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Scientific Reports Apr 2024DNA double-strand breaks (DSBs) activate DNA damage responses (DDRs) in both mitotic and meiotic cells. A single-stranded DNA (ssDNA) binding protein, Replication...
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.
Topics: Replication Protein A; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Meiosis; DNA Breaks, Double-Stranded; Cell Cycle Proteins; DNA-Binding Proteins; Recombination, Genetic; Homologous Recombination; MAP Kinase Kinase 1; DNA, Single-Stranded; Nuclear Proteins; Transcription Factors
PubMed: 38664461
DOI: 10.1038/s41598-024-60082-x -
BioRxiv : the Preprint Server For... Apr 202453BP1 plays a crucial role in regulating DNA damage repair pathway choice and checkpoint signaling in somatic cells; however, its role in meiosis has remained enigmatic....
53BP1 plays a crucial role in regulating DNA damage repair pathway choice and checkpoint signaling in somatic cells; however, its role in meiosis has remained enigmatic. In this study, we demonstrate that the ortholog of 53BP1, HSR-9, associates with chromatin in both proliferating and meiotic germ cells. Notably, HSR-9 is enriched on the chromosome pair in pachytene oogenic germ cells. HSR-9 is also present at kinetochores during both mitotic and meiotic divisions but does not appear to be essential for monitoring microtubule-kinetochore attachments or tension. Using cytological markers of different steps in recombinational repair, we found that HSR-9 influences the processing of a subset of meiotic double strand breaks into COSA-1-marked crossovers. Additionally, HSR-9 plays a role in meiotic chromosome segregation under conditions where chromosomes fail to pair, synapse, and recombine. Together, these results highlight that chromatin-associated HSR-9 has both conserved and unique functions in the regulation of meiotic chromosome behavior.
PubMed: 38659880
DOI: 10.1101/2024.04.12.589267 -
Nature May 2024RAD52 is important for the repair of DNA double-stranded breaks, mitotic DNA synthesis and alternative telomere length maintenance. Central to these functions, RAD52...
RAD52 is important for the repair of DNA double-stranded breaks, mitotic DNA synthesis and alternative telomere length maintenance. Central to these functions, RAD52 promotes the annealing of complementary single-stranded DNA (ssDNA) and provides an alternative to BRCA2/RAD51-dependent homologous recombination repair. Inactivation of RAD52 in homologous-recombination-deficient BRCA1- or BRCA2-defective cells is synthetically lethal, and aberrant expression of RAD52 is associated with poor cancer prognosis. As a consequence, RAD52 is an attractive therapeutic target against homologous-recombination-deficient breast, ovarian and prostate cancers. Here we describe the structure of RAD52 and define the mechanism of annealing. As reported previously, RAD52 forms undecameric (11-subunit) ring structures, but these rings do not represent the active form of the enzyme. Instead, cryo-electron microscopy and biochemical analyses revealed that ssDNA annealing is driven by RAD52 open rings in association with replication protein-A (RPA). Atomic models of the RAD52-ssDNA complex show that ssDNA sits in a positively charged channel around the ring. Annealing is driven by the RAD52 N-terminal domains, whereas the C-terminal regions modulate the open-ring conformation and RPA interaction. RPA associates with RAD52 at the site of ring opening with critical interactions occurring between the RPA-interacting domain of RAD52 and the winged helix domain of RPA2. Our studies provide structural snapshots throughout the annealing process and define the molecular mechanism of ssDNA annealing by the RAD52-RPA complex.
Topics: Humans; Cryoelectron Microscopy; DNA, Single-Stranded; Models, Molecular; Protein Binding; Rad52 DNA Repair and Recombination Protein; Replication Protein A; Multiprotein Complexes; Protein Domains; Binding Sites
PubMed: 38658755
DOI: 10.1038/s41586-024-07347-7 -
BioRxiv : the Preprint Server For... Apr 2024The conserved Rad2/XPG family 5'-3' exonuclease, Exonuclease 1 (Exo1), plays many roles in DNA metabolism including during resolution of DNA double strand breaks (DSBs)...
UNLABELLED
The conserved Rad2/XPG family 5'-3' exonuclease, Exonuclease 1 (Exo1), plays many roles in DNA metabolism including during resolution of DNA double strand breaks (DSBs) via homologous recombination. Prior studies provided evidence that the end-resection activity of Exo1 is downregulated in yeast and mammals by Cdk1/2 family cyclin-dependent and checkpoint kinases, including budding yeast kinase Rad53 which functions in mitotic cells. Here we provide evidence that the master meiotic kinase Mek1, a paralogue of Rad53, limits 5'-3' single strand resection at the sites of programmed meiotic DNA breaks. Mutational analysis suggests that the mechanism of Exo1 suppression by Mek1 differs from that of Rad53.
ARTICLE SUMMARY
Meiotic recombination involves formation of programmed DNA double strand breaks followed by 5' to 3' single strand specific resection by nucleases including Exo1. We find that the activity of budding yeast Exo1 is downregulated during meiotic recombination by the master meiotic kinase Mek1. The mechanism of downregulation of Exo1 by Mek1 in meiosis does not depend on the same phospho-sites as those used by the mitotic kinase Rad53, a relative of Mek1 that downregulates Exo1 in mitosis.
PubMed: 38645032
DOI: 10.1101/2024.04.12.589255 -
EBioMedicine May 2024Poly(ADP-ribose) polymerase (PARP) inhibitors have emerged as promising chemotherapeutic drugs primarily against BRCA1/2-associated tumours, known as synthetic...
BACKGROUND
Poly(ADP-ribose) polymerase (PARP) inhibitors have emerged as promising chemotherapeutic drugs primarily against BRCA1/2-associated tumours, known as synthetic lethality. However, recent clinical trials reported patients' survival benefits from PARP inhibitor treatments, irrelevant to homologous recombination deficiency. Therefore, revealing the therapeutic mechanism of PARP inhibitors beyond DNA damage repair is urgently needed, which can facilitate precision medicine.
METHODS
A CRISPR-based knock-in technology was used to establish stable BRCA1 mutant cancer cells. The effects of PARP inhibitors on BRCA1 mutant cancer cells were evaluated by biochemical and cell biological experiments. Finally, we validated its in vivo effects in xenograft and patient-derived xenograft (PDX) tumour mice.
FINDINGS
In this study, we uncovered that the majority of clinical BRCA1 mutations in breast cancers were in and near the middle of the gene, rather than in essential regions for DNA damage repair. Representative mutations such as R1085I and E1222Q caused transient extra spindle poles during mitosis in cancer cells. PAR, which is synthesized by PARP2 but not PARP1 at mitotic centrosomes, clustered these transient extra poles, independent of DNA damage response. Common PARP inhibitors could effectively suppress PARP2-synthesized PAR and induce cell senescence by abrogating the correction of mitotic extra-pole error.
INTERPRETATION
Our findings uncover an alternative mechanism by which PARP inhibitors efficiently suppress tumours, thereby pointing to a potential new therapeutic strategy for centrosome error-related tumours.
FUNDING
Funded by National Natural Science Foundation of China (NSFC) (T2225006, 82272948, 82103106), Beijing Municipal Natural Science Foundation (Key program Z220011), and the National Clinical Key Specialty Construction Program, P. R. China (2023).
Topics: Poly(ADP-ribose) Polymerase Inhibitors; Humans; Animals; Centrosome; DNA Damage; Cellular Senescence; Mice; BRCA1 Protein; Cell Line, Tumor; Female; Xenograft Model Antitumor Assays; Mutation; DNA Repair; Disease Models, Animal; Breast Neoplasms; Poly(ADP-ribose) Polymerases; Poly (ADP-Ribose) Polymerase-1
PubMed: 38640836
DOI: 10.1016/j.ebiom.2024.105129 -
Cell Reports Apr 2024Overexpression of Cyclin E1 perturbs DNA replication, resulting in DNA lesions and genomic instability. Consequently, Cyclin E1-overexpressing cancer cells increasingly...
Overexpression of Cyclin E1 perturbs DNA replication, resulting in DNA lesions and genomic instability. Consequently, Cyclin E1-overexpressing cancer cells increasingly rely on DNA repair, including RAD52-mediated break-induced replication during interphase. We show that not all DNA lesions induced by Cyclin E1 overexpression are resolved during interphase. While DNA lesions upon Cyclin E1 overexpression are induced in S phase, a significant fraction of these lesions is transmitted into mitosis. Cyclin E1 overexpression triggers mitotic DNA synthesis (MiDAS) in a RAD52-dependent fashion. Chemical or genetic inactivation of MiDAS enhances mitotic aberrations and persistent DNA damage. Mitosis-specific degradation of RAD52 prevents Cyclin E1-induced MiDAS and reduces the viability of Cyclin E1-overexpressing cells, underscoring the relevance of RAD52 during mitosis to maintain genomic integrity. Finally, analysis of breast cancer samples reveals a positive correlation between Cyclin E1 amplification and RAD52 expression. These findings demonstrate the importance of suppressing mitotic defects in Cyclin E1-overexpressing cells through RAD52.
Topics: Humans; Cyclin E; Genomic Instability; Rad52 DNA Repair and Recombination Protein; Mitosis; Oncogene Proteins; DNA Replication; Cell Line, Tumor; DNA Damage; DNA; Breast Neoplasms
PubMed: 38625790
DOI: 10.1016/j.celrep.2024.114116 -
EMBO Reports May 2024ELYS is a nucleoporin that localizes to the nuclear side of the nuclear pore complex (NPC) in interphase cells. In mitosis, it serves as an assembly platform that...
ELYS is a nucleoporin that localizes to the nuclear side of the nuclear pore complex (NPC) in interphase cells. In mitosis, it serves as an assembly platform that interacts with chromatin and then with nucleoporin subcomplexes to initiate post-mitotic NPC assembly. Here we identify ELYS as a major binding partner of the membrane protein VAPB during mitosis. In mitosis, ELYS becomes phosphorylated at many sites, including a predicted FFAT (two phenylalanines in an acidic tract) motif, which mediates interaction with the MSP (major sperm protein)-domain of VAPB. Binding assays using recombinant proteins or cell lysates and co-immunoprecipitation experiments show that VAPB binds the FFAT motif of ELYS in a phosphorylation-dependent manner. In anaphase, the two proteins co-localize to the non-core region of the newly forming nuclear envelope. Depletion of VAPB results in prolonged mitosis, slow progression from meta- to anaphase and in chromosome segregation defects. Together, our results suggest a role of VAPB in mitosis upon recruitment to or release from ELYS at the non-core region of the chromatin in a phosphorylation-dependent manner.
Topics: Mitosis; Humans; Phosphorylation; Protein Binding; HeLa Cells; Chromatin; Transcription Factors; Chromosome Segregation; Nuclear Pore Complex Proteins; Nuclear Envelope; Membrane Proteins; Anaphase
PubMed: 38605278
DOI: 10.1038/s44319-024-00125-6 -
World Journal of Oncology Apr 2024Hepatocellular carcinoma (HCC) with high Ki67 protein expression, the most commonly used cell proliferation marker, is associated with an aggressive biologic phenotype;...
BACKGROUND
Hepatocellular carcinoma (HCC) with high Ki67 protein expression, the most commonly used cell proliferation marker, is associated with an aggressive biologic phenotype; however, conventional immunostaining is hampered by variability in institutional protocol, specific antibody probe, and by assessor subjectivity. To this end, we hypothesized that Ki67 gene () expression would identify highly proliferative HCC, and clarify its association with oncologic outcome, tumor progression, and immune cell population in the tumor microenvironment (TME). Furthermore, we sought to identify the cell-cycle gene expression profile that confers this aggressive phenotype.
METHODS
A total of 473 HCC patients with clinicopathological data associated with transcriptome were selected for this study: 358 patients from The Cancer Genome Atlas (TCGA) as the testing cohort, and 115 from GSE76427 as the validation cohort. Each cohort was divided into a highly proliferative group (MKi67-high) and the low MKi67 group (MKi67-low) by the median of Ki67 gene () expression levels.
RESULTS
MKi67-high HCC patients had worse disease-free survival (DFS), disease-specific survival (DSS), and overall survival (OS) independent of histological grade in the TCGA cohort. MKi67 expression correlated with histological grade and tumor size. MKi67 expression increased throughout the HCC carcinomatous sequence from normal liver, cirrhotic liver, early HCC, and advanced HCC. MKi67-high HCC was associated with higher intratumor heterogeneity, homologous recombination deficiency, and altered fraction as well as intratumoral infiltration of T helper type 1 (Th1) and Th2 cells, but lower interferon-gamma response and M2 macrophage infiltration. Cell proliferation-related gene sets in the Hallmark collection (E2F targets, G2M checkpoint, Myc target v1 and mitotic spindle), MTORC1 signaling, DNA repair, PI3K MTOR signaling, and unfolded protein response were all enriched in the MKi67-high HCC (false discovery rate (FDR) < 0.25).
CONCLUSIONS
High gene expression identified highly proliferative HCC with aggressive biology involving classical pathways in cell cycle regulation and DNA repair, as well as poor overall oncologic outcomes. This suggests potential for personalized treatment strategies, but validation and refinement of these observations require further research to elucidate the underlying mechanisms and validate therapeutic targeting of these pathways in MKi67-high HCC tumors.
PubMed: 38545476
DOI: 10.14740/wjon1751 -
PloS One 2024Cyclin-dependent kinase 1 (Cdk1) complexed with cyclin B phosphorylates multiple sites on hundreds of proteins during mitosis. However, it is not fully understood how...
Cyclin-dependent kinase 1 (Cdk1) complexed with cyclin B phosphorylates multiple sites on hundreds of proteins during mitosis. However, it is not fully understood how multi-site mitotic phosphorylation by cyclin B-Cdk1 controls the structures and functions of individual substrates. Here we develop an easy-to-use protocol to express recombinant vertebrate cyclin B and Cdk1 in insect cells from a single baculovirus vector and to purify their complexes with excellent homogeneity. A series of in-vitro assays demonstrate that the recombinant cyclin B-Cdk1 can efficiently and specifically phosphorylate the SP and TP motifs in substrates. The addition of Suc1 (a Cks1 homolog in fission yeast) accelerates multi-site phosphorylation of an artificial substrate containing TP motifs. Importantly, we show that mitosis-specific multi-subunit and multi-site phosphorylation of the condensin I complex can be recapitulated in vitro using recombinant cyclin B-Cdk1-Suc1. The materials and protocols described here will pave the way for dissecting the biochemical basis of critical mitotic processes that accompany Cdk1-mediated large-scale phosphorylation.
Topics: CDC2 Protein Kinase; Phosphorylation; Cyclin B; Proteins; Mitosis
PubMed: 38527022
DOI: 10.1371/journal.pone.0299003 -
Frontiers in Cellular and Infection... 2024is a globally occurring apicomplexan parasite that infects humans and animals. Globally, different typical and atypical haplotypes of induce varying pathologies in...
is a globally occurring apicomplexan parasite that infects humans and animals. Globally, different typical and atypical haplotypes of induce varying pathologies in hosts. As an obligate intracellular protozoon, was shown to interfere with host cell cycle progression, leading to mitotic spindle alteration, chromosome segregation errors and cytokinesis failure which all may reflect chromosomal instability. Referring to strain-dependent virulence, we here studied the potential of different strains (RH, Me49 and NED) to drive DNA damage in primary endothelial host cells. Utilizing microscopic analyses, comet assays and γ-H2AX quantification, we demonstrated a strain-dependent induction of binucleated host cells, DNA damage and DNA double strand breaks, respectively, in -infected cells with the RH strain driving the most prominent effects. Interestingly, only the NED strain significantly triggered micronuclei formation in -infected cells. Focusing on the RH strain, we furthermore demonstrated that -infected primary host cells showed a DNA damage response by activating the ATM-dependent homologous recombination (HR) pathway. In contrast, key molecules of the nonhomologous DNA end joining (NHEJ) pathway were either not affected or downregulated in RH-infected host cells, suggesting that this pathway is not activated by infection. In conclusion, current finding suggests that infection affects the host cell genome integrity in a strain-dependent manner by causing DNA damage and chromosomal instability.
Topics: Humans; Animals; Toxoplasmosis; Toxoplasma; DNA; DNA Damage; Chromosomal Instability; Homologous Recombination; Ataxia Telangiectasia Mutated Proteins
PubMed: 38524184
DOI: 10.3389/fcimb.2024.1374659