Maintaining Genome Stability Research Articles

SOSSB1 and SOSSB2 mutually regulate protein stability through competitive binding of SOSSA

Human single-stranded DNA-binding protein homologs hSSB1 (SOSSB1) and hSSB2 (SOSSB2) make a vital impact on maintaining genome stability as the B subunits of the sensor of single-stranded DNA complex (SOSS). However, whether and how SOSSB1 and SOSSB2 modulate mutual expression is unclear. This study, demonstrated that the depletion of SOSSB1 in cells enhances the stability of the SOSSB2 protein, and conversely, SOSSB2 depletion enhances the stability of the SOSSB1 protein. The levels of SOSSB1 and SOSSB2 proteins are mutually regulated through their competitive binding with SOSSA which associates with the highly conservative OB-fold domain in SOSSB1 and SOSSB2. The destabilized SOSSB1 and SOSSB2 proteins can be degraded via the proteasome pathway. Additionally, the simultaneous loss of SOSSB1 and SOSSB2 aggravates homologous recombination (HR)-mediated DNA repair defects, enhances cellular radiosensitivity and promotes cell apoptosis. In conclusion, in this study, we showed that SOSSB1 and SOSSB2 positively regulate HR repair and the interaction between SOSSA and SOSSB1 or SOSSB2 prevents the degradation of SOSSB1 and SOSSB2 proteins via the proteasome pathway.

Bioinformatic analysis of the effect of SNPs in the pig TERT gene on the structural and functional characteristics of the enzyme to develop new genetic markers of productivity traits

BackgroundTelomerase reverse transcriptase (TERT) plays a crucial role in synthesizing telomeric repeats that safeguard chromosomes from damage and fusion, thereby maintaining genome stability. Mutations in the TERT gene can lead to a deviation in gene expression, impaired enzyme activity, and, as a result, abnormal telomere shortening. Genetic markers of productivity traits in livestock can be developed based on the TERT gene polymorphism for use in marker-associated selection (MAS). In this study, a bioinformatic-based approach is proposed to evaluate the effect of missense single-nucleotide polymorphisms (SNPs) in the pig TERT gene on enzyme function and structure, with the prospect of developing genetic markers.ResultsA comparative analysis of the coding and amino acid sequences of the pig TERT was performed with corresponding sequences of other species. The distribution of polymorphisms in the pig TERT gene, with respect to the enzyme’s structural-functional domains, was established. A three-dimensional model of the pig TERT structure was obtained through homological modeling. The potential impact of each of the 23 missense SNPs in the pig TERT gene on telomerase function and stability was assessed using predictive bioinformatic tools utilizing data on the amino acid sequence and structure of pig TERT.ConclusionsAccording to bioinformatic analysis of 23 missense SNPs of the pig TERT gene, a predictive effect of rs789641834 (TEN domain), rs706045634 (TEN domain), rs325294961 (TRBD domain) and rs705602819 (RTD domain) on the structural and functional parameters of the enzyme was established. These SNPs hold the potential to serve as genetic markers of productivity traits. Therefore, the possibility of their application in MAS should be further evaluated in associative analysis studies.

Association of ERCC family mutations with prognosis and immune checkpoint inhibitors response in multiple cancers

The proteins encoded by the excision repair cross-complementing (ERCC) family are pivotal in DNA damage repair and maintaining genome stability. However, the precise role of the ERCC family in tumor prognosis and the effectiveness of immune checkpoint inhibitors (ICI) therapy remain uncertain. This study aimed to explore the connection between ERCC mutations and prognosis as well as the response to ICI. We observed that patients with ERCC mutations exhibited enhanced progression-free survival (PFS) and overall survival (OS) in two independent pan-cancer cohorts. Furthermore, this mutant subgroup showed higher tumor mutation burden (TMB) compared to the wild-type subgroup. Notably, ERCC mutations were associated with better OS (HR 0.54, 95% CI 0.42–0.70; P < 0.001) in pan-cancer patients who underwent ICI therapy (N = 1661). These findings were validated in a separate cohort, where patients in the ERCC mutant subgroup demonstrated improved clinical outcomes (HR 0.56, 95% CI 0.37–0.84; P = 0.03) and higher response rates (51.9% vs. 26.8%) than the wild-type subgroup. Further analysis revealed that patients with ERCC mutations displayed elevated tumor neoantigen burden (TNB) levels and increased infiltration of immune-response cells. Our study suggests that ERCC mutations are linked to enhanced immunogenicity and improved ICI efficacy, thus potentially serving as a biomarker for ICI therapy.

The role of Transcription Factor IIH complex in nucleotide excision repair.

DNA damage occurs throughout life from a variety of sources, and it is imperative to repair damage in a timely manner to maintain genome stability. Thus, DNA repair mechanisms are a fundamental part of life. Nucleotide excision repair (NER) plays an important role in the removal of bulky DNA adducts, such as cyclobutane pyrimidine dimers from ultraviolet light or DNA crosslinking damage from platinum-based chemotherapeutics, such as cisplatin. A main component for the NER pathway is transcription factor IIH (TFIIH), a multifunctional, 10-subunit protein complex with crucial roles in both transcription and NER. In transcription, TFIIH is a component of the pre-initiation complex and is important for promoter opening and the phosphorylation of RNA Polymerase II (RNA Pol II). During repair, TFIIH is important for DNA unwinding, recruitment of downstream repair factors, and verification of the bulky lesion. Several different disease states can arise from mutations within subunits of the TFIIH complex. Most strikingly are xeroderma pigmentosum (XP), XP combined with Cockayne syndrome (CS), and trichothiodystrophy (TTD). Here, we summarize the recruitment and functions of TFIIH in the two NER subpathways, global genomic (GG-NER) and transcription-coupled NER (TC-NER). We will also discuss how TFIIH's roles in the two subpathways lead to different genetic disorders.

SOSS-INTAC Regulates R-loop Accumulation and Genome Stability.

The stable association between SOSS and INTAC maintains genome stability by limiting R-loop accumulation.

C16orf72/HAPSTR1/TAPR1 functions with BRCA1/Senataxin to modulate replication-associated R-loops and confer resistance to PARP disruption

While the toxicity of PARP inhibitors to cells with defects in homologous recombination (HR) is well established, other synthetic lethal interactions with PARP1/PARP2 disruption are poorly defined. To inform on these mechanisms we conducted a genome-wide screen for genes that are synthetic lethal with PARP1/2 gene disruption and identified C16orf72/HAPSTR1/TAPR1 as a novel modulator of replication-associated R-loops. C16orf72 is critical to facilitate replication fork restart, suppress DNA damage and maintain genome stability in response to replication stress. Importantly, C16orf72 and PARP1/2 function in parallel pathways to suppress DNA:RNA hybrids that accumulate at stalled replication forks. Mechanistically, this is achieved through an interaction of C16orf72 with BRCA1 and the RNA/DNA helicase Senataxin to facilitate their recruitment to RNA:DNA hybrids and confer resistance to PARP inhibitors. Together, this identifies a C16orf72/Senataxin/BRCA1-dependent pathway to suppress replication-associated R-loop accumulation, maintain genome stability and confer resistance to PARP inhibitors.

Quantitative Investigation of Methylation Heterogeneity by Digital Melting Curve Analysis on a SlipChip for Atrial Fibrillation.

Methylation is an essential epigenetic modification involved in regulating gene expression and maintaining genome stability. Methylation patterns can be heterogeneous, exhibiting variations in both level and density. However, current methods of methylation analysis, including sequencing, methylation-specific PCR, and high-resolution melting curve analysis (HRM), face limitations of high cost, time-consuming workflows, and the difficulty of both accurate heterogeneity analysis and precise quantification. Here, a droplet array SlipChip-based (da-SlipChip-based) digital melting curve analysis (MCA) method was developed for the accurate quantification of both methylation level (ratio of methylated molecules to total molecules) and methylation density (ratio of methylated CpG sites to total CpG sites). The SlipChip-based digital MCA system supplements an in situ thermal cycler with a fluorescence imaging module for real-time MCA. The da-SlipChip can generate 10,656 droplets of 1 nL each, which can be separated into four lanes, enabling the simultaneous analysis of four samples. This method's clinical application was demonstrated by analyzing samples from ten healthy individuals and twenty patients with atrial fibrillation (AF), the most common arrhythmia. This method can distinguish healthy individuals from those with AF of both the paroxysmal and persistent types. It also holds potential for broader application in various research and clinical settings requiring methylation analysis.

FACS-based genome-wide CRISPR screens define key regulators of DNA damage signaling pathways

DNA damage-activated signaling pathways are critical for coordinating multiple cellular processes, which must be tightly regulated to maintain genome stability. To provide a comprehensive and unbiased perspective of DNA damage response (DDR) signaling pathways, we performed 30 fluorescence-activated cell sorting (FACS)-based genome-wide CRISPR screens in human cell lines with antibodies recognizing distinct endogenous DNA damage signaling proteins to identify critical regulators involved in DDR. We discovered that proteasome-mediated processing is an early and prerequisite event for cells to trigger camptothecin- and etoposide-induced DDR signaling. Furthermore, we identified PRMT1 and PRMT5 as modulators that regulate ATM protein level. Moreover, we discovered that GNB1L is a key regulator of DDR signaling via its role as a co-chaperone specifically regulating PIKK proteins. Collectively, these screens offer a rich resource for further investigation of DDR, which may provide insight into strategies of targeting these DDR pathways to improve therapeutic outcomes.

Developing a peptide to disrupt cohesin head domain interactions

Cohesin mediates the 3-D structure of chromatin and is involved in maintaining genome stability and function. The cohesin core comprises Smc1 and Smc3, elongated-shaped proteins that dimerize through globular domains at their edges, called head and hinge. ATP binding to the Smc heads induces their dimerization and the formation of two active sites, while ATP hydrolysis results in head disengagement. This ATPase cycle is essential for driving cohesin activity. We report on the development of the first cohesin-inhibiting peptide (CIP). The CIP binds Smc3 invitro and inhibits the ATPase activity of the holocomplex. Treating yeast cells with the CIP prevents cohesin's tethering activity and, interestingly, leads to the accumulation of cohesin on chromatin. CIP3 also affects cohesin activity in human cells. Altogether, we demonstrate the power of peptides to inhibit cohesin in cells and discuss the potential application of CIPs as a therapeutic approach.

Visualization of direct and diffusion-assisted RAD51 nucleation by full-length human BRCA2 protein

Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA (ssDNA) gaps. To initiate HR, the recombination mediator and tumor suppressor protein BRCA2 facilitates nucleation of RAD51 on ssDNA prior to stimulation of RAD51 filament growth by RAD51 paralogs. Although ssDNA binding by BRCA2 has been implicated in RAD51 nucleation, the function of double-stranded DNA (dsDNA) binding by BRCA2 remains unclear. Here, we exploit single-molecule (SM) imaging to visualize BRCA2-mediated RAD51 nucleation in real time using purified proteins. We report that BRCA2 nucleates and stabilizes RAD51 on ssDNA either directly or through an unappreciated diffusion-assisted delivery mechanism involving binding to and sliding along dsDNA, which requires the cooperative action of multiple dsDNA-binding modules in BRCA2. Collectively, our work reveals two distinct mechanisms of BRCA2-dependent RAD51 loading onto ssDNA, which we propose are critical for its diverse functions in maintaining genome stability and cancer suppression.

Effects of DNA Methylation on Gene Expression and Phenotypic Traits in Cattle: A Review.

Gene expression in cells is determined by the epigenetic state of chromatin. Therefore, the study of epigenetic changes is very important to understand the regulatory mechanism of genes at the molecular, cellular, tissue and organ levels. DNA methylation is one of the most studied epigenetic modifications, which plays an important role in maintaining genome stability and ensuring normal growth and development. Studies have shown that methylation levels in bovine primordial germ cells, the rearrangement of methylation during embryonic development and abnormal methylation during placental development are all closely related to their reproductive processes. In addition, the application of bovine male sterility and assisted reproductive technology is also related to DNA methylation. This review introduces the principle, development of detection methods and application conditions of DNA methylation, with emphasis on the relationship between DNA methylation dynamics and bovine spermatogenesis, embryonic development, disease resistance and muscle and fat development, in order to provide theoretical basis for the application of DNA methylation in cattle breeding in the future.

Revisiting Two Decades of Research Focused on Targeting APE1 for Cancer Therapy: The Pros and Cons.

APE1 is an essential endodeoxyribonuclease of the base excision repair pathway that maintains genome stability. It was identified as a pivotal factor favoring tumor progression and chemoresistance through the control of gene expression by a redox-based mechanism. APE1 is overexpressed and serum-secreted in different cancers, representing a prognostic and predictive factor and a promising non-invasive biomarker. Strategies directly targeting APE1 functions led to the identification of inhibitors showing potential therapeutic value, some of which are currently in clinical trials. Interestingly, evidence indicates novel roles of APE1 in RNA metabolism that are still not fully understood, including its activity in processing damaged RNA in chemoresistant phenotypes, regulating onco-miRNA maturation, and oxidized RNA decay. Recent data point out a control role for APE1 in the expression and sorting of onco-miRNAs within secreted extracellular vesicles. This review is focused on giving a portrait of the pros and cons of the last two decades of research aiming at the identification of inhibitors of the redox or DNA-repair functions of APE1 for the definition of novel targeted therapies for cancer. We will discuss the new perspectives in cancer therapy emerging from the unexpected finding of the APE1 role in miRNA processing for personalized therapy.

Regulation of Rad52-dependent replication fork recovery through serine ADP-ribosylation of PolD3

Although Poly(ADP-ribose)-polymerases (PARPs) are key regulators of genome stability, how site-specific ADP-ribosylation regulates DNA repair is unclear. Here, we describe a novel role for PARP1 and PARP2 in regulating Rad52-dependent replication fork repair to maintain cell viability when homologous recombination is dysfunctional, suppress replication-associated DNA damage, and maintain genome stability. Mechanistically, Mre11 and ATM are required for induction of PARP activity in response to replication stress that in turn promotes break-induced replication (BIR) through assembly of Rad52 at stalled/damaged replication forks. Further, by mapping ADP-ribosylation sites induced upon replication stress, we identify that PolD3 is a target for PARP1/PARP2 and that its site-specific ADP-ribosylation is required for BIR activity, replication fork recovery and genome stability. Overall, these data identify a critical role for Mre11-dependent PARP activation and site-specific ADP-ribosylation in regulating BIR to maintain genome integrity during DNA synthesis.

Preparation of polyclonal antibodies against the Drosophila deacetylases SIRT 6 and SIRT 7

SIRT6 and SIRT7, as members of the Sirtuins family, are indispensable for the growth and development of Drosophila. They play crucial roles in maintaining genome stability, regulating metabolic senescence, and controlling tumorigenesis. To investigate their involvement in the Drosophila life cycle, we focused on describing the expression and purification of recombinant Drosophila SIRT6 and SIRT7 proteins. Subsequently, these proteins were utilized for generating polyclonal antibodies against Drosophila SIRT6 and SIRT7. The recombinant expression plasmid was introduced into E. coli cells to enable the production of SIRT6 and SIRT7 proteins. Following immunizations of New Zealand white rabbits and guinea pigs with the recombinant proteins as antigens, specific polyclonal antisera against both proteins were obtained. After purification, the specificity of SIRT6 and SIRT7 was confirmed using ELISA and western blot analyses, demonstrating strong specificity. These antibodies hold promise for the development of detection assays required for further research.

The nucleolus of Giardia and its ribosomal biogenesis.

Giardia duodenalis is a protozoan intestinal parasite that causes a significant number of infections worldwide each year, particularly in low-income and developing countries. Despite the availability of treatments for this parasitic infection, treatment failures are alarmingly common. As a result, new therapeutic strategies are urgently needed to effectively combat this disease. On the other hand, within the eukaryotic nucleus, the nucleolus stands out as the most prominent structure. It plays a crucial role in coordinating ribosome biogenesis and is involved in vital processes such as maintaining genome stability, regulating cell cycle progression, controlling cell senescence, and responding to stress. Given its significance, the nucleolus presents itself as a valuable target for selectively inducing cell death in undesirable cells, making it a potential avenue for anti-Giardia treatments. Despite its potential importance, the Giardia nucleolus remains poorly studied and often overlooked. In light of this, the objective of this study is to provide a detailed molecular description of the structure and function of the Giardia nucleolus, with a primary focus on its involvement in ribosomal biogenesis. Likewise, it discusses the targeting of the Giardia nucleolus as a therapeutic strategy, its feasibility, and the challenges involved.

Investigation of XPD, miR-145 and miR-770 expression in patients with end-stage renal disease.

The effective maintenance of genome integrity and fidelity is vital for the normal function of our tissues and organs, and the prevention of diseases. DNA repair pathways maintain genome stability, and the adequacy of genes acting in these pathways is essential for disease suppression and direct treatment responses. Chronic kidney disease is characterized by high levels of genomic damage. In this study, we examined the expression levels of the xeroderma pigmentosum group D (XPD) gene, which plays a role in the nucleotide excision repair (NER) repair mechanism, and the expression levels of miR-145 and miR-770 genes, which play a role in the regulation of the expression of the XPD gene, in hemodialysis patients with (n = 42) and without malignancy (n = 9) in pre- and post-dialysis conditions. We also evaluated these values with the clinical findings of the patients. Gene expression analysis was performed by real-time polymerase chain reaction (qRT-PCR). Compared to the individuals with normal kidney function (2.06 ± 0.32), the XPD gene expression was lower in the pre-dialysis condition both in hemodialysis patients without cancer (1.24 ± 0.18; p = 0.02) and in hemodialysis patients with cancer (0.82 ± 0.114; p = 0.001). On the other hand, we found that miR-145 and miR-770 expression levels were high in both groups. We also found that expression levels were affected by dialysis processes. A statistically significant positive correlation was found between miR-145 and mir770 expression levels in the pre-dialysis group of patients with (r=-0.988. p = 0.0001) and without (r=-0.934. p = 0.0001) malignancy. Studies on DNA damage repair in the kidney will help develop strategies to protect kidney function against kidney diseases.

Roles of NRF2 in DNA damage repair.

The transcription factor NF-E2-related factor 2 (NRF2) is a master regulator widely involved in essential cellular functions such as DNA repair. By clarifying the upstream and downstream links of NRF2 to DNA damage repair, we hope that attention will be drawn to the utilization of NRF2 as a target for cancer therapy. Query and summarize relevant literature on the role of NRF2 in direct repair, BER, NER, MMR, HR, and NHEJ in pubmed. Make pictures of Roles of NRF2 in DNA Damage Repair and tables of antioxidant response elements (AREs) of DNA repair genes. Analyze the mutation frequency of NFE2L2 in different types of cancer using cBioPortal online tools. By using TCGA, GTEx and GO databases, analyze the correlation between NFE2L2 mutations and DNA repair systems as well as the degree of changes in DNA repair systems as malignant tumors progress. NRF2 plays roles in maintaining the integrity of the genome by repairing DNA damage, regulating the cell cycle, and acting as an antioxidant. And, it possibly plays roles in double stranded break (DSB) pathway selection following ionizing radiation (IR) damage. Whether pathways such as RNA modification, ncRNA, and protein post-translational modification affect the regulation of NRF2 on DNA repair is still to be determined. The overall mutation frequency of the NFE2L2 gene in esophageal carcinoma, lung cancer, and penile cancer is the highest. Genes (50 of 58) that are negatively correlated with clinical staging are positively correlated with NFE2L2 mutations or NFE2L2 expression levels. NRF2 participates in a variety of DNA repair pathways and plays important roles in maintaining genome stability. NRF2 is a potential target for cancer treatment.

Therapeutic Targeting of DNA Replication Stress in Cancer.

This article reviews the currently used therapeutic strategies to target DNA replication stress for cancer treatment in the clinic, highlighting their effectiveness and limitations due to toxicity and drug resistance. Cancer cells experience enhanced spontaneous DNA damage due to compromised DNA replication machinery, elevated levels of reactive oxygen species, loss of tumor suppressor genes, and/or constitutive activation of oncogenes. Consequently, these cells are addicted to DNA damage response signaling pathways and repair machinery to maintain genome stability and support survival and proliferation. Chemotherapeutic drugs exploit this genetic instability by inducing additional DNA damage to overwhelm the repair system in cancer cells. However, the clinical use of DNA-damaging agents is limited by their toxicity and drug resistance often arises. To address these issues, the article discusses a potential strategy to target the cancer-associated isoform of proliferating cell nuclear antigen (caPCNA), which plays a central role in the DNA replication and damage response network. Small molecule and peptide agents that specifically target caPCNA can selectively target cancer cells without significant toxicity to normal cells or experimental animals.

Arabidopsis lamin-like proteins CRWN1 and CRWN2 interact with SUPPRESSOR OF NPR1-1 INDUCIBLE 1 and RAD51D to prevent DNA damage.

Plants cope with various recurring stress conditions that often induce DNA damage, ultimately affecting plant genome integrity, growth, and productivity. The CROWDED NUCLEI (CRWN) family comprises lamin-like proteins with multiple functions, such as regulating gene expression, genome organization, and DNA damage repair in Arabidopsis (Arabidopsis thaliana). However, the mechanisms and consequences of CRWNs in DNA damage repair are largely unknown. Here, we reveal that CRWNs maintain genome stability by forming repairing nuclear bodies at DNA double-strand breaks. We demonstrate that CRWN1 and CRWN2 physically associate with the DNA damage repair proteins RAD51D and SUPPRESSOR OF NPR1-1 Inducible 1 (SNI1) and act in the same genetic pathway to mediate this process. Moreover, CRWN1 and CRWN2 partially localize at γ-H2AX foci upon DNA damage. Notably, CRWN1 and CRWN2 undergo liquid-liquid phase separation to form highly dynamic droplet-like structures with RAD51D and SNI1 to promote the DNA damage response (DDR). Collectively, our data shed light on the function of plant lamin-like proteins in the DDR and maintenance of genome stability.

SIRT1 regulates DNA damage signaling through the PP4 phosphatase complex.

The Sirtuin family of NAD+-dependent enzymes plays an important role in maintaining genome stability upon stress. Several mammalian Sirtuins have been linked directly or indirectly to the regulation of DNA damage during replication through Homologous recombination (HR). The role of one of them, SIRT1, is intriguing as it seems to have a general regulatory role in the DNA damage response (DDR) that has not yet been addressed. SIRT1-deficient cells show impaired DDR reflected in a decrease in repair capacity, increased genome instability and decreased levels of γH2AX. Here we unveil a close functional antagonism between SIRT1 and the PP4 phosphatase multiprotein complex in the regulation of the DDR. Upon DNA damage, SIRT1 interacts specifically with the catalytical subunit PP4c and promotes its inhibition by deacetylating the WH1 domain of the regulatory subunits PP4R3α/β. This in turn regulates γH2AX and RPA2 phosphorylation, two key events in the signaling of DNA damage and repair by HR. We propose a mechanism whereby during stress, SIRT1 signaling ensures a global control of DNA damage signaling through PP4.