Role In DNA Replication Research Articles

Multifunctional acyltransferase HBO1: a key regulatory factor for cellular functions.

HBO1, also known as KAT7 or MYST2, is a crucial histone acetyltransferase with diverse cellular functions. It typically forms complexes with protein subunits or cofactors such as MEAF6, ING4, or ING5, and JADE1/2/3 or BRPF1/2/3, where the BRPF or JADE proteins serve as the scaffold targeting histone H3 or H4, respectively. The histone acetylation mediated by HBO1 plays significant roles in DNA replication and gene expression regulation. Additionally, HBO1 catalyzes the modification of proteins through acylation with propionyl, butyryl, crotonyl, benzoyl, and acetoacetyl groups. HBO1 undergoes ubiquitination and degradation by two types of ubiquitin complexes and can also act as an E3 ubiquitin ligase for the estrogen receptor α (ERα). Moreover, HBO1 participates in the expansion of medullary thymic epithelial cells (mTECs) and regulates the expression of peripheral tissue genes (PTGs) mediated by autoimmune regulator (AIRE), thus inducing immune tolerance. Furthermore, HBO1 influences the renewal of hematopoietic stem cells and the development of neural stem cells significantly. Importantly, the overexpression of HBO1 in various cancers suggests its carcinogenic role and potential as a therapeutic target. This review summarizes recent advancements in understanding HBO1's involvement in acylation modification, DNA replication, ubiquitination, immunity, and stem cell renewal.

Identification of novel DNA Gyrase inhibitor by combined Pharmacophore modeling, QSAR analysis, Molecular docking, Molecular dynamics, ADMET and DFT approaches

DNA gyrase, an ATP-dependent enzyme, plays a critical role in DNA replication, transcription, and recombination in Mycobacterium tuberculosis (MTB). While fluoroquinolones are effective antibacterial agents targeting DNA gyrase, their clinical use is often limited due to side effects and the emergence of bacterial resistance. In this study, we developed a quantitative structure-activity relationship (QSAR) model to predict the anti-tubercular activity of Quinoline-Aminopiperidine derivatives targeting the DNA gyrase enzyme, using a dataset of 48 compounds obtained from the literature. The QSAR model was validated using both internal and external validation metrics. Model 4, the best predictive model, demonstrated a strong fit with an R² of 0.8393, an adjusted R² (R²adj) of 0.8010, and a lack of fit (LOF) parameter of 0.0626. The QSAR results revealed that DNA gyrase inhibition is significantly influenced by factors such as partition coefficient, molecular flexibility, hydrogen bonding potential, and the presence of fluorine atoms. Twelve quinoline-aminopiperidine derivatives were designed, and their anti-tubercular activity was predicted using QSAR model-4. These compounds were further assessed for pharmacokinetic properties, toxicity, and binding affinity to DNA gyrase. Pharmacophore modeling was also performed and validated using MOE software. The final pharmacophore model includes the features of two aromatic hydrophobic features, one hydrogen bond acceptor, and one hydrogen bond donor. The results indicated that designed compounds QA-3 and dataset compounds C-34 exhibit favorable drug-likeness properties. Molecular dynamics simulations confirmed the stable binding of compounds QA-3 and C-34 to the DNA gyrase protein, highlighting their potential as promising anti-tubercular agents. The developed QSAR Model-4 will facilitate the prediction of anti-tubercular activity in Quinoline-Aminopiperidine derivatives.

The role of prokaryotic argonautes in resistance to type II topoisomerases poison ciprofloxacin.

Argonaute proteins are programmable nucleases found in all domains of life. Eukaryotic argonautes (eAgos) participate in genetic regulation, antiviral response, and transposon silencing during RNA interference. Prokaryotic argonautes (pAgos) are much more diverse than eAgos and have been implicated in defense against invading genetic elements. Recently, it was shown that pAgos protect bacterial cells from a topoisomerase poison ciprofloxacin, raising a possibility that they may play a role in DNA replication and/or repair. Here, we discuss possible models of pAgo-mediated ciprofloxacin resistance. We propose that pAgos could (i) participate in chromosome decatenation as a backup to topoisomerases; (ii) participate in the processing of DNA repair intermediates formed after topoisomerase poisoning, or (iii) induce SOS response that generally affects DNA repair and antibiotic resistance. These hypotheses should guide future investigations of the involvement of pAgos in the emergence of resistance to ciprofloxacin and, possibly, other antibiotics.

Screening of functional maternal-specific chromatin regulators in early embryonic development of zebrafish.

The early stages of embryonic development rely on maternal products for proper regulation. However, screening for functional maternal-specific factors is challenging due to the time- and labor-intensive nature of traditional approaches. Here, we combine a computational pipeline and F0 null mutant technology to screen for functional maternal-specific chromatin regulators in zebrafish embryogenesis and identify Mcm3l, Mcm6l, and Npm2a as playing essential roles in DNA replication and cell division. Our results contribute to understanding the molecular mechanisms underlying early embryo development and highlight the importance of maternal-specific chromatin regulators in this critical stage.

Critical roles of Dpb3-Dpb4 sub-complex of DNA polymerase epsilon in DNA replication, genome stability, and pathogenesis of Candida albicans.

This study explored the role of DNA polymerase epsilon, especially its non-essential structural subunits in Candida albicans biology. Apart from their role in DNA replication and genome stability, the Dpb3-Dpb4 subcomplex regulates morphological switching and virulence. Since the defective strain is locked in filamentous form and is avirulent, the complex may be targeted for anti-fungal drug development.

X-ray crystal structure of proliferating cell nuclear antigen 1 from Aeropyrum pernix.

Proliferating cell nuclear antigen (PCNA) plays a critical role in DNA replication by enhancing the activity of various proteins involved in replication. Inthis study, the crystal structure of ApePCNA1, one of three PCNAs from thethermophilic archaeon Aeropyrum pernix, was elucidated. ApePCNA1 was cloned and expressed in Escherichia coli and the protein was purified and crystallized. The resulting crystal structure determined at 2.00 Å resolution revealed that ApePCNA1 does not form a trimeric ring, unlike PCNAs from other domains of life. It has unique structural features, including a long interdomain-connecting loop and a PIP-box-like sequence at the N-terminus, indicating potential interactions with other proteins. These findings provide insights into the functional mechanisms of PCNAs in archaea and their evolutionary conservation across different domains of life. A modified medium and protocol were used to express recombinant protein containing the lac operon. The expression of the target protein increased and the total incubation time decreased when using this system compared with those of previous expression protocols.

Unveiling the Anticancer Potential: Computational Exploration of Nitrogenated Derivatives of (+)-Pancratistatin as Topoisomerase I Inhibitors.

Cancer poses a substantial global health challenge, driving the need for innovative therapeutic solutions that offer improved effectiveness and fewer side effects. Topoisomerase I (Topo I) has emerged as a validated molecular target in the pursuit of developing anticancer drugs due to its critical role in DNA replication and transcription. (+)-Pancratistatin (PST), a naturally occurring compound found in various Amaryllidaceae plants, exhibits promising anticancer properties by inhibiting Topo I activity. However, its clinical utility is hindered by issues related to limited chemical availability and aqueous solubility. To address these challenges, molecular modelling techniques, including virtual screening, molecular docking, molecular mechanics with generalised born and surface area solvation (MM-GBSA) calculations, and molecular dynamics simulations were utilised to evaluate the binding interactions and energetics of PST analogues with Topo I, comparing them with the well-known Topo I inhibitor, Camptothecin. Among the compounds screened for this study, nitrogenated analogues emerged as the most encouraging drug candidates, exhibiting improved binding affinities, favourable interactions with the active site of Topo I, and stability of the protein-ligand complex. Structural analysis pinpointed key molecular determinants responsible for the heightened potency of nitrogenated analogues, shedding light on essential structural modifications for increased activity. Moreover, in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions highlighted favourable drug-like properties and reduced toxicity profiles for the most prominent nitrogenated analogues, further supporting their potential as effective anticancer agents. In summary, this screening study underscores the significance of nitrogenation in augmenting the anticancer efficacy of PST analogues targeting Topo I. The identified lead compounds exhibit significant potential for subsequent experimental validation and optimisation, thus facilitating the development of novel and efficacious anticancer therapeutics with enhanced pharmacological profiles.

53BP1-the ‘Pandora’s box’ of genome integrity

53BP1 has several functions in the maintenance of genome integrity. It functions as a key mediator involved in double-strand break (DSB) repair, which functions to maintain a balance in the repair pathway choices and in preserving genomic stability. While its DSB repair functions are relatively well-characterized, its role in DNA replication and replication fork protection is less understood. In response to replication stress, 53BP1 contributes to fork protection by regulating fork reversal and restart. It helps maintain replication fork stability and speed, with 53BP1 loss leading to defective fork progression and increased sensitivity to replication stress agents. However, 53BP1's precise role in fork protection remains debated, as some studies have not observed protective effects. Therefore, it is critical to determine the role of 53BP1 in replication to better understand when it promotes replication fork protection, and the underlying mechanisms involved. Moreover, 53BP1's function in replication stress extends beyond its activity at active replication forks; it also forms specialized nuclear bodies (NBs) which protect stretches of under-replicated DNA (UR-DNA) transmitted from a previous cell cycle to daughter cells through mitosis. The mechanism of 53BP1 NBs in the coordination of replication and repair events at UR-DNA loci is not fully understood and warrants further investigation. The present review article focuses on elucidating 53BP1’s functions in replication stress (RS), its role in replication fork protection, and the significance of 53BP1 NBs in this context to provide a more comprehensive understanding of its less well-established role in DNA replication.

MCM4 potentiates evasion of hepatocellular carcinoma from sorafenib-induced ferroptosis through Nrf2 signaling pathway

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. It poses an enormous socioeconomic burden and is a serious public health threat globally due to its poor prognosis. Ferroptosis is a newly identified non-apoptotic form of cell death characterized by lipid peroxidation, iron accumulation, and reactive oxygen species (ROS) generation. However, tumor cells have evolved diverse mechanisms to evade ferroptosis, conferring resistance to drugs. Sorafenib, a first-line therapy for advanced HCC, triggers ferroptosis by selectively targeting solute carrier family 7 member 11 (SLC7A11) to deplete glutathione and inhibit glutathione peroxidase 4 (GPX4), thereby effectively eliminating tumor cells. However, sorafenib resistance has been widely reported, and the precise mechanisms underlying sorafenib drug resistance remain unclear. The minichromosome maintenance (MCM) protein family contains 10 members with vital roles in DNA replication and cell cycle progression. MCM4, a member of the MCM protein family, might be a potential biomarker in pan-cancer analysis. The present study found that MCM4 was upregulated in liver cancer using bioinformatics analysis and sorafenib-treated HCC cells. Moreover, MCM4 might be regarded as a prognostic biomarker for HCC. Further experiments revealed that MCM4-inhibition enhanced the efficacy of sorafenib through elevation of ferroptosis both in vitro and in vivo. Mechanistically, MCM4 potentiates sorafenib-induced ferroptosis evasion in HCC by promoting nuclear factor erythroid 2-related factor 2 (Nrf2) signaling activation. However, no direct interactions were found between Nrf2 and MCM4. Overall, these findings suggest a potential therapeutic strategy for HCC by targeting MCM4 inhibition.

Nsp1 stalls DNA Polymerase α at DNA hairpins.

The human primosome, a four-subunit complex of DNA primase and DNA polymerase alpha (Polα), plays a critical role in DNA replication by initiating RNA and DNA synthesis on both chromosome strands. A recent study has shown that a major virulence factor in the SARS-CoV-2 infection, Nsp1 (non-structural protein 1), forms a stable complex with Polα but does not affect the primosome activity. Here we show that Nsp1 inhibits DNA synthesis across inverted repeats prone to hairpin formation. Analysis of current structural data revealed the overlapping binding sites for Nsp1 and the winged helix-turn-helix domain of RPA (wHTH) on Polα, indicating a competition between them. Comparison of the inhibitory effect of Nsp1 and wHTH on DNA hairpin bypass by Polα showed an 8-fold lower IC 50 value for Nsp1 (1 µM). This study provides a valuable insight into the mechanism of inhibition of human DNA replication by Nsp1 during a SARS-CoV-2 infection.

Histone variant macroH2A1 regulates synchronous firing of replication origins in the inactive X chromosome.

MacroH2A has been linked to transcriptional silencing, cell identity, and is a hallmark of the inactive X chromosome (Xi). However, it remains unclear whether macroH2A plays a role in DNA replication. Using knockdown/knockout cells for each macroH2A isoform, we show that macroH2A-containing nucleosomes slow down replication progression rate in the Xi reflecting the higher nucleosome stability. Moreover, macroH2A1, but not macroH2A2, regulates the number of nano replication foci in the Xi, and macroH2A1 downregulation increases DNA loop sizes corresponding to replicons. This relates to macroH2A1 regulating replicative helicase loading during G1 by interacting with it. We mapped this interaction to a phenylalanine in macroH2A1 that is not conserved in macroH2A2 and the C-terminus of Mcm3 helicase subunit. We propose that macroH2A1 enhances the licensing of pre-replication complexes via DNA helicase interaction and loading onto the Xi.

Essential and multifunctional mpox virus E5 helicase-primase in double and single hexamer.

An outbreak of mpox virus in May 2022 has spread over 110 nonpandemic regions in the world, posing a great threat to global health. Mpox virus E5, a helicase-primase, plays an essential role in DNA replication, but the molecular mechanisms are elusive. Here, we report seven structures of mpox virus E5 in a double hexamer (DH) and six in single hexamer in different conformations, indicating a rotation mechanism for helicase and a coupling action for primase. The DH is formed through the interface of zinc-binding domains, and the central channel density indicates potential double-stranded DNA (dsDNA), which helps to identify dsDNA binding residues Arg249, Lys286, Lys315, and Lys317. Our work is important not only for understanding poxviral DNA replication but also for the development of novel therapeutics for serious poxviral infections including smallpox virus and mpox virus.

Proliferating Cell Nuclear Antigen in the Era of Oncolytic Virotherapy.

Proliferating cell nuclear antigen (PCNA) is a well-documented accessory protein of DNA repair and replication. It belongs to the sliding clamp family of proteins that encircle DNA and acts as a mobile docking platform for interacting proteins to mount and perform their metabolic tasks. PCNA presence is ubiquitous to all cells, and when located in the nucleus it plays a role in DNA replication and repair, cell cycle control and apoptosis in proliferating cells. It also plays a crucial role in the infectivity of some viruses, such as herpes simplex viruses (HSVs). However, more recently it has been found in the cytoplasm of immune cells such as neutrophils and macrophages where it has been shown to be involved in the development of a pro-inflammatory state. PCNA is also expressed on the surface of certain cancer cells and can play a role in preventing immune cells from killing tumours, as well as being associated with cancer virulence. Given the growing interest in oncolytic viruses (OVs) as a novel cancer therapeutic, this review considers the role of PCNA in healthy, cancerous, and immune cells to gain an understanding of how PCNA targeted therapy and oncolytic virotherapy may interact in the future.

Essential Nutrients, Added Sugar Intake, and Epigenetic Age in Midlife Black and White Women

Nutritive compounds play critical roles in DNA replication, maintenance, and repair, and also serve as antioxidant and anti-inflammatory agents. Sufficient dietary intakes support genomic stability and preserve health. To investigate the associations of dietary patterns, including intakes of essential nutrients and added sugar, and diet quality scores of established and new nutrient indices with epigenetic age in a diverse cohort of Black and White women at midlife. This cross-sectional study included analyses (2021-2023) of past women participants of the 1987-1997 National Heart, Lung, and Blood Institute Growth and Health Study (NGHS), which examined cardiovascular health in a community cohort of Black and White females aged between 9 and 19 years. Of these participants who were recruited between 2015 and 2019 from NGHS's California site, 342 females had valid completed diet and epigenetic assessments. The data were analyzed from October 2021 to November 2023. Diet quality scores of established nutrient indices (Alternate Mediterranean Diet [aMED], Alternate Healthy Eating Index [AHEI]-2010); scores for a novel, a priori-developed Epigenetic Nutrient Index [ENI]; and mean added sugar intake amounts were derived from 3-day food records. GrimAge2, a second-generation epigenetic clock marker, was calculated from salivary DNA. Hypotheses were formulated after data collection. Healthier diet indicators were hypothesized to be associated with younger epigenetic age. A total of 342 women composed the analytic sample (mean [SD] age, 39.2 [1.1] years; 171 [50.0%] Black and 171 [50.0%] White participants). In fully adjusted models, aMED (β, -0.41; 95% CI, -0.69 to -0.13), AHEI-2010 (β, -0.05; 95% CI, -0.08 to -0.01), and ENI (β, -0.17; 95% CI, -0.29 to -0.06) scores, and added sugar intake (β, 0.02; 95% CI, 0.01-0.04) were each significantly associated with GrimAge2 in expected directions. In combined analyses, the aforementioned results with GrimAge2 were preserved with the association estimates for aMED and added sugar intake retaining their statistical significance. In this cross-sectional study, independent associations were observed for both healthy diet and added sugar intake with epigenetic age. To our knowledge, these are among the first findings to demonstrate associations between added sugar intake and epigenetic aging using second-generation epigenetic clocks and one of the first to extend analyses to a diverse population of Black and White women at midlife. Promoting diets aligned with chronic disease prevention recommendations and replete with antioxidant or anti-inflammatory and pro-epigenetic health nutrients while emphasizing low added sugar consumption may support slower cellular aging relative to chronological age, although longitudinal analyses are needed.

Early postovulatory aging reveals the first proteomic markers of egg quality in pikeperch

This study explored the molecular mechanisms underlying egg quality deterioration due to in vivo postovulatory aging in pikeperch (Sander lucioperca L.), a key species in aquaculture. We employed tandem mass tag (TMT) peptide labeling coupled with LC–MS/MS quantitative proteomics to analyze eggs collected at various postovulation intervals. Our research revealed four distinct proteomic markers (Gins4, Atrx, DnaJB14, and Mrpl10) that are differentially expressed in response to early aging, shedding light on their potential roles in DNA replication, chromatin organization, protein folding, and mitochondrial function. The study confirmed that eggs maintain morphological integrity up to 5 h postovulation but exhibit compromised fertilization capacity, underscoring the importance of timely egg utilization in aquaculture practices. These findings enhance the understanding of egg aging at the molecular level, offering insights for improving reproductive success and larval quality in pikeperch aquaculture. The data are available via ProteomeXchange with the identifier PXD048349.

Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage

CTC1-STN1-TEN1 (CST) is a single-stranded DNA binding protein vital for telomere length maintenance with additional genome-wide roles in DNA replication and repair. While CST was previously shown to function in double-strand break repair and promote replication restart, it is currently unclear whether it has specialized roles in other DNA repair pathways. Proper and efficient repair of DNA is critical to protecting genome integrity. Telomeres and other G-rich regions are strongly predisposed to oxidative DNA damage in the form of 8-oxoguanines, which are typically repaired by the base-excision repair (BER) pathway. Moreover, recent studies suggest that CST functions in the repair of oxidative DNA lesions. Therefore, we tested whether CST interacts with and regulates BER protein activity. Here, we show that CST robustly stimulates proteins involved in BER, including OGG1, Pol β, APE1, and LIGI, on both telomeric and non-telomeric DNA substrates. Biochemical reconstitution of the pathway indicates that CST stimulates BER. Finally, knockout of STN1 or CTC1 leads to increased levels of 8-oxoguanine, suggesting defective BER in the absence of CST. Combined, our results define an undiscovered function of CST in BER, where it acts as a stimulatory factor to promote efficient genome-wide oxidative repair.

Abstract PO1-15-06: Serum thymidine kinase activity as a “real-time” clinical biomarker of tumor response to CDK4/6 inhibition in hormone receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer

Abstract Background: CDK4/6 inhibitors (CDK4/6i) plus endocrine therapy (ET) are standard first-line (1L) treatment for hormone receptor positive (HR+), HER2-negative (HER2-) metastatic breast cancer (MBC). Despite general improvements in long-term outcome, not all patients benefit equally from these drugs. The average duration of 1L CDK4/6i therapy is ~2 years. However, 10-15% of patients will progress within 6 months of therapy initiation, while others will derive durable clinical benefit of years. Differences in treatment efficacy cannot be easily explained by clinical-pathological features. No predictive biomarkers are currently available to determine tumor sensitivity to CDK4/6i. There is an unmet need for a complementary diagnostic to identify patients that will or will not respond to these treatments in order to better inform therapy management. Thymidine kinase (TK) is an E2F-regulated enzyme that plays a key role in DNA replication during cell division. The expression and activity of TK is strongly linked to the cell cycle. TK is first expressed in late G1, peaks during S-phase, and then is degraded during mitosis. Because of the restricted expression of TK (only during S phase of the cell cycle) combined with the mechanism of action of CDK4/6i, the measurement of TK activity (TKa) may serve as an ideal pharmacodynamic biomarker to assess a tumor’s response to CDK4/6i-based therapy. DiviTum® TKa is an FDA cleared assay that can quantitate TKa from human serum (sTKa) and is intended to be used as a disease progression monitoring tool for patients with HR+, HER2- MBC. TK IMPACT is a prospective, single arm trial designed to assess the impact of incorporation of DiviTum® TKa on the physician’s decision regarding subsequent timing of routine disease monitoring modalities in patients with advanced HR+, HER2- MBC receiving 1L ET plus CDK 4/6i (NCT04968964). As a part of this ongoing study, DiviTum® TKa values are serially assessed relative to imaging findings and other circulating tumor biomarkers, such as CA15-3 and ctDNA. The primary endpoint is any physician-reported intended change in imaging testing interval post TKa by study cohort, within the first 48-week period of study participation, assessments for which are ongoing. Here we report real-world data and examples of the clinical utility of sTKa as a companion diagnostic from patients treated at Washington University Siteman Cancer Center, St. Louis, MO. Methods: Patients with HR+, HER2- MBC receiving 1L ET + any FDA-approved CDK 4/6i (palbociclib, ribociclib, or abemaciclib) had blood serum samples collected at baseline, weeks 2, 4, 6, 8, every 4 weeks up to week 24, and then every 12 weeks thereafter until disease progression. Samples were analyzed for TKa levels in real-time using the DiviTum® TKa assay. Optional repeat TKa within 2-4 wks (+/- 3 days) is permitted in case of rising TKa. Other standard monitoring assessments were carried out according to institutional guidelines and/or treating clinician discretion. Results: Pre-treatment and early on-therapy TKa levels predicted response to CDK4/6i therapy more accurately than a patient’s clinical characteristics. Rising TKa levels also identified patients with clinically occult disease progression ahead of imaging, and in some cases where radiographic interpretation and serum tumor markers such as CA15-3 were ambiguous. Measurement of TKa levels following CDK4/6i dose reduction was able to confirm continued optimal therapy response at the lower dose. A rise in TKa levels aligned with the re-emergence of ESR1 mutation as identified by ctDNA in one patient and may herald molecular progression. Conclusion: sTKa was able to predict tumor sensitivity and resistance (both intrinsic and acquired) to CDK4/6i + ET therapy in patients with HR+, HER2- MBC. We believe these findings help support the role of sTKa as a non-invasive pharmacodynamic biomarker that can assess in real-time a tumor’s response to CDK4/6i based therapy. Citation Format: Nusayba Bagegni, Amy Williams, Isabella Grigsby, Mattias Bergqvist, Katherine Clifton, Cynthia Ma. Serum thymidine kinase activity as a “real-time” clinical biomarker of tumor response to CDK4/6 inhibition in hormone receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-15-06.

The MCM2-7 Complex: Roles beyond DNA Unwinding.

The MCM2-7 complex is a hexameric protein complex that serves as a DNA helicase. It unwinds the DNA double helix during DNA replication, thereby providing the single-stranded replication template. In recent years, it has become clear that the MCM2-7 complex has additional functions that extend well beyond its role in DNA replication. Through physical and functional interactions with different pathways, it impacts other nuclear events and activities, including folding of the genome, histone inheritance, chromosome segregation, DNA damage sensing and repair, and gene transcription. Collectively, the diverse roles of the MCM2-7 complex suggest it plays a critical role in maintaining genome integrity by integrating the regulation of DNA replication with other pathways in the nucleus.

A nucleoid-associated protein is involved in the emergence of antibiotic resistance by promoting the frequent exchange of the replicative DNA polymerase in Mycobacterium smegmatis.

Unlike many other pathogens, Mycobacterium tuberculosis has limited ability for horizontal gene transfer, a major mechanism for developing antibiotic resistance. Thus, the mechanisms that facilitate chromosomal mutagenesis are of particular importance in mycobacteria. Here, we show that Lsr2, a nucleoid-associated protein, has a novel role in DNA replication and mutagenesis in the model mycobacterium Mycobacterium smegmatis. We find that Lsr2 promotes the fast exchange rate of the replicative DNA polymerase, DnaE1, at the replication fork and is important for the effective loading of the DnaE2-ImuA'-ImuB translesion complex. Without lsr2, M. smegmatis replicates its chromosome more faithfully and acquires resistance to rifampin at a lower rate, but at the cost of impaired survival to DNA damaging agents. Together, our work establishes Lsr2 as a potential factor in the emergence of mycobacterial antibiotic resistance.

Abstract 3363: LIG1 inactivation selectively inhibits growth of BRCA1 mutant cells in vitro and in vivo

Abstract Women harboring a mutation in the BRCA1 gene have a >7X increased risk of developing breast cancer and >35X increased risk of developing ovarian cancer in their lifetime compared to women lacking such mutations. While PARP1/2 inhibitors are efficacious in the BRCA mutant setting, this efficacy is limited and resistance develops readily. Additional therapeutic targets beyond PARP1/2 are needed to treat cancer patients with BRCA mutations. We performed CRISPR screens in 11 BRCA1/2 wild-type and 2 BRCA1 mutant cancer cell lines to identify targets that are synthetic lethal with BRCA1 mutations. In addition to USP1, PARP1 and POLQ, we identified the DNA ligase gene LIG1 as a novel target that when knocked out kills BRCA1 mutant cells selectively. Internal analysis of BRCA mutations in breast and ovarian cancer cell lines in the Project Achilles database further validated the hyperdependence of BRCA1 mutant cells on LIG1. Single-gene perturbation of LIG1 using CRISPRn, CRISPRi, and RNAi confirm the lethal effect of LIG1 inactivation in BRCA1 mutant cell lines, but not BRCA1/2 wild-type cell lines. This loss of viability could be rescued by complementing with an exogenous wild-type LIG1 cDNA, demonstrating the on-target nature of the genetic tools. Using a degradable DNA Ligase I fusion protein, we demonstrate a strong correlation between DNA Ligase I protein level and viability in BRCA1 mutant cells. Enzymatically inactive DNA Ligase I mutant protein (LIG1K568A) was unable to rescue the loss of viability caused by endogenous LIG1 depletion supporting the tractability of this target from a small molecule inhibitor perspective. These data were reproduced in vivo using BRCA1 mutant MDA-MB-436 derived tumors, in which tumor growth was inhibited >80% upon loss of LIG1.Mechanistically, DNA ligase I plays a critical role in DNA replication and damage repair by sealing nicks in the phosphodiester backbone of DNA. When these nicks are not repaired, they are marked by the addition of PAR chains. Consistent with this mechanism, we demonstrated that inactivation of LIG1 leads to increased PARylation. The induction of PARylation was proportional to the level of inactivation of LIG1 protein, supporting a correlation between LIG1 activity and DNA nick repair. Together, these data support DNA ligase I as a synthetic lethal target in the context of BRCA1 mutations. Citation Format: Lauren Martires, Leanne G. Ahronian, Charlotte Pratt, Nikitha Das, Xiaobin Zhang, Douglas A. Whittington, Hongxiang Zhang, Binzhang Shen, Jon Come, Patrick McCarren, Mu-Sen Liu, Chengyin Min, Tianshu Feng, Haris Jahic, Eric Brophy, Daniel Aird, Jannik N. Andersen, Alan Huang, Fang Li, William D. Mallender, Hilary E. Nicholson. LIG1 inactivation selectively inhibits growth of BRCA1 mutant cells in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3363.