce thinking states of the patient’s demand for assistance. Therefore, in our research, according to the movement of wiping the table in human daily life, we designed a three-level-force MI paradigm under a unilateral upper-limb dynamic state. Based on the event-related de-synchronization (ERD) feature analysis of the electroencephalography (EEG) signals generated by the brain’s force change motor imagination, we proposed a multi-scale temporal convolutional network with attention mechanism (MSTCN-AM) algorithm to recognize ERD features of MI-EEG signals. Aiming at the slight feature differences of single-trial MI-EEG signals among different levels of force, the MSTCN module was designed to extract fine-grained features of different dimensions in the time–frequency domain. The spatial convolution module was then used to learn the area differences of space domain features. Finally, the attention mechanism dynamically weighted the time–frequency–space domain features to improve the algorithm’s sensitivity. The results showed that the accuracy of the algorithm was 86.4 ± 14.0% for the three-level-force MI-EEG data collected experimentally. Compared with the baseline algorithms (OVR-CSP+SVM (77.6 ± 14.5%), Deep ConvNet (75.3 ± 12.3%), Shallow ConvNet (77.6 ± 11.8%), EEGNet (82.3 ± 13.8%), and SCNN-BiLSTM (69.1 ± 16.8%)), our algorithm had higher classification accuracy with significant differences and better fitting performance.

Visuospatial selective attention can focus attention on a certain spatial area and rationally allocate attentional resources during visual target perception. Previous studies investigated the behavioral advantages of subjects when the target appeared in the upper and lower visual fields. However, the neurophysiological characteristics of the brain are not clear, and there is a lack of comprehensive analysis of the external behavior and the internal neurophysiological characteristics. We designed two task paradigms containing a spatial location orientation task and a visual search task. We used event-related potentials (ERP) components (P1 and P2) and electroencephalogram (EEG) rhythms (theta and alpha) to analyze the attention level and allocation of attention resources of the brain. The results showed that when the target appeared in the lower visual field in the spatial location orientation task, subjects consumed fewer attention resources and demonstrated better behavioral performance. In the visual search task, when the target appeared in the upper left visual field, subjects could better mobilize attention resources and behaved more advantageously. The study provides a basis for the design of the target in the upper and lower visual fields in the rehabilitation task, especially for stroke patients with low attention levels due to attention disorders such as spatial attention deficit.

Protein sumoylation, especially when catalyzed by the Mms21 SUMO E3 ligase, plays a major role in suppressing duplication-mediated gross chromosomal rearrangements (dGCRs). How Mms21 targets its substrates in the cell is insufficiently understood. Here, we demonstrate that Esc2, a protein with SUMO-like domains (SLDs), recruits the Ubc9 SUMO conjugating enzyme to specifically facilitate Mms21-dependent sumoylation and suppress dGCRs. The D430R mutation in Esc2 impairs its binding to Ubc9 and causes a synergistic growth defect and accumulation of dGCRs with mutations that delete the Siz1 and Siz2 E3 ligases. By contrast, esc2-D430R does not appreciably affect sensitivity to DNA damage or the dGCRs caused by the catalytically inactive mms21-CH. Moreover, proteome-wide analysis of intracellular sumoylation demonstrates that esc2-D430R specifically down-regulates sumoylation levels of Mms21-preferred targets, including the nucleolar proteins, components of the SMC complexes and the MCM complex that acts as the catalytic core of the replicative DNA helicase. These effects closely resemble those caused by mms21-CH, and are relatively unaffected by deleting Siz1 and Siz2. Thus, by recruiting Ubc9, Esc2 facilitates Mms21-dependent sumoylation to suppress the accumulation of dGCRs independent of Siz1 and Siz2.

High-efficiency dechlorination processes are crucial for the clean utilization of CuCl waste residue from zinc hydrometallurgical processes. A new method of oxygen-enriched microwave roasting has been proposed to improve the dechlorination process. The cavity perturbation method was used in this paper to measure the permittivity of a CuCl residue at various temperatures and apparent densities. The results show that temperature had a more significant effect on the loss tangent when it exceeded 400 °C more than that of apparent densities. The degree of dechlorination of CuCl residue exceeded 93% after 90 min of microwave roasting at 450 °C and 150 mL/min oxygen flow, which was 12% higher than that of conventional calcination. The elemental distribution in samples was studied using single-point, linear, and lateral scans at the microscale, and the microstructure and phase changes of the CuCl residue under a microwave field were characterized by XRD and SEM-EDS. This study shows that microwaves can promote the dechlorination reaction by decreasing the activation energy from 52.69 kJ/mol to 42.36 kJ/mol.

Wireless powered techniques have been recognized as promising techniques in future wireless communication systems, especially in cognitive radios (CRs) with energy-limit devices. However, most of the existing works focus on CRs with an ideal linear energy harvesting model. In this paper, a wireless powered wideband CR network is considered, and a practical non-linear energy harvesting model is adopted. To maximize the sum throughput of the secondary users, the energy harvesting time, channel allocation, and transmit power are jointly optimized. The closed-form expressions for the optimal transmit power and channel allocation are given. Simulation results show that there is a tradeoff between the harvesting energy and the sum throughput of the secondary users. It is also shown that the performance achieved under the non-linear energy harvesting model may equal to that achieved under the linear energy harvesting model.

Rotary ultrasonic machining has been widely used for machining of hard and brittle materials due to the advantages of low cutting force, high machining accuracy, and high surface integrity. Focusing on the development of specialized rotary ultrasonic machining systems, this article summarizes the advances in the functional components and key technologies of rotary ultrasonic machining systems for hard and brittle materials, including the ultrasonic generator, power transfer structure, transducer, ultrasonic horn, and cutting tool. Developments on the automatic frequency tracking method, the establishment of an electrical compensation model for power transfer, the energy conversion characteristics of piezoelectric materials and giant magnetostrictive materials, and the design methods for the ultrasonic horn and cutting tool were elaborated. The principle of magnetostrictive energy conversion, output amplitude characteristics of a giant magnetostrictive transducer, and high-power giant magnetostrictive rotary ultrasonic machining systems were also presented. Future research and developments of rotary ultrasonic machining systems regarding the ultrasonic generator, amplitude stability, energy conversion efficiency, vibration mode, and system integration were finally discussed.

The kinetochore is the central molecular machine that drives chromosome segregation in all eukaryotes. Genetic studies have suggested that protein sumoylation plays a role in regulating the inner kinetochore; however, the mechanism remains elusive. Here, we show that Saccharomyces cerevisiae Ulp2, an evolutionarily conserved SUMO specific protease, contains a previously uncharacterized kinetochore-targeting motif that recruits Ulp2 to the kinetochore via the Ctf3CENP-I-Mcm16CENP-H-Mcm22CENP-K complex (CMM). Once recruited, Ulp2 selectively targets multiple subunits of the kinetochore, specifically the Constitutive Centromere-Associated Network (CCAN), via its SUMO-interacting motif (SIM). Mutations that impair the kinetochore recruitment of Ulp2 or its binding to SUMO result in an elevated rate of chromosome loss, while mutations that affect both result in a synergistic increase of chromosome loss rate, hyper-sensitivity to DNA replication stress, along with a dramatic accumulation of hyper-sumoylated CCAN. Notably, sumoylation of CCAN occurs at the kinetochore and is perturbed by DNA replication stress. These results indicate that Ulp2 utilizes its dual substrate recognition to prevent hyper-sumoylation of CCAN, ensuring accurate chromosome segregation during cell division.

In this paper, we present a novel muscle synergy extraction method based on multivariate curve resolution–alternating least squares (MCR-ALS) to overcome the limitation of the nonnegative matrix factorization (NMF) method for extracting non-sparse muscle synergy, and we study its potential application for evaluating motor function of stroke survivors. Nonnegative matrix factorization (NMF) is the most widely used method for muscle synergy extraction. However, NMF is susceptible to components’ sparseness and usually provides inferior reliability, which significantly limits the promotion of muscle synergy. In this study, MCR-ALS was employed to extract muscle synergy from electromyography (EMG) data. Its performance was compared with two other matrix factorization algorithms, NMF and self-modeling mixture analysis (SMMA). Simulated data sets were utilized to explore the influences of the sparseness and noise on the extracted synergies. As a result, the synergies estimated by MCR-ALS were the most similar to true synergies as compared with SMMA and NMF. MCR-ALS was used to analyze the muscle synergy characteristics of upper limb movements performed by healthy (n = 11) and stroke (n = 5) subjects. The repeatability and intra-subject consistency were used to evaluate the performance of MCR-ALS. As a result, MCR-ALS provided much higher repeatability and intra-subject consistency as compared with NMF, which were important for the reliability of the motor function evaluation. The stroke subjects had lower intra-subject consistency and seemingly had more synergies as compared with the healthy subjects. Thus, MCR-ALS is a promising muscle synergy analysis method for motor function evaluation of stroke patients.

Mms21, a subunit of the Smc5/6 complex, possesses an E3 ligase activity for the Small Ubiquitin-like MOdifier (SUMO). Here we show that the mms21-CH mutation, which inactivates Mms21 ligase activity, causes increased accumulation of gross chromosomal rearrangements (GCRs) selected in the dGCR assay. These dGCRs are formed by non-allelic homologous recombination between divergent DNA sequences mediated by Rad52-, Rrm3- and Pol32-dependent break-induced replication. Combining mms21-CH with sgs1Δ caused a synergistic increase in GCRs rates, indicating the distinct roles of Mms21 and Sgs1 in suppressing GCRs. The mms21-CH mutation also caused increased rates of accumulating uGCRs mediated by breakpoints in unique sequences as revealed by whole genome sequencing. Consistent with the accumulation of endogenous DNA lesions, mms21-CH mutants accumulate increased levels of spontaneous Rad52 and Ddc2 foci and had a hyper-activated DNA damage checkpoint. Together, these findings support that Mms21 prevents the accumulation of spontaneous DNA lesions that cause diverse GCRs.

A nonlinear inversion scheme is proposed for electromagnetic inverse scattering imaging. It exploits inexact Newton (IN) and least square QR factorization (LSQR) methods to tackle the nonlinearity and ill-posedness of the electromagnetic inverse scattering problem. A nonlinear model of the inverse scattering in functional form is developed. At every IN iteration, the sparse storage method is adopted to solve the storage and computational bottleneck of Fréchet derivative matrix, a large-scale sparse Jacobian matrix. Moreover, to address the slow convergence problem encountered in the inexact Newton solution via Landweber iterations, an LSQR algorithm is proposed for obtaining a better solution of the internal large-scale sparse linear equations in the IN step. Numerical results demonstrate the applicability of the proposed IN-LSQR method to quantitative inversion of scatterer electric performance parameters. Moreover, compared with the inexact Newton method based on Landweber iterations, the proposed method significantly improves the convergence rate with less computational and storage cost.

In an integrated radar-communication network, multiuser access techniques with minimal performance degradation and without range-Doppler ambiguities are required, especially in a dense user environment. In this paper, a multiuser access scheme with random subcarrier allocation mechanism is proposed for orthogonal frequency division multiplexing (OFDM) based integrated radar-communication networks. The expression of modulation Symbol-Domain method combined with sparse representation (SR) for range-Doppler estimation is introduced and a parallel reconstruction algorithm is employed. The radar target detection performance is improved with less spectrum occupation. Additionally, a Doppler frequency detector is exploited to decrease the computational complexity. Numerical simulations show that the proposed method outperforms the traditional modulation Symbol-Domain method under ideal and realistic nonideal scenarios.

[This corrects the article DOI: 10.1371/journal.pgen.1003670.].

Meiotic recombination plays a key role in sexual reproduction as it generates crossovers that, in combination with sister chromatid cohesion, physically connect homologous chromosomes, thereby promoting their proper segregation at the first meiotic division. Meiotic recombination is initiated by programmed double strand breaks (DSBs) catalyzed by the evolutionarily conserved, topoisomerase-like protein Spo11. Repair of these DSBs is highly regulated to create crossovers between homologs that are distributed throughout the genome. This repair requires the presence of the mitotic recombinase, Rad51, as well as the strand exchange activity of the meiosis-specific recombinase, Dmc1. A key regulator of meiotic DSB repair in Saccharomyces cerevisiae is the meiosis-specific kinase Mek1, which promotes interhomolog strand invasion and is required for the meiotic recombination checkpoint and the crossover/noncrossover decision. Understanding how Mek1 regulates meiotic recombination requires the identification of its substrates. Towards that end, an unbiased phosphoproteomic approach utilizing Stable Isotope Labeling by Amino Acids in Cells (SILAC) was utilized to generate a list of potential Mek1 substrates, as well as proteins containing consensus phosphorylation sites for cyclin-dependent kinase, the checkpoint kinases, Mec1/Tel1, and the polo-like kinase, Cdc5. These experiments represent the first global phosphoproteomic dataset for proteins in meiotic budding yeast.

In order to preserve genome integrity, extrinsic or intrinsic DNA damages must be repaired before they accumulate in cells and trigger other mutations and genome rearrangements. Eukaryotic cells are able to respond to different genotoxic stresses as well as to single DNA double strand breaks (DSBs), suggesting highly sensitive and robust mechanisms to detect lesions that trigger a signal transduction cascade which, in turn, controls the DNA damage response (DDR). Furthermore, cells must be able to distinguish natural chromosomal ends from DNA DSBs in order to prevent inappropriate checkpoint activation, DDR and chromosomal rearrangements. Since the original discovery of RAD9, the first DNA damage checkpoint gene identified in Saccharomyces cerevisiae, many genes that have a role in this pathway have been identified, including MRC1, MEC3, RAD24, RAD53, DUN1, MEC1 and TEL1. Extensive studies have established most of the genetic basis of the DNA damage checkpoint and uncovered its different functions in cell cycle regulation, DNA replication and repair, and telomere maintenance. However, major questions concerning the regulation and functions of the DNA damage checkpoint remain to be answered. First, how is the checkpoint activity coupled to DNA replication and repair? Second, how do cells distinguish natural chromosome ends from deleterious DNA DSBs? In this review we will examine primarily studies performed using Saccharomyces cerevisiae as a model system.

Interhomolog crossovers promote proper chromosome segregation during meiosis and are formed by the regulated repair of programmed double-strand breaks. This regulation requires components of the synaptonemal complex (SC), a proteinaceous structure formed between homologous chromosomes. In yeast, SC formation requires the "ZMM" genes, which encode a functionally diverse set of proteins, including the transverse filament protein, Zip1. In wild-type meiosis, Zmm proteins promote the biased resolution of recombination intermediates into crossovers that are distributed throughout the genome by interference. In contrast, noncrossovers are formed primarily through synthesis-dependent strand annealing mediated by the Sgs1 helicase. This work identifies a conserved region on the C terminus of Zip1 (called Zip1 4S), whose phosphorylation is required for the ZMM pathway of crossover formation. Zip1 4S phosphorylation is promoted both by double-strand breaks (DSBs) and the meiosis-specific kinase, MEK1/MRE4, demonstrating a role for MEK1 in the regulation of interhomolog crossover formation, as well as interhomolog bias. Failure to phosphorylate Zip1 4S results in meiotic prophase arrest, specifically in the absence of SGS1. This gain of function meiotic arrest phenotype is suppressed by spo11Δ, suggesting that it is due to unrepaired breaks triggering the meiotic recombination checkpoint. Epistasis experiments combining deletions of individual ZMM genes with sgs1-md zip1-4A indicate that Zip1 4S phosphorylation functions prior to the other ZMMs. These results suggest that phosphorylation of Zip1 at DSBs commits those breaks to repair via the ZMM pathway and provides a mechanism by which the crossover/noncrossover decision can be dynamically regulated during yeast meiosis.

A variety of cellular pathways are regulated by protein modifications with ubiquitin-family proteins. SUMO, the Small Ubiquitin-like MOdifier, is covalently attached to lysine on target proteins via a cascade reaction catalyzed by E1, E2, and E3 enzymes. A major barrier to understanding the diverse regulatory roles of SUMO has been a lack of suitable methods to identify protein sumoylation sites. Here we developed a mass-spectrometry (MS) based approach combining chemical and enzymatic modifications to identify sumoylation sites. We applied this method to analyze the auto-sumoylation of the E1 enzyme in vitro and compared it to the GG-remnant method using Smt3-I96R as a substrate. We further examined the effect of smt3-I96R mutation in vivo and performed a proteome-wide analysis of protein sumoylation sites in Saccharomyces cerevisiae. To validate these findings, we confirmed several sumoylation sites of Aos1 and Uba2 in vivo. Together, these results demonstrate that our chemical and enzymatic method for identifying protein sumoylation sites provides a useful tool and that a combination of methods allows a detailed analysis of protein sumoylation sites.

Checkpoint kinase 2 (Chk2) is a major regulator of DNA damage response and can induce alternative cellular responses: cell cycle arrest and DNA repair or programmed cell death. Here, we report the identification of a new role of Chk2 in transcriptional regulation that also contributes to modulating the balance between survival and apoptosis following DNA damage. We found that Chk2 interacts with members of the NCoR/SMRT transcriptional co-regulator complexes and serves as a functional component of the repressor complex, being required for recruitment of SMRT on the promoter of pro-apoptotic genes upon DNA damage. Thus, the co-repressor SMRT exerts a critical protective action against genotoxic stress-induced caspase activation, repressing a functionally important cohort of pro-apoptotic genes. Amongst them, SMRT is responsible for basal repression of Wip1, a phosphatase that de-phosphorylates and inactivates Chk2, thus affecting a feedback loop responsible for licensing the correct timing of Chk2 activation and the proper execution of the DNA repair process.

Suppression of duplication-mediated gross chromosomal rearrangements (GCRs) is essential to maintain genome integrity in eukaryotes. Here we report that SUMO ligase Mms21 has a strong role in suppressing GCRs in Saccharomyces cerevisiae, while Siz1 and Siz2 have weaker and partially redundant roles. Understanding the functions of these enzymes has been hampered by a paucity of knowledge of their substrate specificity in vivo. Using a new quantitative SUMO-proteomics technology, we found that Siz1 and Siz2 redundantly control the abundances of most sumoylated substrates, while Mms21 more specifically regulates sumoylation of RNA polymerase-I and the SMC-family proteins. Interestingly, Esc2, a SUMO-like domain-containing protein, specifically promotes the accumulation of sumoylated Mms21-specific substrates and functions with Mms21 to suppress GCRs. On the other hand, the Slx5-Slx8 complex, a SUMO-targeted ubiquitin ligase, suppresses the accumulation of sumoylated Mms21-specific substrates. Thus, distinct SUMO ligases work in concert with Esc2 and Slx5-Slx8 to control substrate specificity and sumoylation homeostasis to prevent GCRs.

The Whi5 transcriptional repressor is a negative regulator of G1 cell cycle progression in Saccharomyces cerevisiae and is functionally equivalent to the Retinoblastoma (Rb) tumor suppressor protein in mammals. In early G1, Whi5 binds to and inhibits SBF (Swi4/Swi6) transcriptional complexes. At Start, Cln:Cdc28 kinases phosphorylate and inactivate Whi5, causing its dissociation from SBF promoters and nuclear export, allowing activation of SBF transcription and entry into late G1. In an analysis of Whi5 phosphorylation, we found that 10 of the 12 putative CDK phosphorylation sites on Whi5 were occupied in vivo in asynchronously growing cells. In addition, we identified 6 non-CDK Whi5 phosphorylation sites. Whi5 CDK and non-CDK phosphorylation mutants were functional and able to rescue the small cell size of whi5Delta cells. However, the Whi5 CDK mutant with all 12 putative CDK sites changed to alanine causes a dramatic cell cycle phenotype when expressed with a Swi6 CDK phosphorylation mutant. Mutational analysis of Whi5 determined that only four C-terminal CDK sites were necessary and sufficient for Whi5 inactivation when Swi6 CDK sites were also mutated. Although these four Whi5 CDK sites do not wholly determine Whi5 nuclear export, they do impact regulation of cell size. Taken together, these observations begin to dissect the regulatory role of specific phosphorylation sites on Whi5.