Human AAA+ ATPase FIGNL1 suppresses RAD51-mediated ultra-fine bridge formation.

RAD51 filament is crucial for the homology-dependent repair of DNA double-strand breaks and stalled DNA replication fork protection. Positive and negative regulators control RAD51 filament assembly and disassembly. RAD51 is vital for genome integrity but excessive accumulation of RAD51 on chromatin causes genome instability and growth defects. However, the detailed mechanism underlying RAD51 disassembly by negative regulators and the physiological consequence of abnormal RAD51 persistence remain largely unknown. Here, we report the role of the human AAA+ ATPase FIGNL1 in suppressing a novel type of RAD51-mediated genome instability. FIGNL1 knockout human cells were defective in RAD51 dissociation after replication fork restart and accumulated ultra-fine chromosome bridges (UFBs), whose formation depends on RAD51 rather than replication fork stalling. FIGNL1 suppresses homologous recombination intermediate-like UFBs generated between sister chromatids at genomic loci with repeated sequences such as telomeres and centromeres. These data suggest that RAD51 persistence per se induces the formation of unresolved linkage between sister chromatids resulting in catastrophic genome instability. FIGNL1 facilitates post-replicative disassembly of RAD51 filament to suppress abnormal recombination intermediates and UFBs. These findings implicate FIGNL1 as a key factor required for active RAD51 removal after processing of stalled replication forks, which is essential to maintain genome stability.

Replication protein-A, RPA, plays a pivotal role in the maintenance of recombination checkpoint in yeast meiosis.

DNA double-strand breaks (DSBs) activate DNA damage responses (DDRs) in both mitotic and meiotic cells. A single-stranded DNA (ssDNA) binding protein, Replication protein-A (RPA) binds to the ssDNA formed at DSBs to activate ATR/Mec1 kinase for the response. Meiotic DSBs induce homologous recombination monitored by a meiotic DDR called the recombination checkpoint that blocks the pachytene exit in meiotic prophase I. In this study, we further characterized the essential role of RPA in the maintenance of the recombination checkpoint during Saccharomyces cerevisiae meiosis. The depletion of an RPA subunit, Rfa1, in a recombination-defective dmc1 mutant, fully alleviates the pachytene arrest with the persistent unrepaired DSBs. RPA depletion decreases the activity of a meiosis-specific CHK2 homolog, Mek1 kinase, which in turn activates the Ndt80 transcriptional regulator for pachytene exit. These support the idea that RPA is a sensor of ssDNAs for the activation of meiotic DDR. Rfa1 depletion also accelerates the prophase I delay in the zip1 mutant defective in both chromosome synapsis and the recombination, consistent with the notion that the accumulation of ssDNAs rather than defective synapsis triggers prophase I delay in the zip1 mutant.

RPA interacts with Rad52 to promote meiotic crossover and noncrossover recombination.

Meiotic recombination is initiated by programmed double-strand breaks (DSBs). Studies in Saccharomyces cerevisiae have shown that, following rapid resection to generate 3' single-stranded DNA (ssDNA) tails, one DSB end engages a homolog partner chromatid and is extended by DNA synthesis, whereas the other end remains associated with its sister. Then, after regulated differentiation into crossover- and noncrossover-fated types, the second DSB end participates in the reaction by strand annealing with the extended first end, along both pathways. This second-end capture is dependent on Rad52, presumably via its known capacity to anneal two ssDNAs. Here, using physical analysis of DNA recombination, we demonstrate that this process is dependent on direct interaction of Rad52 with the ssDNA binding protein, replication protein A (RPA). Furthermore, the absence of this Rad52-RPA joint activity results in a cytologically-prominent RPA spike, which emerges from the homolog axes at sites of crossovers during the pachytene stage of the meiotic prophase. Our findings suggest that this spike represents the DSB end of a broken chromatid caused by either the displaced leading DSB end or the second DSB end, which has been unable to engage with the partner homolog-associated ssDNA. These and other results imply a close correspondence between Rad52-RPA roles in meiotic recombination and mitotic DSB repair.

DNA double-strand breaks regulate the cleavage-independent release of Rec8-cohesin during yeast meiosis.

The mitotic cohesin complex necessary for sister chromatid cohesion and chromatin loop formation shows local and global association to chromosomes in response to DNA double-strand breaks (DSBs). Here, by genome-wide binding analysis of the meiotic cohesin with Rec8, we found that the Rec8-localization profile along chromosomes is altered from middle to late meiotic prophase I with cleavage-independent dissociation. Each Rec8-binding site on the chromosome axis follows a unique alternation pattern with dissociation and probably association. Centromeres showed altered Rec8 binding in late prophase I relative to mid-prophase I, implying chromosome remodeling of the regions. Rec8 dissociation ratio per chromosome is correlated well with meiotic DSB density. Indeed, the spo11 mutant deficient in meiotic DSB formation did not change the distribution of Rec8 along chromosomes in late meiotic prophase I. These suggest the presence of a meiosis-specific regulatory pathway for the global binding of Rec8-cohesin in response to DSBs.

Heterozygosity alters Msh5 binding to meiotic chromosomes in the baker's yeast.

Meiotic crossovers are initiated from programmed DNA double-strand breaks. The Msh4-Msh5 heterodimer is an evolutionarily conserved mismatch repair-related protein complex that promotes meiotic crossovers by stabilizing strand invasion intermediates and joint molecule structures such as Holliday junctions. In vivo studies using homozygous strains of the baker's yeast Saccharomyces cerevisiae (SK1) show that the Msh4-Msh5 complex associates with double-strand break hotspots, chromosome axes, and centromeres. Many organisms have heterozygous genomes that can affect the stability of strand invasion intermediates through heteroduplex rejection of mismatch-containing sequences. To examine Msh4-Msh5 function in a heterozygous context, we performed chromatin immunoprecipitation and sequencing (ChIP-seq) analysis in a rapidly sporulating hybrid S. cerevisiae strain (S288c-sp/YJM789, containing sporulation-enhancing QTLs from SK1), using SNP information to distinguish reads from homologous chromosomes. Overall, Msh5 localization in this hybrid strain was similar to that determined in the homozygous strain (SK1). However, relative Msh5 levels were reduced in regions of high heterozygosity, suggesting that high mismatch densities reduce levels of recombination intermediates to which Msh4-Msh5 binds. Msh5 peaks were also wider in the hybrid background compared to the homozygous strain (SK1). We determined regions containing heteroduplex DNA by detecting chimeric sequence reads with SNPs from both parents. Msh5-bound double-strand break hotspots overlap with regions that have chimeric DNA, consistent with Msh5 binding to heteroduplex-containing recombination intermediates.

Positive and negative regulators of RAD51/DMC1 in homologous recombination and DNA replication.

RAD51 recombinase plays a central role in homologous recombination (HR) by forming a nucleoprotein filament on single-stranded DNA (ssDNA) to catalyze homology search and strand exchange between the ssDNA and a homologous double-stranded DNA (dsDNA). The catalytic activity of RAD51 assembled on ssDNA is critical for the DNA-homology-mediated repair of DNA double-strand breaks in somatic and meiotic cells and restarting stalled replication forks during DNA replication. The RAD51-ssDNA complex also plays a structural role in protecting the regressed/reversed replication fork. Two types of regulators control RAD51 filament formation, stability, and dynamics, namely positive regulators, including mediators, and negative regulators, so-called remodelers. The appropriate balance of action by the two regulators assures genome stability. This review describes the roles of positive and negative RAD51 regulators in HR and DNA replication and its meiosis-specific homolog DMC1 in meiotic recombination. We also provide future study directions for a comprehensive understanding of RAD51/DMC1-mediated regulation in maintaining and inheriting genome integrity.

Postoperative Spinal Subdural Extra-arachnoid Hygroma Because of Trauma: Resolution with Lumbar Puncture: A Case Report.

CASE: A 73-year-old woman, after spinal surgery, presented with symptomatic spinal subdural extra-arachnoid hygroma (SSEH) because of a fall on the third postoperative day. The hygroma was diagnosed by magnetic resonance imaging (MRI). Lumbar puncture was performed under local anesthesia, after which the leg pain disappeared immediately. MRI obtained immediately after puncture and 1 week later confirmed disappearance of the hygroma. CONCLUSION: Although dural transection is mentioned in most of the reports on treatment of symptomatic postoperative SSEH, we were able to treat this entity by epidural puncture. In the absence of paraplegia or cystorectal disturbance, puncture can be an effective and minimally invasive treatment option.

Fanconi anemia-associated mutation in RAD51 compromises the coordinated action of DNA-binding and ATPase activities.

Fanconi anemia (FA) is a rare genetic disease caused by a defect in DNA repair pathway for DNA interstrand crosslinks. These crosslinks can potentially impede the progression of the DNA replication fork, consequently leading to DNA double-strand breaks. Heterozygous RAD51-Q242R mutation has been reported to cause FA-like symptoms. However, the molecular defect of RAD51 underlying the disease is largely unknown. In this study, we conducted a biochemical analysis of RAD51-Q242R protein, revealing notable deficiencies in its DNA-dependent ATPase activity and its ATP-dependent regulation of DNA-binding activity. Interestingly, although RAD51-Q242R exhibited the filament instability and lacked the ability to form displacement loop, it efficiently stimulated the formation of displacement loops mediated by wild-type RAD51. These findings facilitate understanding of the biochemical properties of the mutant protein and how RAD51 works in the FA patient cells.

FIGNL1 AAA+ ATPase remodels RAD51 and DMC1 filaments in pre-meiotic DNA replication and meiotic recombination.

The formation of RAD51/DMC1 filaments on single-stranded (ss)DNAs essential for homology search and strand exchange in DNA double-strand break (DSB) repair is tightly regulated. FIGNL1 AAA+++ ATPase controls RAD51-mediated recombination in human cells. However, its role in gametogenesis remains unsolved. Here, we characterized a germ line-specific conditional knockout (cKO) mouse of FIGNL1. Fignl1 cKO male mice showed defective chromosome synapsis and impaired meiotic DSB repair with the accumulation of RAD51/DMC1 on meiotic chromosomes, supporting a positive role of FIGNL1 in homologous recombination at a post-assembly stage of RAD51/DMC1 filaments. Fignl1 cKO spermatocytes also accumulate RAD51/DMC1 on chromosomes in pre-meiotic S-phase. These RAD51/DMC1 assemblies are independent of meiotic DSB formation. We also showed that purified FIGNL1 dismantles RAD51 filament on double-stranded (ds)DNA as well as ssDNA. These results suggest an additional role of FIGNL1 in limiting the non-productive assembly of RAD51/DMC1 on native dsDNAs during pre-meiotic S-phase and meiotic prophase I.

Characterizing interaction between the juxtamembrane region of the single transmembrane protein and membrane using chemically synthesized peptides.

In membrane proteins, a transmembrane region and a juxtamembrane region play important roles in its function. Here, we present a protocol for characterizing membrane protein dynamics between the juxtamembrane region of the single transmembrane protein and acidic membrane. We describe steps for solid-phase peptide synthesis, peptide purification, and labeling. We then detail reconstitution of the transmembrane peptide into lipid bilayers and its evaluation and structural analysis. For complete details on the use and execution of this protocol, please refer to Prasada Rao et al.1.

The Msh5 complex shows homeostatic localization in response to DNA double-strand breaks in yeast meiosis.

Meiotic crossing over is essential for the segregation of homologous chromosomes. The formation and distribution of meiotic crossovers (COs), which are initiated by the formation of double-strand break (DSB), are tightly regulated to ensure at least one CO per bivalent. One type of CO control, CO homeostasis, maintains a consistent level of COs despite fluctuations in DSB numbers. Here, we analyzed the localization of proteins involved in meiotic recombination in budding yeast xrs2 hypomorphic mutants which show different levels of DSBs. The number of cytological foci with recombinases, Rad51 and Dmc1, which mark single-stranded DNAs at DSB sites is proportional to the DSB numbers. Among the pro-CO factor, ZMM/SIC proteins, the focus number of Zip3, Mer3, or Spo22/Zip4, was linearly proportional to reduced DSBs in the xrs2 mutant. In contrast, foci of Msh5, a component of the MutSγ complex, showed a non-linear response to reduced DSBs. We also confirmed the homeostatic response of COs by genetic analysis of meiotic recombination in the xrs2 mutants and found a chromosome-specific homeostatic response of COs. Our study suggests that the homeostatic response of the Msh5 assembly to reduced DSBs was genetically distinct from that of the Zip3 assembly for CO control.

The role of conserved amino acid residues of Sae3 in Mei5-Sae3 complex for Dmc1 assembly in meiotic recombination.

Meiotic recombination between homologous chromosomes is promoted by the collaborative action of two RecA homologs, Rad51 and meiosis-specific Dmc1. The filament assembly of Dmc1 is promoted by meiosis-specific Mei5-Sae3 in budding yeast. Mei5-Sae3 shows sequence similarity to fission yeast Sfr1-Swi5, which stimulates DNA strand exchanges by Rad51 as well as Dmc1. Sae3 and Swi5 share a conserved motif with the amino acid sequence YNEI/LK/RD. In this study, we analyzed the role of the YNEL residues in the Sae3 sequence in meiotic recombination and found that these residues are critical for Sae3 function in Dmc1 assembly. L59 substitution in the Sae3 protein disrupts complex formation with Mei5, while Y56 and N57 substitutions do not. These observations reveal the differential contribution of conserved YNEL residues to Sae3 activities in meiotic recombination.

Exo1 protects DNA nicks from ligation to promote crossover formation during meiosis.

In most sexually reproducing organisms crossing over between chromosome homologs during meiosis is essential to produce haploid gametes. Most crossovers that form in meiosis in budding yeast result from the biased resolution of double Holliday junction (dHJ) intermediates. This dHJ resolution step involves the actions of Rad2/XPG family nuclease Exo1 and the Mlh1-Mlh3 mismatch repair endonuclease. Here, we provide genetic evidence in baker's yeast that Exo1 promotes meiotic crossing over by protecting DNA nicks from ligation. We found that structural elements in Exo1 that interact with DNA, such as those required for the bending of DNA during nick/flap recognition, are critical for its role in crossing over. Consistent with these observations, meiotic expression of the Rad2/XPG family member Rad27 partially rescued the crossover defect in exo1 null mutants, and meiotic overexpression of Cdc9 ligase reduced the crossover levels of exo1 DNA-binding mutants to levels that approached the exo1 null. In addition, our work identified a role for Exo1 in crossover interference. Together, these studies provide experimental evidence for Exo1-protected nicks being critical for the formation of meiotic crossovers and their distribution.

Singlet Oxygen Generation Driven by Sulfide Ligand Exchange on Porphyrin-Gold Nanoparticle Conjugates.

Here, we report a switching method of singlet oxygen (1O2) generation based on the adsorption/desorption of porphyrins to gold nanoparticles driven by sulfide (thiol or disulfide) compounds. The generation of 1O2 by photosensitization is effectively suppressed by the gold nanoparticles and can be restored by a sulfide ligand exchange reaction. The on/off ratio of 1O2 quantum yield (ΦΔ) reached 7.4. By examining various incoming sulfide compounds, it was found that the ligand exchange reaction on the gold nanoparticle surface could be thermodynamically or kinetically controlled. The remaining gold nanoparticles in the system still suppress the generation of 1O2, which can be precipitated out simultaneously with porphyrin desorption by the proper polarity choice of the incoming sulfide to restore the 1O2 generation.

Clinical study of preoperative skeletal muscle mass as a predictor of physical performance recovery following palliative surgery for spinal metastases.

BACKGROUND: Surgical treatment of spinal metastases has been associated with high morbidity and mortality in patients with sarcopenia based on low skeletal muscle mass. We assessed physical performance using the Eastern Cooperative Oncology Group performance status scale and the Barthel Index on the 30th day after palliative surgery for spinal metastases and investigated the effectiveness of surgery according to sarcopenia assessed by skeletal muscle mass. METHODS: We retrospectively analyzed 78 consecutive patients with thoracic and lumbar spinal metastases who underwent palliative surgery. The value of the area of the psoas major muscle at the L3 level normalized by the vertebral area was divided into first, middle, and third tertiles. Clinical variables were compared by tertile. Variables affecting the 30-day good performance status were investigated with univariate and multivariate analyses. RESULTS: The 30-day morbidity rates were 50%, 38.5%, and 15.4% by tertile. The 30-day mortality rate was 2%; all were in the first tertile. Good preoperative performance status scores were seen in 15.4% of first and 50% of third tertile patients. Postoperatively, the performance status improved in all groups, with 30.8%, 65.4%, and 92.3% by tertile. Multivariate regression analysis revealed that a good preoperative performance status (OR: 15.50, 95% CI: 1.610-149.00, P < 0.05) and the value of the area of the psoas major muscle at the L3 level normalized by the vertebral area not in the first tertile (OR: 0.22, 95% CI: 0.06-0.82, P < 0.05) were significant predictors of a good postoperative 30-day performance status. CONCLUSIONS: A good preoperative performance status and exclusion from the first tertile were clinical factors predicting a good postoperative 30-day performance status. In patients with large psoas muscle mass (third tertile), a good 30-day performance status can be expected after surgery, suggesting that surgery in this population should be pursued aggressively.

Cancer-Chemotherapy-Related Regimen Checks Performed by Pharmacists of General Hospitals Other than Cancer Treatment Collaborative Base Hospitals: A Multicenter, Prospective Survey.

Although prescription review is an important role for pharmacists in anticancer drug therapy, there are no guidelines in Japan that specify what pharmacists should check for in chemotherapy regimens. This prospective multicenter survey aimed to investigate the implementation of chemotherapy regimen checks by pharmacists in general hospitals by focusing on 19 recommended confirmation items designed to enhance chemotherapy safety. This study involved 14 hospitals within the National Hospital Organization in different regions of Japan. The top five cancers in Japan (gastric, colorectal, lung, breast, and gynecological) were targeted and specific chemotherapy regimens were analyzed. This study assessed the amount of time required for regimen checks, the number of confirmation items completed, the number and the content of inquiries raised regarding prescriptions, and the pharmacists' opinions using a questionnaire that had a maximum score of 10 points. Pharmacists checked 345 and 375 chemotherapies of patients in the control group (CG) and recommended items group (RIG), respectively. The mean time periods required for completing a chemotherapy regimen check were 4 min and 14 s (SD ±1 min and 50 s) and 6 min and 18 s (SD, ±1 min and 7 s) in the CG and RIG, respectively. The mean of the recommended items for the CG = 12.4 and for the RIG = 18.6. The items that the pharmacists did not confirm included urine protein (sixty-nine cases, 18.4%), allergy history (four cases, 1%), previous history (two cases, 0.5%), and a previous history of hepatitis B virus (sixty-nine cases, 18.4%). The number of inquiries for a doctor's prescription order was higher in the RIG than in the CG (41 vs. 27 cases). This multicenter survey demonstrated the potential effectiveness of implementing 19 recommended confirmation items in the regimen checks by pharmacists in general hospitals other than cancer treatment collaborative base hospitals.

Tension Pneumocephalus Associated with Negative Pressure Wound Therapy with Instillation and Dwell Time for Methicillin-resistant Staphylococcus aureus Infection After Spinal Deformity Surgery.

PURPOSE: Surgical site infection (SSI) is a serious complication after spine surgery. Recently, it has become possible to perform negative pressure wound therapy with instillation and dwell time (NPWTi-d) for postoperative infected wounds. We report the first rare case of symptomatic pneumoencephalopathy following NPWTi-d for methicillin-resistant Staphylococcus aureus (MRSA) infection after spinal deformity surgery. METHODS: Retrospective review of a patient's medical record and imaging. RESULTS: A 77-year-old female patient underwent posterior corrective fixation with no intraoperative complications. On the 10th postoperative day, SSI was diagnosed, and debridement was performed. Since MRSA was detected in the wound culture, and a prolonged inflammatory reaction was observed, NPWTi-d was started to preserve the instrumentation. Gradually, good granulation was observed, and the extensive soft tissue defect decreased. On the 29th day after the start of NPWTi-d, the patient experienced sudden headache and neck pain while standing, and head computed tomography led to the diagnosis of symptomatic pneumoencephalopathy. NPWTi-d was discontinued, and when surgery was performed to close the wound, dural injury was found, which was not present at the time of the initial surgery, and dural repair was performed. After 2 weeks of bed rest, the patient's pneumoencephalopathy improved. Three years have passed since the surgery, and no recurrence of cerebrospinal fluid leakage or infection has been observed. CONCLUSIONS: Although NPWTi-d is a useful treatment for SSI, it is always necessary to pay attention to the development of pneumoencephalopathy and promptly diagnose and treat it because of the risk of life-threatening complications.

Minimally Invasive Spinal Treatment (MIST)-A New Concept in the Treatment of Spinal Diseases: A Narrative Review.

In the past two decades, minimally invasive spine surgery (MISS) techniques have been developed for spinal surgery. Historically, minimizing invasiveness in decompression surgery was initially reported as a MISS technique. In recent years, MISS techniques have also been applied for spinal stabilization techniques, which were defined as minimally invasive spine stabilization (MISt), including percutaneous pedicle screws (PPS) fixation, lateral lumbar interbody fusion, balloon kyphoplasty, percutaneous vertebroplasty, cortical bone trajectory, and cervical total disc replacement. These MISS techniques typically provide many advantages such as preservation of paraspinal musculature, less blood loss, a shorter operative time, less postoperative pain, and a lower infection rate as well as being more cost-effective compared to traditional open techniques. However, even MISS techniques are associated with several limitations including technical difficulty, training opportunities, surgical cost, equipment cost, and radiation exposure. These downsides of surgical treatments make conservative treatments more feasible option. In the future, medicine must become "minimally invasive" in the broadest sense-for all patients, conventional surgeries, medical personnel, hospital management, nursing care, and the medical economy. As a new framework for the treatment of spinal diseases, the concept of minimally invasive spinal treatment (MIST) has been proposed.