Rad54 Drives ATP Hydrolysis-Dependent DNA Sequence Alignment during Homologous Recombination.

Homologous recombination (HR) helps maintain genome integrity, and HR defects give rise to disease, especially cancer. During HR, damaged DNA must be aligned with an undamaged template through a process referred to as the homology search. Despite decades of study, key aspects of this search remain undefined. Here, we use single-molecule imaging to demonstrate that Rad54, a conserved Snf2-like protein found in all eukaryotes, switches the search from the diffusion-based pathways characteristic of the basal HR machinery to an active process in which DNA sequences are aligned via an ATP-dependent molecular motor-driven mechanism. We further demonstrate that Rad54 disrupts the donor template strands, enabling the search to take place within a migrating DNA bubble-like structure that is bound by replication protein A (RPA). Our results reveal that Rad54, working together with RPA, fundamentally alters how DNA sequences are aligned during HR.

Dynamic Processing of Displacement Loops during Recombinational DNA Repair.

Displacement loops (D-loops) are pivotal intermediates of homologous recombination (HR), a universal DNA double strand break (DSB) repair pathway. We developed a versatile assay for the physical detection of D-loops in vivo, which enabled studying the kinetics of their formation and defining the activities controlling their metabolism. Nascent D-loops are detected within 2 h of DSB formation and extended in a delayed fashion in a genetic system designed to preclude downstream repair steps. The majority of nascent D-loops are disrupted by two pathways: one supported by the Srs2 helicase and the other by the Mph1 helicase and the Sgs1-Top3-Rmi1 helicase-topoisomerase complex. Both pathways operate without significant overlap and are delineated by the Rad54 paralog Rdh54 in an ATPase-independent fashion. This study uncovers a layer of quality control of HR relying on nascent D-loop dynamics.

The TRAF3-DYRK1A-RAD54L2 complex maintains ACE2 expression to promote SARS-CoV-2 infection.

Angiotensin converting enzyme 2 (ACE2), the host receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is differentially expressed in a wide variety of tissues and cell types. The expression of ACE2 is under tight regulation, but the mechanisms regulating ACE2 expression have not yet been well defined. Through a genome-wide CRISPR knockout screen, we discovered that host factors TRAF3, DYRK1A, and RAD54L2 (TDR) form a complex to regulate the expression of ACE2. Knockout of TRAF3, DYRK1A, or RAD54L2 reduces the mRNA levels of ACE2 and inhibits the cellular entry of SARS-CoV-2. On the other hand, SARS-CoV-2 continuously evolves by genetic mutations for the adaption to the host. We have identified mutations in spike (S) (P1079T) and nucleocapsid (N) (S194L) that enhance the replication of SARS-CoV-2 in cells that express ACE2 at a low level. Our results have revealed the mechanisms for the transcriptional regulation of ACE2 and the adaption of SARS-CoV-2. IMPORTANCE: The expression of ACE2 is essential for the entry of SARS-CoV-2 into host cells. We identify a new complex-the TDR complex-that acts to maintain the abundance of ACE2 in host cells. The identification and characterization of the TDR complex provide new targets for the development of therapeutics against SARS-CoV-2 infection. By analysis of SARS-CoV-2 virus replicating in cells expressing low levels of ACE2, we identified mutations in spike (P1079T) and nucleocapsid (S194L) that overcome the restriction of limited ACE2. Functional analysis of these key amino acids in S and N extends our knowledge of the impact of SARS-CoV-2 variants on virus infection and transmission.

Rad54 and Rdh54 prevent Srs2-mediated disruption of Rad51 presynaptic filaments.

Srs2 is a superfamily 1 (SF1) helicase that participates in several pathways necessary for the repair of damaged DNA. Srs2 regulates formation of early homologous recombination (HR) intermediates by actively removing the recombinase Rad51 from single-stranded DNA (ssDNA). It is not known whether and how Srs2 itself is down-regulated to allow for timely HR progression. Rad54 and Rdh54 are two closely related superfamily 2 (SF2) motor proteins that promote the formation of Rad51-dependent recombination intermediates. Rad54 and Rdh54 bind tightly to Rad51-ssDNA and act downstream of Srs2, suggesting that they may affect the ability of Srs2 to dismantle Rad51 filaments. Here, we used DNA curtains to determine whether Rad54 and Rdh54 alter the ability of Srs2 to disrupt Rad51 filaments. We show that Rad54 and Rdh54 act synergistically to greatly restrict the antirecombinase activity of Srs2. Our findings suggest that Srs2 may be accorded only a limited time window to act and that Rad54 and Rdh54 fulfill a role of prorecombinogenic licensing factors.

SOG1 and BRCA1 Interdependently Regulate RAD54 Expression for Repairing Salinity-Induced DNA Double-Strand Breaks in Arabidopsis.

As sessile organisms, land plants experience various forms of environmental stresses throughout their life span. Therefore, plants have developed extensive and complicated defense mechanisms, including a robust DNA damage response (DDR) and DNA repair systems for maintaining genome integrity. In Arabidopsis, the NAC [NO APICAL MERISTEM (NAM), ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR (ATAF), CUP-SHAPED COTYLEDON (CUC)] domain family transcription factor SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) plays an important role in regulating DDR. Here, we show that SOG1 plays a key role in regulating the repair of salinity-induced DNA double-strand breaks (DSBs) via the homologous recombination (HR) pathway in Arabidopsis. The sog1-1 mutant seedlings display a considerably slower rate of repair of salinity-induced DSBs. Accumulation of SOG1 protein increases in wild-type Arabidopsis under salinity stress, and it enhances the expression of HR pathway-related genes, including RAD51, RAD54 and BReast CAncer gene 1 (BRCA1), respectively, as found in SOG1 overexpression lines. SOG1 binds specifically to the AtRAD54 promoter at the 5'-(N)4GTCAA(N)3C-3' consensus sequence and positively regulates its expression under salinity stress. The phenotypic responses of sog1-1/atrad54 double mutants suggest that SOG1 functions upstream of RAD54, and both these genes are essential in regulating DDR under salinity stress. Furthermore, SOG1 interacts directly with BRCA1, an important component of the HR-mediated DSB repair pathway in plants, where BRCA1 appears to facilitate the binding of SOG1 to the RAD54 promoter. At the genetic level, SOG1 and BRCA1 function interdependently in modulating RAD54 expression under salinity-induced DNA damage. Together, our results suggest that SOG1 regulates the repair of salinity-induced DSBs via the HR-mediated pathway through genetic interactions with RAD54 and BRCA1 in Arabidopsis.

Rad54 and Rdh54 occupy spatially and functionally distinct sites within the Rad51-ssDNA presynaptic complex.

Rad54 and Rdh54 are closely related ATP-dependent motor proteins that participate in homologous recombination (HR). During HR, these enzymes functionally interact with the Rad51 presynaptic complex (PSC). Despite their importance, we know little about how they are organized within the PSC, or how their organization affects PSC function. Here, we use single-molecule optical microscopy and genetic analysis of chimeric protein constructs to evaluate the binding distributions of Rad54 and Rdh54 within the PSC. We find that Rad54 and Rdh54 have distinct binding sites within the PSC, which allow these proteins to act cooperatively as DNA sequences are aligned during homology search. Our data also reveal that Rad54 must bind to a specific location within the PSC, whereas Rdh54 retains its function in the repair of MMS-induced DNA damage even when recruited to the incorrect location. These findings support a model in which the relative binding sites of Rad54 and Rdh54 help to define their functions during mitotic HR.

RAD54B inhibits vascular endothelial senescence via suppression of CHK1/p53/p21 pathway.

RAD54B belongs to the SNF2/SWI2 superfamily, participating in homologous recombination repair. DNA damage is the central driver of aging, but there is no direct evidence of an association between RAD54B and vascular aging. The present study sought to investigate the role and mechanisms of RAD54B in endothelial senescence. In senescent animal models, including spontaneously hypertensive rats, normal aging mice, and D-gal-induced senescent mice, and senescent cell models induced by H2O2, D-gal, and culture, RAD54B was remarkably downregulated. Knockdown of RAD54B increased the expression of p53 and p21, increased the ratio of SA-ß-gal-positive cells, and decreased the proportion of EdU-positive cells. Conversely, overexpression of RAD54B reversed the senescent phenotypes stimulated by H2O2 and delayed replicative endothelial senescence. Mechanistically, silencing RAD54B compensatorily increased the expression of RAD51/XRCC4, which remained unchanged in H2O2-induced senescence. RAD54B lacking the SNF2 domain could still reverse the increasing expression of p53/p21 induced by H2O2. RAD54B reduced γH2A.X expression and inhibited the expression and phosphorylation of CHK1. In conclusion, RAD54B exerts a direct protective effect against DNA damage through enhancing homologous recombination repair in endothelial senescence, resulting in inhibition of the downstream CHK1/p53/p21 pathway, suggesting that RAD54B may be a potential therapeutic target for vascular aging-associated diseases.

RAD54B mutations enhance the sensitivity of ovarian cancer cells to poly(ADP-ribose) polymerase (PARP) inhibitors.

Synthetic lethal targeting of homologous recombination (HR)-deficient ovarian cancers (OvCas) with poly(ADP-ribose) polymerase inhibitors (PARPis) has attracted considerable attention. Olaparib was the first PARPi approved by the Food and Drug Administration, offering significant clinical benefits in BRCA1/2-deficient OvCas. However, only approximately 20% of OvCa patients harbor BRCA1/2 mutations. Given the shared roles that BRCA1/2 have with other HR regulators, alterations in HR genes may also contribute to "BRCAness profiles" in OvCas. RAD54B has been considered a key player in HR repair, although its roles and therapeutic potential in cancers need further investigation. Here, we identified 22 frequently mutated HR genes by whole-exome sequencing of OvCa tissues from 82 patients. To our surprise, 7.3% of patients were found to harbor mutations of RAD54B, the third-highest mutated gene among patients. We determined that RAD54B-mutated tumor tissues harbored more DNA double-strand breaks than normal tissues. Additionally, we found that RAD54B knockdown inhibited HR repair, enhanced sensitivities of OvCa cells with increased DNA double-strand breaks to olaparib, and induced apoptosis. Enhanced inhibitory effects of olaparib on the growth of ES2 xenograft tumors were further demonstrated by RAD54B knockdown. Finally, we show that restoration with wildtype RAD54B rather than RAD54BN593S and RAD54BH219Y, identified in patients, abolished the effects of RAD54B knockdown, indicating these RAD54B mutants probably malfunctioned in HR repair. Our investigations may offer insight into the contributions of RAD54B mutations to synthetic lethality with olaparib treatment in OvCas, enrich the gene list for "HR deficiency scoring," and expand the applications of PARPis.

RAD54L promotes progression of hepatocellular carcinoma via the homologous recombination repair pathway.

Hepatocellular carcinoma (HCC) is a malignant tumor with high incidence worldwide. The underlying mechanisms remain poorly understood. The DNA metabolic process of homologous recombination repair (HRR) has been linked to a high probability of tumorigenesis and drug resistance. This study aimed to determine the role of HRR in HCC and identify critical HRR-related genes that affect tumorigenesis and prognosis. A total of 613 tumor and 252 para-carcinoma tissue samples were collected from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) to obtain differentially expressed genes (DEGs). HRR-related genes were assessed using gene enrichment and pathway analyses. Survival analysis was performed using the Kaplan-Meier method in the Gene Expression Profiling Interactive Analysis portal. The levels of RAD54L in the HRR pathway were detected by RT-qPCR and western blotting in para-carcinoma and HCC tissues and in L02 normal human liver cells and Huh7 HCC cells. Immunohistochemistry (IHC) was performed on the clinical specimens to determine the connection between gene expression and clinical features. Bioinformatics analysis revealed that the HRR pathway was enriched in HCC tissues. Upregulation of HRR pathway DEGs in HCC tissues was positively correlated with tumor pathological staging and negatively associated with patient overall survival. RAD54B, RAD54L, and EME1 genes in the HRR pathway were screened as markers for predicting HCC prognosis. RT-qPCR identified RAD54L as the most significantly expressed of the three genes. Western blotting and IHC quantitative analyses further demonstrated that RAD54L protein levels were higher in HCC tissues. IHC analysis of 39 pairs of HCC and para-carcinoma tissue samples also revealed an association between RAD54L and Edmondson-Steiner grade and the proliferation-related gene Ki67. The collective findings positively correlate RAD54L in the HRR signaling pathway with HCC staging and implicate RAD54L as a marker to predict HCC progression.

RNA-sequencing analysis reveals the potential molecular mechanism of RAD54B in the proliferation of inflamed human dental pulp cells.

AIM: To investigate the role of RAD54B in the proliferation of inflamed human dental pulp cells (hDPCs) induced by lipopolysaccharide (LPS). METHODOLOGY: Normal, carious and pulpitic human dental pulp tissues were collected. Total RNA was subjected to RNA-sequencing (seq) and gene expression profiles were studied by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Differentially expressed genes (DEGs) in homologous recombination repair (HRR) were validated with qRT-PCR. The expression of RAD54B and TNF-α in human dental pulp tissues was detected using immunohistochemistry. HDPCs were cultured and RAD54B level in hDPCs was detected after LPS stimulation using western blot. CCK-8 was used to investigate the proliferation of hDPCs transfected with negative control (Nc) small interfering RNA (siRNA), RAD54B siRNA, P53 siRNA or both siRNAs with or without LPS stimulation. Flow cytometry was used to detect the cell cycle distribution, and western blot and immunofluorescence were used to analyse the expression of RAD54B, P53 and P21 under the above treatments. One-way and two-way anova followed by least significant difference posttest were used for statistical analysis. RESULTS: RNA-seq results identified DEGs amongst the three groups. KEGG pathway analysis revealed enrichment of DEGs in the replication and repair pathway. HRR and non-homologous end joining (NHEJ) components were further verified and qRT-PCR results were basically consistent with the sequencing data. RAD54B, an HRR accessory factor highly expressed in carious and pulpitic tissues as compared to that in normal pulps, was chosen as our gene of interest. High RAD54B expression was confirmed in inflamed human dental pulp tissues and LPS-stimulated hDPCs. Upon RAD54B knockdown, P53 and P21 expressions in hDPCs were upregulated whereas the proliferation was significantly downregulated, accompanied by increased G2/M phase arrest. After inhibiting P53 expression in RAD54B-knockdown hDPCs, P21 expression and cell proliferation were reversed. CONCLUSIONS: Gene expression profiles of normal, carious and pulpitic human dental pulp tissues were revealed. HRR components were elucidated to function in dental pulp inflammation. Amongst the DEGs in HRR, RAD54B regulated the proliferation of inflamed hDPCs via P53/P21 signalling. This research deepens our understanding of dental pulp inflammation and provides new insight to clarify the underlying mechanisms.

Arsenite Impairs BRCA1-Dependent DNA Double-Strand Break Repair, a Mechanism Potentially Contributing to Genomic Instability.

BRCA1 is a key player in maintaining genomic integrity with multiple functions in DNA damage response (DDR) mechanisms. Due to its thiol-rich zinc-complexing domain, the protein may also be a potential target for redox-active and/or thiol-reactive (semi)metal compounds. The latter includes trivalent inorganic arsenic, which is indirectly genotoxic via induction of oxidative stress and inhibition of DNA repair pathways. In the present study, we investigated the effect of NaAsO2 on the transcriptional and functional DDR. Particular attention was paid to the potential impairment of BRCA1-mediated DDR mechanisms by arsenite by comparing BRCA1-deficient and -proficient cells. At the transcriptional level, arsenite itself activated several DDR mechanisms, including a pronounced oxidative stress and DNA damage response, mostly independent of BRCA1 status. However, at the functional level, a clear BRCA1 dependency was observed in both cell cycle regulation and cell death mechanisms after arsenite exposure. Furthermore, in the absence of arsenite, the lack of functional BRCA1 impaired the largely error-free homologous recombination (HR), leading to a shift towards the error-prone non-homologous end-joining (NHEJ). Arsenic treatment also induced this shift in BRCA1-proficient cells, indicating BRCA1 inactivation. Although BRCA1 bound to DNA DSBs induced via ionizing radiation, its dissociation was impaired, similarly to the downstream proteins RAD51 and RAD54. A shift from HR to NHEJ by arsenite was further supported by corresponding reporter gene assays. Taken together, arsenite appears to negatively affect HR via functional inactivation of BRCA1, possibly by interacting with its RING finger structure, which may compromise genomic stability.

Impact of Manganese and Chromate on Specific DNA Double-Strand Break Repair Pathways.

Manganese is an essential trace element; nevertheless, on conditions of overload, it becomes toxic, with neurotoxicity being the main concern. Chromate is a well-known human carcinogen. The underlying mechanisms seem to be oxidative stress as well as direct DNA damage in the case of chromate, but also interactions with DNA repair systems in both cases. However, the impact of manganese and chromate on DNA double-strand break (DSB) repair pathways is largely unknown. In the present study, we examined the induction of DSB as well as the effect on specific DNA DSB repair mechanisms, namely homologous recombination (HR), non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ). We applied DSB repair pathway-specific reporter cell lines, pulsed field gel electrophoresis as well as gene expression analysis, and investigated the binding of specific DNA repair proteins via immunoflourescence. While manganese did not seem to induce DNA DSB and had no impact on NHEJ and MMEJ, HR and SSA were inhibited. In the case of chromate, the induction of DSB was further supported. Regarding DSB repair, no inhibition was seen in the case of NHEJ and SSA, but HR was diminished and MMEJ was activated in a pronounced manner. The results indicate a specific inhibition of error-free HR by manganese and chromate, with a shift towards error-prone DSB repair mechanisms in both cases. These observations suggest the induction of genomic instability and may explain the microsatellite instability involved in chromate-induced carcinogenicity.

Studying the mechanism of sperm DNA damage caused by folate deficiency.

Sperm DNA injury is one of the common causes of male infertility. Folic acid deficiency would increase the methylation level of the important genes, including those involved in DNA double-strand break (DSB) repair pathway. In the early stages, we analysed the correlation between seminal plasma folic acid concentration and semen parameters in 157 infertility patients and 91 sperm donor volunteers, and found that there was a significant negative correlation between seminal folic acid concentration and sperm DNA Fragmentation Index (DFI; r = -0.495, p < 0.01). Then through reduced representation bisulphite sequencing, global DNA methylation of sperm of patients in the low folic acid group and the high folic acid group was analysed, it was found that the methylation level in Rad54 promoter region increased in the folic acid deficiency group compared with the normal folic acid group. Meanwhile, the results of animal model and spermatocyte line (GC-2) also found that folic acid deficiency can increase the methylation level in Rad54 promoter region, increased sperm DFI in mice, increased the expression of γ-H2AX, that is, DNA injury marker protein, and increased sensitivity of GC-2 to external damage and stimulation. The study indicates that the expression of Rad54 is downregulated by folic acid deficiency via DNA methylation. This may be one of the mechanisms of sperm DNA damage caused by folate deficiency.

Impact of GTF2H1 and RAD54L2 polymorphisms on the risk of lung cancer in the Chinese Han population.

BACKGROUND: Repair pathway genes play an important role in the development of lung cancer. The study aimed to assess the correlation between single nucleotide polymorphisms (SNPs) in DNA repair gene (GTF2H1 and RAD54L2) and the risk of lung cancer. METHODS: Five SNPs in GTF2H1 and four SNPs in RAD54L2 in 506 patients with lung cancer and 510 age-and gender-matched healthy controls were genotyped via the Agena MassARRAY platform. The influence of GTF2H1 and RAD54L2 polymorphisms on lung cancer susceptibility was assessed using logistic regression analysis by calculating odds ratios (ORs) and their corresponding 95% confidence intervals (CIs). RESULTS: RAD54L2 rs9864693 GC genotype increased the risk of lung cancer (OR = 1.33, 95%CI: 1.01-1.77, p = 0.045). Stratified analysis found that associations of RAD54L2 rs11720298, RAD54L2 rs4687592, RAD54L2 rs9864693 and GTF2H1 rs4150667 with lung cancer risk were found in subjects aged ≤ 59 years. Precisely, a protective effect of RAD54L2 rs11720298 on the occurrence of lung cancer was observed in non-smokers and drinkers. GTF2H1 rs4150667 was associated with a decreased risk of lung cancer in subjects with BMI ≤ 24 kg/m2. RAD54L2 rs4687592 was associated with an increased risk of lung cancer in drinkers. In addition, GTF2H1 rs3802967 was associated with a reduced risk of lung squamous cell carcinoma. CONCLUSION: Our study first revealed that RAD54L2 rs9864693 was associated with an increased risk of lung cancer in the Chinese Han population. This study may increase the understanding of the effect of RAD54L2 and GTF2H1 polymorphisms on lung cancer occurrence.

RAD54 promotes alternative lengthening of telomeres by mediating branch migration.

Cancer cells can activate the alternative lengthening of telomeres (ALT) pathway to promote replicative immortality. The ALT pathway promotes telomere elongation through a homologous recombination pathway known as break-induced replication (BIR), which is often engaged to repair single-ended double-stranded breaks (DSBs). Single-ended DSBs are resected to promote strand invasion and facilitate the formation of a local displacement loop (D-loop), which can trigger DNA synthesis, and ultimately promote telomere elongation. However, the exact proteins involved in the maturation, migration, and resolution of D-loops at ALT telomeres are unclear. In vitro, the DNA translocase RAD54 both binds D-loops and promotes branch migration suggesting that RAD54 may function to promote ALT activity. Here, we demonstrate that RAD54 is enriched at ALT telomeres and promotes telomeric DNA synthesis through its ATPase-dependent branch migration activity. Loss of RAD54 leads to the formation of unresolved recombination intermediates at telomeres that form ultra-fine anaphase bridges in mitosis. These data demonstrate an important role for RAD54 in promoting ALT-mediated telomere synthesis.

Integrated study of miR-215 promoting breast cancer cell apoptosis by targeting RAD54B.

BACKGROUND: MicroRNAs (miRNAs) are widely distributed in cells and participate in the regulation of the pathophysiological process of many diseases. As an important part of non-coding RNA, miRNAs regulate a variety of molecules and signal pathways in tumour cells. However, the evidence for regulatory mechanisms of specific miRNAs in tumour cells is still lacking. METHODS: In this study, we used transcriptomics analysis and integrated a variety of public databases to screen miRNAs that have key regulatory effects on breast cancer (BC). In addition, we used in vitro and in vivo studies and combined clinical samples to verify its regulatory mechanism. RESULTS: We found that among the specific miRNAs, miR-215-5p is a key regulator in BC. Compared with normal adjacent tissues, miR-215-5p has a lower expression level in BC tissues. Patients with high expression levels of miR-215-5p have a longer survival time. miR-215-5p can specifically target the 3'UTR region of RAD54B mRNA and down-regulate the expression of RAD54B, thereby inhibiting the proliferation of BC cells and promoting the apoptosis of BC cells. CONCLUSIONS: Finally, we found that miR-215-5p can be used as an important biomarker for BC. We have clarified its function and revealed its mechanism of targeting RAD54B mRNA for the first time. This may provide important clues to reveal the deeper molecular regulation mechanism of BC.

Irradiation-Induced Upregulation of miR-711 Inhibits DNA Repair and Promotes Neurodegeneration Pathways.

Radiotherapy for brain tumors induces neuronal DNA damage and may lead to neurodegeneration and cognitive deficits. We investigated the mechanisms of radiation-induced neuronal cell death and the role of miR-711 in the regulation of these pathways. We used in vitro and in vivo models of radiation-induced neuronal cell death. We showed that X-ray exposure in primary cortical neurons induced activation of p53-mediated mechanisms including intrinsic apoptotic pathways with sequential upregulation of BH3-only molecules, mitochondrial release of cytochrome c and AIF-1, as well as senescence pathways including upregulation of p21WAF1/Cip1. These pathways of irradiation-induced neuronal apoptosis may involve miR-711-dependent downregulation of pro-survival genes Akt and Ang-1. Accordingly, we demonstrated that inhibition of miR-711 attenuated degradation of Akt and Ang-1 mRNAs and reduced intrinsic apoptosis after neuronal irradiation; likewise, administration of Ang-1 was neuroprotective. Importantly, irradiation also downregulated two novel miR-711 targets, DNA-repair genes Rad50 and Rad54l2, which may impair DNA damage responses, amplifying the stimulation of apoptotic and senescence pathways and contributing to neurodegeneration. Inhibition of miR-711 rescued Rad50 and Rad54l2 expression after neuronal irradiation, enhancing DNA repair and reducing p53-dependent apoptotic and senescence pathways. Significantly, we showed that brain irradiation in vivo persistently elevated miR-711, downregulated its targets, including pro-survival and DNA-repair molecules, and is associated with markers of neurodegeneration, not only across the cortex and hippocampus but also specifically in neurons isolated from the irradiated brain. Our data suggest that irradiation-induced miR-711 negatively modulates multiple pro-survival and DNA-repair mechanisms that converge to activate neuronal intrinsic apoptosis and senescence. Using miR-711 inhibitors to block the development of these regulated neurodegenerative pathways, thus increasing neuronal survival, may be an effective neuroprotective strategy.

E2F1 Promotes Progression of Bladder Cancer by Modulating RAD54L Involved in Homologous Recombination Repair.

DNA repair defects are important factors in cancer development. High DNA repair activity can affect cancer progression and chemoresistance. DNA double-strand breaks in cancer cells caused by anticancer agents can be restored by non-homologous end joining (NHEJ) and homologous recombination repair (HRR). Our previous study has identified E2F1 as a key gene in bladder cancer progression. In this study, DNA repair genes related to E2F1 were analyzed, and RAD54L involved in HRR was identified. In gene expression analysis of bladder cancer patients, the survival of patients with high RAD54L expression was shorter with cancer progression than in patients with low RAD54L expression. This study also revealed that E2F1 directly binds to the promoter region of RAD54L and regulates the transcription of RAD54L related to the HRR pathway. This study also confirmed that DNA breaks are repaired by RAD54L induced by E2F1 in bladder cancer cells treated with MMC. In summary, RAD54L was identified as a new target directly regulated by E2F1. Our results suggest that, E2F1 and RAD54L could be used as diagnostic markers for bladder cancer progression and represent potential therapeutic targets.

RAD54 forms DNA repair foci in response to DNA damage in living plant cells.

Plants have various defense mechanisms against environmental stresses that induce DNA damage. Genetic and biochemical analyses have revealed the sensing and signaling of DNA damage, but little is known about subnuclear dynamics in response to DNA damage in living plant cells. Here, we observed that the chromatin remodeling factor RAD54, which is involved in DNA repair via the homologous recombination pathway, formed subnuclear foci (termed RAD54 foci) in Arabidopsis thaliana after induction of DNA double-strand breaks. The appearance of RAD54 foci was dependent on the ATAXIA-TELANGIECTASIA MUTATED-SUPPRESSOR OF GAMMA RESPONSE 1 pathway, and RAD54 foci were co-localized with γH2AX signals. Laser irradiation of a subnuclear area demonstrated that in living cells RAD54 was specifically accumulated at the damaged site. In addition, the formation of RAD54 foci showed specificity for cell type and region. We conclude that RAD54 foci correspond to DNA repair foci in A. thaliana.

Association between PTCH1 and RAD54B single-nucleotide polymorphisms and non-syndromic orofacial clefts in a northern Chinese population.

BACKGROUND: Non-syndromic orofacial clefts (NSOC) is one of the most common congenital malformations, and its etiology involves both genetic and environmental factors. The present aimed to investigate the association of six single nucleotide polymorphisms (SNPs) (rs10512248 in PTCH1, rs12681366 and rs958447 in RAD54B, rs13317 in FGFR1, rs1838105 and rs4968247 in WNT9B) with NSOC in a Northern Chinese population. METHODS: In the present study, HI-SNP technology was used to conduct genotyping of the six SNPs (rs10512248, rs12681366, rs957448, rs13317, rs1838105 and rs4968247) in 596 patients with NSOC and 466 healthy individuals from a Northern Chinese population. RESULTS: The results obtained indicated that rs10512248 and rs12681366 minor allele frequencies were statistically significant (p = 0.020 and 0.015, respectively). Statistical analysis confirmed that the CT genotype of RAD54B rs12681366 was associated with a decreased risk of NSOC (odds ratio = 0.62, 95% confidence interval = 0.46-0.82, P = 0.001). After correcting for multiple testing, the associations remained significant. By contrast, nonsignificant differences were found for the rs957448, rs13317, rs1838105 and rs4968247 allele and genotype frequencies between cases and controls. CONCLUSIONS: These results demonstrate that the PTCH1 rs10512248 and RAD54B rs12681366 were significantly associated with NSOC in a Northern Chinese population. Additionally, the RAD54B rs12381366 CT genotype could decrease the risk of NSOC in a Northern Chinese population. We provide novel evidence for the development of NSOC in a Northern Chinese population.