Role In DNA Replication Research Articles

P-056: HUWE1 orchestrates DNA Repair in response to Replicative Stress in Multiple Myeloma

P-056: HUWE1 orchestrates DNA Repair in response to Replicative Stress in Multiple Myeloma

Topoisomerase Assays.

Topoisomerases are enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of DNA topoisomerases: type I enzymes, which make single-stranded cuts in DNA, and type II enzymes, which cut and decatenate double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. Provided in this article are protocols to assess activities of topoisomerases and their inhibitors. Included are an assay for topoisomerase I activity based on relaxation of supercoiled DNA; an assay for topoisomerase II based on the decatenation of double-stranded DNA; and approaches for enriching and quantifying DNA-protein covalent complexes formed as obligatory intermediates in the reactions of type I and II topoisomerases with DNA; and assays for measuring DNA cleavage in vitro. Topoisomerases are not the only proteins that form covalent adducts with DNA in living cells, and the approaches described here are likely to find use in characterizing other protein-DNA adducts and exploring their utility as targets for therapy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Assay of topoisomerase I activity Basic Protocol 2: Assay of topoisomerase II activity Basic Protocol 3: In vivo determination of topoisomerase covalent complexes using the in vivo complex of enzyme (ICE) assay Support Protocol 1: Preparation of mouse tissue for determination of topoisomerase covalent complexes using the ICE assay Support Protocol 2: Using recombinant topoisomerase standard for absolute quantification of cellular TOP2CC Basic Protocol 4: Quantification of topoisomerase-DNA covalent complexes by RADAR/ELISA: The rapid approach to DNA adduct recovery (RADAR) combined with the enzyme-linked immunosorbent assay (ELISA) Basic Protocol 5: Analysis of protein-DNA covalent complexes by RADAR/Western Support Protocol 3: Adduct-Seq to characterize adducted DNA Support Protocol 4: Nuclear fractionation and RNase treatment to reduce sample complexity Basic Protocol 6: Determination of DNA cleavage by purified topoisomerase I Basic Protocol 7: Determination of inhibitor effects on DNA cleavage by topoisomerase II using a plasmid linearization assay Alternate Protocol: Gel electrophoresis determination of topoisomerase II cleavage.

Variations in Nucleosome Structure and Organization among Eukaryotes

Folding eukaryotic DNA by chromatin is a vital process necessary for the proper function of DNA. This is achieved by the fundamental unit of chromatin, known as a nucleosome. The position of a nucleosome and its interaction with DNA plays a crucial role in regulating the vital processes involved in DNA function. Factors such as variations in nucleosome and its core structure and histone fold variations will help to understand nucleosome functions and their role in DNA replication, transcription, translation, posttranslational modifications, re-combinations and repair. The present review focuses on recent findings in understanding the variations in the structure and functions of nucleosomes across eukaryotes. Variations in the nucleosome organization and its assembly have also been discussed by stating the contribution of histone binding factors and chromatin assembly factors.

Single-Molecule Insights Into the Dynamics of Replicative Helicases.

Helicases are molecular motors that translocate along single-stranded DNA and unwind duplex DNA. They rely on the consumption of chemical energy from nucleotide hydrolysis to drive their translocation. Specialized helicases play a critically important role in DNA replication by unwinding DNA at the front of the replication fork. The replicative helicases of the model systems bacteriophages T4 and T7, Escherichia coli and Saccharomyces cerevisiae have been extensively studied and characterized using biochemical methods. While powerful, their averaging over ensembles of molecules and reactions makes it challenging to uncover information related to intermediate states in the unwinding process and the dynamic helicase interactions within the replisome. Here, we describe single-molecule methods that have been developed in the last few decades and discuss the new details that these methods have revealed about replicative helicases. Applying methods such as FRET and optical and magnetic tweezers to individual helicases have made it possible to access the mechanistic aspects of unwinding. It is from these methods that we understand that the replicative helicases studied so far actively translocate and then passively unwind DNA, and that these hexameric enzymes must efficiently coordinate the stepping action of their subunits to achieve unwinding, where the size of each step is prone to variation. Single-molecule fluorescence microscopy methods have made it possible to visualize replicative helicases acting at replication forks and quantify their dynamics using multi-color colocalization, FRAP and FLIP. These fluorescence methods have made it possible to visualize helicases in replication initiation and dissect this intricate protein-assembly process. In a similar manner, single-molecule visualization of fluorescent replicative helicases acting in replication identified that, in contrast to the replicative polymerases, the helicase does not exchange. Instead, the replicative helicase acts as the stable component that serves to anchor the other replication factors to the replisome.

Abstract 1970: Preclinical candidate TQB3824, a small molecule inhibitor of CDC7, shows strong antitumor efficacy in colorectal and pancreatic tumor models

Abstract Objective: Cell division cycle 7 (CDC7), a receptor serine/threonine kinase which features high-affinity with protein DBF4, plays an important role in DNA replication and DNA damage response via MCM2 phosphorylation. CDC7 has been reported to be up-regulated by about 50% in various human cancers and is being investigated as a therapeutic target in clinical trials. The purpose of this study is to investigate the in-vitro and in-vivo antitumor activities of TQB3824, a small molecule of CDC7 kinase inhibitor, in preclinical models with CDC7 overexpression. Method: Kinase inhibition activities of TQB3824 were determined with CDC7/DBF4 kinase assays. Cellular anti-proliferative activities were evaluated in COLO205 cell line and NCI-H226 cell line, which features CDC7 overexpression and no CDC7 expression, respectively. Three CDX models, colorectal cancer COLO205/SW620 and pancreatic cancer CAPAN-1 with KRAS mutation, were used to test the efficacy of TQB-3824 in vivo. Result: TQB3824 showed potent activities in both kinase inhibition and COLO205 cell anti-proliferation, with IC50 of 2.9 nM and 14.5 nM, separately. In contrast, TQB3824 showed much weaker anti-proliferative activity in NCI H226 cells with IC50 of 8154 nM. TQB3824 displayed strong antitumor efficacy in CDC7 highly expressed colorectal cancer CDX model COLO205 (TGI = 77% @5 mpk, BID), SW620 CDX model (TGI = 100% @5 mpk, BID), and pancreatic cancer CAPAN-1 CDX model (TGI = 115% @6 mpk, BID). Conclusion: We have identified a novel potent CDC7 inhibitor TQB3824, which shows high antitumor efficacy in CDC7 overexpressed solid tumor models. TQB3824 represents a promising clinical candidate for treating solid tumors with high CDC7 expression. Citation Format: Gang Li, Xiquan Zhang, Ling Yang, Xin Tian, Zhong xiong, Lihong Hu, Yuanfeng xia, Chi-chung chan, Charles Z. Ding, Shuhui Chen. Preclinical candidate TQB3824, a small molecule inhibitor of CDC7, shows strong antitumor efficacy in colorectal and pancreatic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1970.

Set2-mediated H3K36 methylation states redundantly repress the production of antisense transcripts: role in transcription regulation.

Methyltransferase Set2‐mediated methylation of histone H3 lysine 36 (H3K36), which involves the addition of up to three methyl groups at this site, has been demonstrated to function in many chromatin‐coupled events. The methylation of H3K36 is known to recruit different chromatin effector proteins, affecting transcription, mRNA splicing and DNA repair. In this study, we engineered two yeast set2 mutants that lack H3K36 mono/dimethylation (H3K36me1/2) and trimethylation (H3K36me3), respectively, and characterized their roles in the production of antisense transcripts under nutrient‐rich conditions. Using our new bioinformatics identification pipeline analysis, we are able to identify a larger number of antisense transcripts in set2∆ cells than has been published previously. We further show that H3K36me1/2 or H3K36me3 redundantly repressed the production of antisense transcripts. Moreover, gene ontology (GO) analysis implies that H3K36me3‐mediated antisense transcription might play a role in DNA replication and DNA damage repair, which is independent of regulation of the corresponding sense gene expression. Overall, our results validate a coregulatory mechanism of different H3K36 methylation states, particularly in the repression of antisense transcription.

A CDC7 inhibitor sensitizes DNA-damaging chemotherapies by suppressing homologous recombination repair to delay DNA damage recovery.

Cell division cycle 7 (CDC7), a serine/threonine kinase, plays important roles in DNA replication. We developed a highly specific CDC7 inhibitor, TAK-931, as a clinical cancer therapeutic agent. This study aimed to identify the potential combination partners of TAK-931 for guiding its clinical development strategies. Unbiased high-throughput chemical screening revealed that the highest synergistic antiproliferative effects observed were the combinations of DNA-damaging agents with TAK-931. Functional phosphoproteomic analysis demonstrated that TAK-931 suppressed homologous recombination repair activity, delayed recovery from double-strand breaks, and led to accumulation of DNA damages in the combination. Whole-genome small interfering RNA library screening identified sensitivity-modulating molecules, which propose the experimentally predicted target cancer types for the combination, including pancreatic, esophageal, ovarian, and breast cancers. The efficacy of combination therapy in these cancer types was preclinically confirmed in the corresponding primary-derived xenograft models. Thus, our findings would be helpful to guide the future clinical strategies for TAK-931.

The coordination between DNA polymerase β and ligase governs the formation of mutagenic repair intermediates as an important determinant of faithful base excision repair

Base excision repair (BER) is a critical process for preventing the mutagenic and lethal consequences of DNA damage that arises from endogenous and environmental agents and underlies disease and aging. BER pathway involves a series of sequential enzymatic steps that are tightly coordinated in a process known as the substrate-product channeling between repair proteins so that release of cytotoxic BER intermediates is minimized. DNA ligase I and III catalyze the ultimate ligation step following DNA polymerase (pol) β nucleotide insertion at downstream steps of BER pathway. DNA ligases and DNA polymerases that play roles in DNA replication and repair transactions have long been prime candidates for key regulators of genome stability. However, the ligation efficiency of pol β-promoted mutagenesis products with 3′-DNA mismatches or 8-oxo-2'-deoxyguanosine (8-oxodG) at the downstream steps of BER pathway remains undefined. We show that pol β 8-oxo-2'-deoxyribonucleoside 5'-triphosphate (8-oxodGTP) insertion confounds DNA ligases leading to the formation of ligation failure products with a 5'-adenylate (AMP) block. However, pol β Watson-Crick-like dGTP mismatch insertion opposite T can be efficiently ligated by the BER DNA ligases in vitro. Moreover, we report the mutagenic ligation of pol β 8-oxodGTP insertion products by the LIG1 MgHiFimutant with a perturbed fidelity and enhanced ligation failure by the variants of LIG1 deficiency disease that exhibits the symptoms of developmental abnormalities, immunodeficiency, lymphoma, and predisposition to cancer. Our results also demonstrate that the BER DNA ligases are compromised by subtle changes in all 12 possible noncanonical base pairs at the 3′-end of the nicked repair intermediate. Finally, we found that Aprataxin and Flap Endonuclease 1, as compensatory DNA-end processing enzymes, can remove the 5'-AMP block from the abortive ligation products harboring 3′-8-oxodG or all possible mispairs. These findings contribute to understanding of how the identity of the damaged or mismatch affects the substrate channeling of the repair pathway, importance of mechanism underlying the interplay between pol β and DNA ligases for faithful BER, and how a multi-protein complex can coordinate to maintain the repair efficiency.

Folding and persistence times of intramolecular G-quadruplexes transiently embedded in a DNA duplex.

G-quadruplex (G4) DNA structures have emerged as important regulatory elements during DNA metabolic transactions. While many in vitro studies have focused on the kinetics of G4 formation within DNA single-strands, G4 are found in vivo in double-stranded DNA regions, where their formation is challenged by the complementary strand. Since the energy of hybridization of Watson-Crick structures dominates the energy of G4 folding, this competition should play a critical role on G4 persistence. To address this, we designed a single-molecule assay allowing to measure G4 folding and persistence times in the presence of the complementary strand. We quantified both folding and unfolding rates of biologically relevant G4 sequences, such as the cMYC and cKIT oncogene promoters, human telomeres and an avian replication origin. We confirmed that G4s are found much more stable in tested replication origin and promoters than in human telomere repeats. In addition, we characterized how G4 dynamics was affected by G4 ligands and showed that both folding rate and persistence time increased. Our assay opens new perspectives for the measurement of G4 dynamics in double-stranded DNA mimicking a replication fork, which is important to understand their role in DNA replication and gene regulation at a mechanistic level.

RRM2B Is Frequently Amplified Across Multiple Tumor Types: Implications for DNA Repair, Cellular Survival, and Cancer Therapy.

RRM2B plays a crucial role in DNA replication, repair and oxidative stress. While germline RRM2B mutations have been implicated in mitochondrial disorders, its relevance to cancer has not been established. Here, using TCGA studies, we investigated RRM2B alterations in cancer. We found that RRM2B is highly amplified in multiple tumor types, particularly in MYC-amplified tumors, and is associated with increased RRM2B mRNA expression. We also observed that the chromosomal region 8q22.3–8q24, is amplified in multiple tumors, and includes RRM2B, MYC along with several other cancer-associated genes. An analysis of genes within this 8q-amplicon showed that cancers that have both RRM2B-amplified along with MYC have a distinct pattern of amplification compared to cancers that are unaltered or those that have amplifications in RRM2B or MYC only. Investigation of curated biological interactions revealed that gene products of the amplified 8q22.3–8q24 region have important roles in DNA repair, DNA damage response, oxygen sensing, and apoptosis pathways and interact functionally. Notably, RRM2B-amplified cancers are characterized by mutation signatures of defective DNA repair and oxidative stress, and at least RRM2B-amplified breast cancers are associated with poor clinical outcome. These data suggest alterations in RR2MB and possibly the interacting 8q-proteins could have a profound effect on regulatory pathways such as DNA repair and cellular survival, highlighting therapeutic opportunities in these cancers.

The Genome Stability Maintenance DNA Helicase DDX11 and Its Role in Cancer.

DDX11/ChlR1 is a super-family two iron–sulfur cluster containing DNA helicase with roles in DNA replication and sister chromatid cohesion establishment, and general chromosome architecture. Bi-allelic mutations of the DDX11 gene cause a rare hereditary disease, named Warsaw breakage syndrome, characterized by a complex spectrum of clinical manifestations (pre- and post-natal growth defects, microcephaly, intellectual disability, heart anomalies and sister chromatid cohesion loss at cellular level) in accordance with the multifaceted, not yet fully understood, physiological functions of this DNA helicase. In the last few years, a possible role of DDX11 in the onset and progression of many cancers is emerging. Herein we summarize the results of recent studies, carried out either in tumoral cell lines or in xenograft cancer mouse models, suggesting that DDX11 may have an oncogenic role. The potential of DDX11 DNA helicase as a pharmacological target for novel anti-cancer therapeutic interventions, as inferred from these latest developments, is also discussed.

Differential Regulation of Cancer Progression by CDK4/6 Plays a Central Role in DNA Replication and Repair Pathways.

Although the cyclin-dependent kinases CDK4 and CDK6 play fundamental roles in cancer, the specific pathways and downstream targets by which they exert their tumorigenic effects remain elusive. In this study, we uncover distinct and novel functions for these kinases in regulating tumor formation and metastatic colonization in various solid tumors, including those of the breast, prostate, and pancreas. Combining in vivo CRISPR-based CDK4 and CDK6 gene editing with pharmacologic inhibition approaches in orthotopic transplantation and patient-derived xenograft preclinical models, we defined clear functions for CDK4 and CDK6 in facilitating tumor growth and progression in metastatic cancers. Transcriptomic profiling of CDK4/6 CRISPR knockouts in breast cancer revealed these two kinases to regulate cancer progression through distinct mechanisms. CDK4 regulated prometastatic inflammatory cytokine signaling, whereas CDK6 mainly controlled DNA replication and repair processes. Inhibition of CDK6 but not CDK4 resulted in defective DNA repair and increased DNA damage. Multiple CDK6 DNA replication/repair genes were not only associated with cancer subtype, grades, and poor clinical outcomes, but also facilitated primary tumor growth and metastasis in vivo. CRISPR-based genomic deletion of CDK6 efficiently blocked tumor formation and progression in preestablished cell- and patient-derived xenograft preclinical models of breast cancer, providing a potential novel targeted therapy for these deadly tumors. SIGNIFICANCE: In-depth transcriptomic analysis identifies cyclin-dependent kinases CDK4 and CDK6 as regulators of metastasis through distinct signaling pathways and reveals the DNA replication/repair pathway as central in promoting these effects.

Abstract PS4-31: Characteristics of HER2/neu positive breast cancer among patients with and without germline BRCAmutations

Abstract Introduction: Breast cancer in BRCA 1/2 carriers is a well-characterized disease process, and its association with triple negative breast cancer has been extensively studied. In contrast, there is limited data on BRCA carriers and HER2/neu positive breast cancer, which identifies this topic as an area for further investigation. The BRCA protein plays an important role in DNA replication, whereas HER2/neu is a proto-oncogene that, when overexpressed, correlates with aggressive cancers. The relationship between these two mutations is poorly understood. There have been major strides with anti-HER2/neu therapies for HER2/neu positive disease and PARP inhibitors for BRCA germline positive patients, however there is a clinical knowledge gap in BRCA carriers who also have HER2/neu positive breast cancer. Further research into the characteristics of this population, treatment strategies, and outcomes is needed. More specifically, determining the utility of anti-HER2/neu therapies, PARP inhibitors, or the combination in this population could prove to have clinical utility. We aim to characterize the clinical characteristics, treatment strategies, and outcomes within this population. Methods: Using a prospective research patient database at the UT MD Anderson Cancer Center 1038 patients were identified to have HER2/neu associated breast cancer who had undergone genetic counseling and testing for hereditary breast cancer syndromes between 1996 and 2019. This population was descriptively characterized to evaluate the prevalence of BRCA 1/2 positivity. Clinical and pathological characteristics as well as treatment, and recurrence were collected. Chi-square was used for statistical analysis. The Kaplan-Meier method was used to compare overall survival based on BRCA status. Results: Amongst 1038 patients, germline BRCA mutation was detected in 49 (4.7%) of the patients. The median age at diagnosis for patients with BRCA mutations was significantly younger, 41.7 years (27-76), compared to 44.9 years (21-83) in the BRCA negative group (p= 0.0147). More patients were premenopausal in the BRCA positive group (p=0.027). Further characteristics are shown in Table 1. Overall survival was 71.6 months for the BRCA1 population, 81.3 months for the BRCA2 population, and 70.7 months for the BRCA negative population (p = 0.63). Recurrence free survival was 71 months for the BRCA1, 51.2 months for the BRCA2, and 59.6 months for the BRCA negative cohort (p = 0.4). Conclusion: HER2/neu positive breast cancer patients with and without germline BRCA mutations have different clinical presentations. While we did not detect a survival difference due to small number of patients, this presents an opportunity to evaluate whether new treatment strategies, such as combining anti-HER2/neu therapies with PARP inhibitors can further improve outcomes for these patients. Table 1:BRCA negativeBRCA positivep valuen%n%RaceASIAN666.724.10.0122BLACK979.8510.2HISPANIC16716.936.1NATIVE AMERICAN50.500.0OTHER111.148.2WHITE64365.03571.4Menopause Status at DxN/A40.400.00.0268POST17818.036.1PRE53454.03265.3Unknown27327.61428.6Age at Dxavg (range)44.9 (21-83)41.7 (27-76)0.0147Histology Typeductal93694.64591.80.3741lobular181.812.0mixed262.636.1other90.900.0StageI23523.81632.70.3293II46747.22040.8III28228.51224.5Unknown50.512.0Hormone Receptor StatusER and/or PR positive62663.33061.20.7271ER/PR negative35736.11938.8Unknown60.600.0Nuclear GradeI90.912.00.3474II22222.4714.3III69370.13673.5Unknown656.6510.2Ki67avg (range)40.5 (0-99)46 (10-90)0.1858Chemotypeadj32733.11632.70.2932neoadj56757.32551.0no chemo959.6816.3Herceptin Trxadj27027.31428.60.4093neoadj50651.22142.9none21121.31428.6unknown20.200.0Adj HormoneN44244.72449.00.5558Y54755.32551.0XRTN38038.42244.90.3637Y60961.62755.1989100.049100.0 Citation Format: Mihir A Shukla, Angelica Gutierrez Barrera, Stephen Karl Gruschkus, Rachel M Layman, Rashmi K Murthy, Banu K Arun. Characteristics of HER2/neu positive breast cancer among patients with and without germline BRCAmutations [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-31.

Prognostic implications of cell division cycle protein 45 expression in hepatocellular carcinoma.

BackgroundThe overall prognosis of hepatocellular carcinoma (HCC) is poor and novel prognostic biomarkers might better monitor the progression of HCC. Cell division cycle protein 45 (CDC45) plays a key role in DNA replication and considered to be involved in tumorigenesis. This study investigated CDC45 expression in tumour tissues and defined its prognostic value in HCC patients.MethodsWe used immunohistochemistry (IHC) staining to examine the expression of CDC45 in tumour tissue specimens and compare them with adjacent normal tissue specimens using a constructed tissue microarray (TMA) and analyzed how clinical features are related to HCC prognosis. Functional enrichment analyses were used to describe significantly involved hallmark pathways of differentially expressed genes (DEGs, which were screened out according to the high or low expression of CDC45 in tumour tissues).ResultsOur findings showed that the proteome expression of CDC45 was evidently downregulated in HCC tissues compared with matched normal tissues (P < 0.0001). Although we did not find any differences in terms of vascular invasion, metastasis, lymphatic infiltration, or Edmondson grade between patients with high and low CDC45 expression, low CDC45 expression was significantly correlated with microvascular invasion (P = 0.046). Multivariate analysis indicated that CDC45 expression (P = 0.035) was an independent prognostic factor for the overall survival (OS) rate of HCC patients. Patients with CDC45 expression was positively correlated with OS rates among HCC patients (P < 0.05). Functional annotations indicated that CDC45 is involved in the most significant pathways, including the cell cycle, DNA replication, chemical carcinogenesis and drug metabolism–cytochrome P450 pathways.DiscussionOur findings showed that low proteomic level of CDC45 was associated with a poor prognosis in HCC patients, indicating that CDC45 might be a novel prognostic marker.

Role of Amygdalin in Blocking DNA Replication in Breast Cancer In Vitro.

Amygdalin has anticancer benefits because of its active component, hydrocyanic acid. However, the underlying molecular mechanism is unclear. This study aimed to investigate the molecular mechanism by which amygdalin exerts antiproliferative effects in the human Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line. MCF-7 cells were exposed to amygdalin at a particular IC50 value for 24 and 48 hours and compared to non-treated cells. An Affymetrix whole-transcript expression array was used to analyze the expression of 32 genes related to DNA replication. Among the 32 genes, amygdalin downregulated the expression of 16 genes and 19 genes by >1.5-fold at 24 and 48 hours, respectively. At 24 hours, the downregulated genes from the DNA polymerase α-primase complex were POLA1, POLA2, PRIM1, and PRIM2; DNA polymerase δ complex: POLD3; DNA polymerase ε complex: POLE4, Minichromosome Maintenance protein (MCM) complex (helicase): MCM2, MCM3, MCM4, MCM6, and MCM7; clamp and clamp loader: PCNA; nuclease: FEN1; and DNA ligase: LIG1. At 48 hours, the downregulated genes from the DNA polymerase α-primase complex were POLA1, POLA2, and PRIM1; DNA polymerase δ complex: POLD3; DNA polymerase ε complex: POLE and POLE2; MCM complex (helicase): MCM2, MCM3, MCM4, MCM5, MCM6, and MCM7; clamp and clamp loader: PCNA, RFC2, and RFC3; RNase H: RNASEH2A; nucleases: DNA2 and FEN1; and DNA ligase: LIG1. Amygdalin treatment caused downregulation of several genes that play critical roles in DNA replication in the MCF-7 cell line. Thus, it might be useful as an anticancer agent.

ClpX Is Essential and Activated by Single-Strand DNA Binding Protein in Mycobacteria.

The ClpP1P2 proteolytic complex is essential in Mycobacterium tuberculosis Proteolysis by ClpP1P2 requires an associated ATPase, either ClpX or ClpC1. Here, we sought to define the unique contributions of the ClpX ATPase to mycobacterial growth. We formally demonstrated that ClpX is essential for mycobacterial growth, and to understand its essential functions, we identified ClpX-His-interacting proteins by pulldown and tandem mass spectrometry. We found an unexpected association between ClpX and proteins involved in DNA replication, and we confirm a physical association between ClpX and the essential DNA maintenance protein single-stranded-DNA binding protein (SSB). Purified SSB is not degraded by ClpXP1P2; instead, SSB enhances ATP hydrolysis by ClpX and degradation of the model substrate GFP-SsrA by ClpXP1P2. This activation of ClpX is mediated by the C-terminal tail of SSB, which had been implicated in the activation of other ATPases associated with DNA replication. Consistent with the predicted interactions, depletion of clpX transcript perturbs DNA replication. These data reveal that ClpX participates in DNA replication and identify the first activator of ClpX in mycobacteria.IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis, imposes a major global health burden, surpassing HIV and malaria in annual deaths. The ClpP1P2 proteolytic complex and its cofactor ClpX are attractive drug targets, but their precise cellular functions are unclear. This work confirms ClpX's essentiality and describes a novel interaction between ClpX and SSB, a component of the DNA replication machinery. Further, we demonstrate that a loss of ClpX is sufficient to interrupt DNA replication, suggesting that the ClpX-SSB complex may play a role in DNA replication in mycobacteria.

Unraveling the characterization of minichromosome maintenance complex component 2 (MCM2) gene and its SNPs associated with cold-tolerance trait in Pacific white shrimp (Litopenaeus vannamei)

Minichromosome maintenance complex component 2 (MCM2) plays an important role in DNA replication induced by cold stress. In this study, MCM2 cDNA and genomic DNA (gDNA) sequences were isolated from Pacific white shrimp (Litopenaeus vannamei), and the gene was designated Lv-MCM2. The full-length Lv-MCM2 cDNA was 3,138 bp in length, with an open reading frame (ORF) of 2,721 bp encoding 906 amino acids. The Lv-MCM2 gDNA sequence was 7,322 bp in length and consisted of 16 coding segments in the L. vannamei genome. The expression of Lv-MCM2 mRNA was detected in all selected tissues, with the highest levels being observed in the stomach and gills of the shrimp. The transcript levels of Lv-MCM2 in the stomach and gills were found to be significantly affected by low-temperature, high-pH, low-pH, poly(I:C), and lipopolysaccharide (LPS) stimuli, indicating that Lv-MCM2 might play roles in coping with various stresses. Specifically, under low-temperature stress (13 °C), the knockdown of Lv-MCM2 with siRNA significantly decreased the shrimp survival rate, indicating its key roles in the response to low-temperature stress. Additionally, four single-nucleotide polymorphisms (SNPs) in the Lv-MCM2 gene were identified as being significantly associated (P < 0.001) with the cold tolerance trait in a full-sib family of L. vannamei. Our study suggests that the Lv-MCM2 gene itself or closely linked genes might play important roles in the cold tolerance trait in L. vannamei, and the SNPs from the Lv-MCM2 gene provide a basis for developing molecular markers for breeding cold-tolerant Pacific white shrimp seeds in the future.

STAT3 plays an important role in DNA replication by turning on WDHD1

BackgroundSignal transducers and activators of transcription 3 (STAT3) is a transcription factor that plays a key role in many cellular processes such as cell growth and cancer. However, the functions and mechanisms by which STAT3 regulates cellular processes are not fully understood.ResultsHere we describe a novel function of STAT3. We demonstrated that STAT3 plays an important role in DNA replication. Specifically, knockdown of STAT3 reduced DNA replication while activation and ectopic expression of STAT3 promoted DNA replication. We further identified the WD repeat and HMG-box DNA-binding protein 1 (WDHD1), which plays an important role in DNA replication initiation, as a novel STAT3 target gene that mediated the DNA replication function of STAT3. We showed that STAT3 bind the promoter/up regulatory region of WDHD1 gene.ConclusionsThese studies identified a novel function of STAT3 that is mediated by its newly identified target gene WDHD1 and have important implications.

A comparative study of protein-ssDNA interactions.

Single-stranded DNA-binding proteins (SSBs) play crucial roles in DNA replication, recombination and repair, and serve as key players in the maintenance of genomic stability. While a number of SSBs bind single-stranded DNA (ssDNA) non-specifically, the others recognize and bind specific ssDNA sequences. The mechanisms underlying this binding discrepancy, however, are largely unknown. Here, we present a comparative study of protein–ssDNA interactions by annotating specific and non-specific SSBs and comparing structural features such as DNA-binding propensities and secondary structure types of residues in SSB–ssDNA interactions, protein–ssDNA hydrogen bonding and π–π interactions between specific and non-specific SSBs. Our results suggest that protein side chain-DNA base hydrogen bonds are the major contributors to protein–ssDNA binding specificity, while π–π interactions may mainly contribute to binding affinity. We also found the enrichment of aspartate in the specific SSBs, a key feature in specific protein–double-stranded DNA (dsDNA) interactions as reported in our previous study. In addition, no significant differences between specific and non-specific groups with respect of conformational changes upon ssDNA binding were found, suggesting that the flexibility of SSBs plays a lesser role than that of dsDNA-binding proteins in conferring binding specificity.

Characterization of the T4 gp32-ssDNA complex by native, cross-linking, and ultraviolet photodissociation mass spectrometry.

Protein–DNA interactions play crucial roles in DNA replication across all living organisms. Here, we apply a suite of mass spectrometry (MS) tools to characterize a protein-ssDNA complex, T4 gp32·ssDNA, with results that both support previous studies and simultaneously uncover novel insight into this non-covalent biological complex. Native mass spectrometry of the protein reveals the co-occurrence of Zn-bound monomers and homodimers, while addition of differing lengths of ssDNA generates a variety of protein:ssDNA complex stoichiometries (1 : 1, 2 : 1, 3 : 1), indicating sequential association of gp32 monomers with ssDNA. Ultraviolet photodissociation (UVPD) mass spectrometry allows characterization of the binding site of the ssDNA within the protein monomer via analysis of holo ions, i.e. ssDNA-containing protein fragments, enabling interrogation of disordered regions of the protein which are inaccessible via traditional crystallographic techniques. Finally, two complementary cross-linking (XL) approaches, bottom-up analysis of the crosslinked complexes as well as MS1 analysis of the intact complexes, are used to showcase the absence of ssDNA binding with the intact cross-linked homodimer and to generate two homodimer gp32 model structures which highlight that the homodimer interface overlaps with the monomer ssDNA-binding site. These models suggest that the homodimer may function in a regulatory capacity by controlling the extent of ssDNA binding of the protein monomer. In sum, this work underscores the utility of a multi-faceted mass spectrometry approach for detailed investigation of non-covalent protein-DNA complexes.