Transcriptional repressor ZBTB1 promotes chromatin remodeling and translesion DNA synthesis.

Timely DNA replication across damaged DNA is critical for maintaining genomic integrity. Translesion DNA synthesis (TLS) allows bypass of DNA lesions using error-prone TLS polymerases. The E3 ligase RAD18 is necessary for proliferating cell nuclear antigen (PCNA) monoubiquitination and TLS polymerase recruitment; however, the regulatory steps upstream of RAD18 activation are less understood. Here, we show that the UBZ4 domain-containing transcriptional repressor ZBTB1 is a critical upstream regulator of TLS. The UBZ4 motif is required for PCNA monoubiquitination and survival after UV damage. ZBTB1 associates with KAP-1, a transcriptional repressor whose phosphorylation relaxes chromatin after DNA damage. ZBTB1 depletion impairs formation of phospho-KAP-1 at UV damage sites and reduces RAD18 recruitment. Furthermore, phosphorylation of KAP-1 is necessary for efficient PCNA modification. We propose that ZBTB1 is required for localizing phospho-KAP-1 to chromatin and enhancing RAD18 accessibility. Collectively, our study implicates a ubiquitin-binding protein in orchestrating chromatin remodeling during DNA repair.

A DNA repair player, ring finger protein 43, relieves etoposide-induced topoisomerase II poisoning.

Ring finger protein 43 (RNF43) is an E3 ubiquitin ligase which is well-known for its role in negative regulation of the Wnt-signaling pathway. However, the function in DNA double-strand break repairs has not been investigated. In this study, we used a lymphoblast cell line, DT40, and mouse embryonic fibroblast as cellular models to study DNA double-strand break (DSB) repairs. For this purpose, we created RNF43 knockout, RNF43-/- DT40 cell line to investigate DSB repairs. We found that deletion of RNF43 does not interfere with cell proliferation. However, after exposure to various types of DNA-damaging agents, RNF43-/- cells become more sensitive to topoisomerase II inhibitors, etoposide, and ICRF193, than wild type cells. Our results also showed that depletion of RNF43 results in apoptosis upon etoposide-mediated DNA damage. The delay in resolution of γH2AX and 53BP1 foci formation after etoposide treatment, as well as epistasis analysis with DNAPKcs, suggested that RNF43 might participate in DNA repair of etoposide-induced DSB via non-homologous end joining. Disturbed γH2AX foci formation in MEFs following pulse etoposide treatment supported the notion that RNF43 also functions DNA repair in mammalian cells. These findings propose two possible functions of RNF43, either participating in NHEJ or removing the blockage of 5' topo II adducts from DSB ends.

Inhibition of homologous recombination by the PCNA-interacting protein PARI.

Inappropriate homologous recombination (HR) causes genomic instability and cancer. In yeast, the UvrD family helicase Srs2 is recruited to sites of DNA replication by SUMO-modified PCNA, where it acts to restrict HR by disassembling toxic RAD51 nucleofilaments. How human cells control recombination at replication forks is unknown. Here, we report that the protein PARI, containing a UvrD-like helicase domain, is a PCNA-interacting partner required for preservation of genome stability in human and DT40 chicken cells. Using cell-based and biochemical assays, we show that PARI restricts unscheduled recombination by interfering with the formation of RAD51-DNA HR structures. Finally, we show that PARI knockdown suppresses the genomic instability of Fanconi Anemia/BRCA pathway-deficient cells. Thus, we propose that PARI is a long sought-after factor that suppresses inappropriate recombination events at mammalian replication forks.

Carrier frequency of spinal muscular atrophy in Thailand.

Spinal muscular atrophy (SMA) is one of the leading causes of death in infants and young children from heritable diseases. Patients diagnosed with SMA develop symmetrical progressive muscle weakness and atrophy from degeneration of alpha motor neurons. Approximately 95% of patients have a homozygous deletion of survival motor neuron 1 (SMN1) gene in exon 7 and inherited in autosomal recessive pattern. Considering the high prevalence of SMA carrier in many population, it is possible that SMA is one of the most common autosomal recessive disorders in Thailand and Southeast Asia. In this study, we analyzed DNA from peripheral blood of 505 healthy Thai adults using quantitative PCR-based for SMN1 gene exon 7 copy number analysis. Individual samples with heterozygous deletion of SMN1 gene were confirmed with MLPA. The result identified 9 samples (1.78%) with heterozygous deletion and 39 samples as more than 2 copies of SMN1. No homozygous deletion was detected in the samples. In conclusion, we established carrier frequency of SMA in selected Thai population at 1.8% from 505 participants. The prevalence coincides with prevalence in East Asia and Caucasian population. The result could be implemented for SMA carrier screening in couples at risk in the region.

The epistatic relationship between BRCA2 and the other RAD51 mediators in homologous recombination.

RAD51 recombinase polymerizes at the site of double-strand breaks (DSBs) where it performs DSB repair. The loss of RAD51 causes extensive chromosomal breaks, leading to apoptosis. The polymerization of RAD51 is regulated by a number of RAD51 mediators, such as BRCA1, BRCA2, RAD52, SFR1, SWS1, and the five RAD51 paralogs, including XRCC3. We here show that brca2-null mutant cells were able to proliferate, indicating that RAD51 can perform DSB repair in the absence of BRCA2. We disrupted the BRCA1, RAD52, SFR1, SWS1, and XRCC3 genes in the brca2-null cells. All the resulting double-mutant cells displayed a phenotype that was very similar to that of the brca2-null cells. We suggest that BRCA2 might thus serve as a platform to recruit various RAD51 mediators at the appropriate position at the DNA-damage site.

A comprehensive Thai pharmacogenomics database (TPGxD-1): Phenotype prediction and variants identification in 942 whole-genome sequencing data.

Computational methods analyze genomic data to identify genetic variants linked to drug responses, thereby guiding personalized medicine. This study analyzed 942 whole-genome sequences from the Electricity Generating Authority of Thailand (EGAT) cohort to establish a population-specific pharmacogenomic database (TPGxD-1) in the Thai population. Sentieon (version 201808.08) implemented the GATK best workflow practice for variant calling. We then annotated variant call format (VCF) files using Golden Helix VarSeq 2.5.0 and employed Stargazer v2.0.2 for star allele analysis. The analysis of 63 very important pharmacogenes (VIPGx) reveals 85,566 variants, including 13,532 novel discoveries. Notably, we identified 464 known PGx variants and 275 clinically relevant novel variants. The phenotypic prediction of 15 VIPGx demonstrated a varied metabolic profile for the Thai population. Genes like CYP2C9 (9%), CYP3A5 (45.2%), CYP2B6 (9.4%), NUDT15 (15%), CYP2D6 (47%) and CYP2C19 (43%) showed a high number of intermediate metabolizers; CYP3A5 (41%), and CYP2C19 (9.9%) showed more poor metabolizers. CYP1A2 (52.7%) and CYP2B6 (7.6%) were found to have a higher number of ultra-metabolizers. The functional prediction of the remaining 10 VIPGx genes reveals a high frequency of decreased functional alleles in SULT1A1 (12%), NAT2 (84%), and G6PD (12%). SLCO1B1 reports 20% poor functional alleles, while PTGIS (42%), SLCO1B1 (4%), and TPMT (5.96%) showed increased functional alleles. This study discovered new variants and alleles in the 63 VIPGx genes among the Thai population, offering insights into advancing clinical pharmacogenomics (PGx). However, further validation is needed using other computational and genotyping methods.

ATR Inhibitor Synergizes PARP Inhibitor Cytotoxicity in Homologous Recombination Repair Deficiency TK6 Cell Lines.

The inhibition of poly(ADP-ribose) polymerases (PARPs) and ataxia telangiectasia and Rad3-related (ATR) would be an alternative approach for cancer treatments. The aim of this study is to investigate the synergy of the different combinations of PARP inhibitors (olaparib, talazoparib, or veliparib) and ATR inhibitor AZD6738. A drug combinational synergy screen that combines olaparib, talazoparib, or veliparib with AZD6738 was performed to identify the synergistic interaction, and the combination index was calculated to verify synergy. TK6 isogenic cell lines with defects in different DNA repair genes were used as a model. Cell cycle analysis, micronucleus induction, and focus formation assays of serine-139 phosphorylation of the histone variant H2AX demonstrated that AZD6738 diminished G2/M checkpoint activation induced by PARP inhibitors and allowed DNA damage-containing cells to continue dividing, leading to greater increases in micronuclei as well as double-strand DNA breaks in mitotic cells. We also found that AZD6738 was likely to potentiate cytotoxicity of PARP inhibitors in homologous recombination repair deficiency cell lines. AZD6738 sensitized more genotypes of DNA repair-deficient cell lines to talazoparib than to olaparib and veliparib, respectively. The combinational approach of PARP and ATR inhibition to enhance response to PARP inhibitors could expand the utility of PARP inhibitors to cancer patients without BRCA1/2 mutations.

PARP1pred: a web server for screening the bioactivity of inhibitors against DNA repair enzyme PARP-1.

Cancer is the leading cause of death worldwide, resulting in the mortality of more than 10 million people in 2020, according to Global Cancer Statistics 2020. A potential cancer therapy involves targeting the DNA repair process by inhibiting PARP-1. In this study, classification models were constructed using a non-redundant set of 2018 PARP-1 inhibitors. Briefly, compounds were described by 12 fingerprint types and built using the random forest algorithm concomitant with various sampling approaches. Results indicated that PubChem with an oversampling approach yielded the best performance, with a Matthews correlation coefficient > 0.7 while also affording interpretable molecular features. Moreover, feature importance, as determined from the Gini index, revealed that the aromatic/cyclic/heterocyclic moiety, nitrogen-containing fingerprints, and the ether/aldehyde/alcohol moiety were important for PARP-1 inhibition. Finally, our predictive model was deployed as a web application called PARP1pred and is publicly available at https://parp1pred.streamlitapp.com, allowing users to predict the biological activity of query compounds using their SMILES notation as the input. It is anticipated that the model described herein will aid in the discovery of effective PARP-1 inhibitors.

An essential role for Cdk1 in S phase control is revealed via chemical genetics in vertebrate cells.

In vertebrates Cdk1 is required to initiate mitosis; however, any functionality of this kinase during S phase remains unclear. To investigate this, we generated chicken DT40 mutants, in which an analog-sensitive mutant cdk1 as replaces the endogenous Cdk1, allowing us to specifically inactivate Cdk1 using bulky ATP analogs. In cells that also lack Cdk2, we find that Cdk1 activity is essential for DNA replication initiation and centrosome duplication. The presence of a single Cdk2 allele renders S phase progression independent of Cdk1, which suggests a complete overlap of these kinases in S phase control. Moreover, we find that Cdk1 inhibition did not induce re-licensing of replication origins in G2 phase. Conversely, inhibition during mitosis of Cdk1 causes rapid activation of endoreplication, depending on proteolysis of the licensing inhibitor Geminin. This study demonstrates essential functions of Cdk1 in the control of S phase, and exemplifies a chemical genetics approach to target cyclin-dependent kinases in vertebrate cells.

Noonan syndrome, metabolic syndrome and stroke-in-the-young: coincidence, causal or contribution?

A 34-year-old Thai woman developed acute left hemiparesis with dysarthria from subcortical infarction of the right MCA territory eighteen months after being diagnosed with Noonan syndrome, diabetes mellitus, dyslipidaemia, and hypertension. Further investigations suggested atherosclerosis as a cause. Modifying her risk factors was difficult, partly because of limited adherence. Three years later, she had another attack of ischaemic stroke in the same area. Unlike the three previously reported cases, the causation of strokes in this patient appeared to be a more 'complex' interaction between genetic defect and environment including possible subtle arterial abnormalities, metabolic risk factors, and mental insufficiency.

TRIM29 is required for efficient recruitment of 53BP1 in response to DNA double-strand breaks in vertebrate cells.

Tripartite motif-containing protein 29 (TRIM29) is involved in DNA double-strand break (DSB) repair. However, the specific roles of TRIM29 in DNA repair are not clearly understood. To investigate the involvement of TRIM29 in DNA DSB repair, we disrupted TRIM29 in DT40 cells by gene targeting with homologous recombination (HR). The roles of TRIM29 were investigated by clonogenic survival assays and immunofluorescence analyses. TRIM29 triallelic knockout (TRIM29-/-/-/+) cells were sensitive to etoposide, but resistant to camptothecin. Foci formation assays to assess DNA repair activities showed that the dissociation of etoposide-induced phosphorylated H2A histone family member X (É£-H2AX) foci was retained in TRIM29-/-/-/+ cells, and the formation of etoposide-induced tumor suppressor p53-binding protein 1 (53BP1) foci in TRIM29-/-/-/+ cells was slower compared with wild-type (WT) cells. Interestingly, the kinetics of camptothecin-induced RAD51 foci formation of TRIM29-/-/-/+ cells was higher than that of WT cells. These results indicate that TRIM29 is required for efficient recruitment of 53BP1 to facilitate the nonhomologous end-joining (NHEJ) pathway and thereby suppress the HR pathway in response to DNA DSBs. TRIM29 regulates the choice of DNA DSB repair pathway by facilitating 53BP1 accumulation to promote NHEJ and may have potential for development into a therapeutic target to sensitize refractory cancers or as biomarker of personalized therapies.

DNA Repair Biosensor-Identified DNA Damage Activities of Endophyte Extracts from Garcinia cowa.

Recent developments in chemotherapy focus on target-specific mechanisms, which occur only in cancer cells and minimize the effects on normal cells. DNA damage and repair pathways are a promising target in the treatment of cancer. In order to identify novel compounds targeting DNA repair pathways, two key proteins, 53BP1 and RAD54L, were tagged with fluorescent proteins as indicators for two major double strand break (DSB) repair pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). The engineered biosensor cells exhibited the same DNA repair properties as the wild type. The biosensor cells were further used to investigate the DNA repair activities of natural biological compounds. An extract from Phyllosticta sp., the endophyte isolated from the medicinal plant Garcinia cowa Roxb. ex Choisy, was tested. The results showed that the crude extract induced DSB, as demonstrated by the increase in the DNA DSB marker γH2AX. The damaged DNA appeared to be repaired through NHEJ, as the 53BP1 focus formation in the treated fraction was higher than in the control group. In conclusion, DNA repair-based biosensors are useful for the preliminary screening of crude extracts and biological compounds for the identification of potential targeted therapeutic drugs.

Frequencies of spinocerebellar ataxia subtypes in Thailand: window to the population history?

Spinocerebellar ataxias (SCAs) are a heterogeneous group of disorders with almost 30 subtypes. The prevalence and relative frequency of each subtype vary among different populations. In this article, we report the relative frequency of six SCA subtypes in the Thai population and attempt to explain the observed pattern when compared with other populations in this region. We searched for SCA type 1, SCA2, SCA3, SCA6, SCA7 and dentatorubral-pallidoluysian atrophy mutations using GeneScan analysis in 340 patients from 182 families, in which at least one person had a clinical diagnosis of SCA. We analyzed the relative frequencies of SCA subtypes on a family basis, and compared these with the data in the Chinese and Indian populations. SCA3 was found in 19.2% of the patients (Agresti-Coull 95% confidence interval: 14.1-25.6%), SCA1 in 11.5% (7.6-17.1%) and SCA2 in 10.4% (6.7-15.8%). SCA6 was found in three families, with a relative frequency of 1.6% (0.3-5.0%). Compared with the related populations, the Thai SCA3 frequency was less than that of the Chinese, whereas it was higher than that in most of the Indian studies. The reverse is true for the SCA1/SCA2 frequency. A similar study in Singapore, where there was a clear history of population admixture, also showed the frequencies between those of the Chinese and the Indian populations. Although SCA3 was the most common identifiable SCA subtype in Thailand, SCA1 and SCA2 were also relatively common. Our results also supported some degree of admixture with the Indians in the Thai population and justify further study in the area.

Compensatory functions and interdependency of the DNA-binding domain of BRCA2 with the BRCA1-PALB2-BRCA2 complex.

BRCA1, BRCA2, and PALB2 are key players in cellular tolerance to chemotherapeutic agents, including camptothecin, cisplatin, and PARP inhibitor. The N-terminal segment of BRCA2 interacts with PALB2, thus contributing to the formation of the BRCA1-PALB2-BRCA2 complex. To understand the role played by BRCA2 in this complex, we deleted its N-terminal segment and generated BRCA2(Δ)(N) mutant cells. Although previous studies have suggested that BRCA1-PALB2 plays a role in the recruitment of BRCA2 to DNA-damage sites, BRCA2(Δ)(N) mutant cells displayed a considerably milder phenotype than did BRCA2(-/-) null-deficient cells. We hypothesized that the DNA-binding domain (DBD) of BRCA2 might compensate for a defect in BRCA2(ΔN) that prevented stable interaction with PALB2. To test this hypothesis, we disrupted the DBD of BRCA2 in wild-type and BRCA2(Δ)(N) cells. Remarkably, although the resulting BRCA2(Δ)(DBD) cells displayed a moderate phenotype, the BRCA2(Δ)(N+ΔDBD) cells displayed a very severe phenotype, as did the BRCA2(-/-) cells, suggesting that the N-terminal segment and the DBD play a substantially overlapping role in the functionality of BRCA2. We also showed that the formation of both the BRCA1-PALB2-BRCA2 complex and the DBD is required for efficient recruitment of BRCA2 to DNA-damage sites. Our study revealed the essential role played by both the BRCA1-PALB2-BRCA2 complex and the DBD in the functionality of BRCA2, as each can compensate for the other in the recruitment of BRCA2 to DNA-damage sites. This knowledge adds to our ability to accurately predict the efficacy of antimalignant therapies for patients carrying mutations in the BRCA2 gene.

PARI overexpression promotes genomic instability and pancreatic tumorigenesis.

Treatment options for patients with pancreatic ductal adenocarcinoma (PDAC) remain limited. Therapeutic targets of interest include mutated molecules that predispose to pancreatic cancer such as KRAS and TP53. Here, we show that an element of the homologous recombination pathway of DNA repair, the PARP-binding protein C12orf48/PARI (PARPBP), is overexpressed specifically in pancreatic cancer cells where it is an appealing candidate for targeted therapy. PARI upregulation in pancreatic cancer cells or avian DT40 cells conferred DNA repair deficiency and genomic instability. Significantly, PARI silencing compromised cancer cell proliferation in vitro, leading to cell-cycle alterations associated with S-phase delay, perturbed DNA replication, and activation of the DNA damage response pathway in the absence of DNA damage stimuli. Conversely, PARI overexpression produced tolerance to DNA damage by promoting replication of damaged DNA. In a mouse xenograft model of pancreatic cancer, PARI silencing was sufficient to reduce pancreatic tumor growth in vivo. Taken together, our findings offered a preclinical proof-of-concept for PARI as candidate therapeutic target to treat PDAC.

Regulation of Rev1 by the Fanconi anemia core complex.

The 15 known Fanconi anemia proteins cooperate in a pathway that regulates DNA interstrand cross-link repair. Recent studies indicate that the Fanconi anemia pathway also controls Rev1-mediated translesion DNA synthesis (TLS). We identified Fanconi anemia-associated protein (FAAP20), an integral subunit of the multisubunit Fanconi anemia core complex. FAAP20 binds to FANCA subunit and is required for stability of the complex and monoubiquitination of FANCD2. FAAP20 contains a ubiquitin-binding zinc finger 4 domain and binds to the monoubiquitinated form of Rev1. FAAP20 binding stabilizes Rev1 nuclear foci and promotes interaction of the Fanconi anemia core with PCNA-Rev1 DNA damage bypass complexes. FAAP20 therefore provides a critical link between the Fanconi anemia pathway and TLS polymerase activity. We propose that the Fanconi anemia core complex regulates cross-link repair by channeling lesions to damage bypass pathways and preventing large DNA insertions and deletions.

The USP1/UAF1 complex promotes double-strand break repair through homologous recombination.

Protein ubiquitination plays a key role in the regulation of a variety of DNA repair mechanisms. Protein ubiquitination is controlled by the coordinate activity of ubiquitin ligases and deubiquitinating enzymes (DUBs). The deubiquitinating enzyme USP1 regulates DNA repair and the Fanconi anemia pathway through its association with its WD40 binding partner, UAF1, and through its deubiquitination of two critical DNA repair proteins, FANCD2-Ub and PCNA-Ub. To investigate the function of USP1 and UAF1, we generated USP1⁻/⁻, UAF1⁻/⁻/⁻, and USP1⁻/⁻ UAF1⁻/⁻/⁻ chicken DT40 cell clones. These three clones showed similar sensitivities to chemical cross-linking agents, to a topoisomerase poison, camptothecin, and to an inhibitor of poly(ADP-ribose) polymerase (PARP), indicating that the USP1/UAF1 complex is a regulator of the cellular response to DNA damage. The hypersensitivity to both camptothecin and a PARP inhibitor suggests that the USP1/UAF1 complex promotes homologous recombination (HR)-mediated double-strand break (DSB) repair. To gain insight into the mechanism of the USP1/UAF1 complex in HR, we inactivated the nonhomologous end-joining (NHEJ) pathway in UAF1-deficient cells. Disruption of NHEJ in UAF1-deficient cells restored cellular resistance to camptothecin and the PARP inhibitor. Our results indicate that the USP1/UAF1 complex promotes HR, at least in part by suppressing NHEJ.

An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63.

Activation of the protein kinases ATM and ATR following chromosomal breakage prevents initiation of DNA replication and entry into mitosis. However, the effects of ATM and ATR activation in cells already progressing through mitosis are poorly understood. Here we report that ATM and ATR activation induced by DNA double-strand breaks (DSBs) inhibits centrosome-driven spindle assembly in Xenopus laevis mitotic egg extract and somatic cells, delaying mitotic progression. Using a cDNA expression library to screen for ATM and ATR substrates, we identified centrosomal protein CEP63 as an ATM and ATR target required for normal spindle assembly. ATM and ATR phosphorylate Xenopus CEP63 (XCEP63) on Ser 560 and promote its delocalization from the centrosome. Suppression of ATM and ATR activity or mutation of XCEP63 Ser 560 to Ala prevented spindle assembly defects. Consistently, inactivation of the CEP63 gene in avian DT40 cells impaired spindle assembly and prevented ATM- and ATR-dependent effects on mitosis. These data indicate that ATM and ATR control mitotic events in vertebrate cells by targeting CEP63 and centrosome dependent spindle assembly.

Parp-1 protects homologous recombination from interference by Ku and Ligase IV in vertebrate cells.

Parp-1 and Parp-2 are activated by DNA breaks and have been implicated in the repair of DNA single-strand breaks (SSB). Their involvement in double-strand break (DSB) repair mediated by homologous recombination (HR) or nonhomologous end joining (NHEJ) remains unclear. We addressed this question using chicken DT40 cells, which have the advantage of carrying only a PARP-1 gene but not a PARP-2 gene. We found that PARP-1(-/-) DT40 mutants show reduced levels of HR and are sensitive to various DSB-inducing genotoxic agents. Surprisingly, this phenotype was strictly dependent on the presence of Ku, a DSB-binding factor that mediates NHEJ. PARP-1/KU70 double mutants were proficient in the execution of HR and displayed elevated resistance to DSB-inducing drugs. Moreover, we found deletion of Ligase IV, another NHEJ gene, suppressed the camptothecin of PARP-1(-/-) cells. Our results suggest a new critical function for Parp in minimizing the suppressive effects of Ku and the NHEJ pathway on HR.