Characterization of the Role of Acetylated APE1 in DNA Damage Repair and Transcriptional Regulation

Apurinic/apyrimidinic (AP) sites are the most frequently formed DNA lesions in the genome. The primary enzyme to repair AP sites in mammalian cells is the AP endonuclease (APE1), which functions through the base excision repair (BER) pathway. Mammalian APE1 has a unique N-terminal unstructured tail and has both DNA repair and transcriptional regulatory activities. Our lab discovered that APE1 can be regulated via post-translational acetylation of lysine residues 6, 7, 27, 31, and 32. The role of mammalian APE1 in repair has been extensively studied and well characterized. However, the regulatory role of APE1 acetylation (AcAPE1) in the context of both DNA damage repair and transcriptional regulation has not been elucidated. We show that APE1 is acetylated after binding to the AP sites in chromatin and that AcAPE1 is exclusively present on chromatin throughout the cell cycle. Positive charges of acetylable Lysine residues in the N-terminal domain of APE1 are essential for chromatin association. Acetylation-mediated neutralization of positive charges of Lysine residues in the N-terminal domain of APE1 induces a conformation change; this, in turn, enhances the AP-endonuclease activity of APE1. In the absence of APE1 acetylation, cells accumulated AP sites in the genome and showed higher sensitivity to DNA-damaging agents. Our study reveals that APE1 acetylation is an integral part of the BER pathway for maintaining genomic integrity. By mapping genome-wide occurrence of endogenous AP site damages and binding of repair proteins APE1 and AcAPE1, we demonstrate that oxidative base damages predominantly occur in transcriptionally active regions, particularly G-quadruplex (G4) sequences and activation of APE1-mediated BER pathway promotes the formation of G4 structures in the genome. Loss of APE1 or its acetylation abrogates the formation of G4 structures in cells. Acetylation of APE1 enhances its residence time and facilitates transcription factor loading, providing mechanistic insight into the role of APE1 in G4-mediated gene expression. Our study unravels an acquired function of endogenous base damage and AcAPE1-mediated BER in regulating transcription. Together this study highlights role of AcAPE1 in coordinating potential functional overlap between DNA damage repair activity and transcriptional regulation

Red Panda: A Novel Method for Detecting Variants in Single-Cell RNA Sequencing

BACKGROUND: Single-cell sequencing enables us to better understand genetic diseases, such as cancer or autoimmune disorders, which are often affected by changes in rare cells. Currently, no existing software is aimed at identifying single nucleotide variations or micro (1-50 bp) insertions and deletions in single-cell RNA sequencing (scRNA-seq) data. Generating high-quality variant data is vital to the study of the aforementioned diseases, among others. RESULTS: In this study, we report the design and implementation of Red Panda, a novel method to accurately identify variants in scRNA-seq data. Variants were called on scRNA-seq data from human articular chondrocytes, mouse embryonic fibroblasts (MEFs), and simulated data stemming from the MEF alignments. Red Panda had the highest Positive Predictive Value at 45.0%, while other tools-FreeBayes, GATK HaplotypeCaller, GATK UnifiedGenotyper, Monovar, and Platypus-ranged from 5.8-41.53%. From the simulated data, Red Panda had the highest sensitivity at 72.44%. CONCLUSIONS: We show that our method provides a novel and improved mechanism to identify variants in scRNA-seq as compared to currently existing software. However, methods for identification of genomic variants using scRNA-seq data can be still improved

Distinct distribution pattern of hepatitis B virus genotype C and D in liver tissue and serum of dual genotype infected liver cirrhosis and hepatocellular carcinoma patients.

The impact of co-infection of several hepatitis B virus (HBV) genotypes on the clinical outcome remains controversial. This study has for the first time investigated the distribution of HBV genotypes in the serum and in the intrahepatic tissue of liver cirrhotic (LC) and hepatocellular carcinoma (HCC) patients from India. In addition, the genotype-genotype interplay and plausible mechanism of development of HCC has also been explored.The assessment of HBV genotypes was performed by nested PCR using either surface or HBx specific primers from both the circulating virus in the serum and replicative virus that includes covalently closed circular DNA (cccDNA) and relaxed circular DNA (rcDNA) of HBV from the intrahepatic tissue. The integrated virus within the host chromosome was genotyped by Alu-PCR method. Each PCR products were cloned and sequences of five randomly selected clones were subsequently analysed.HBV/genotype D was detected in the serum of all LC and HCC patients whereas the sequences of the replicative HBV DNA (cccDNA and rcDNA) from the intrahepatic tissue of the same patients revealed the presence of both HBV/genotype C and D. The sequences of the integrated viruses exhibited the solo presence of HBV/genotype C in the majority of LC and HCC tissues while both HBV/genotype C and D clones were found in few patients in which HBV/genotype C was predominated. Moreover, compared to HBV/genotype D, genotype C had higher propensity to generate double strand breaks, ER stress and reactive oxygen species and it had also showed higher cellular homologous-recombination efficiency that engendered more chromosomal rearrangements, which ultimately led to development of HCC.Our study highlights the necessity of routine analysis of HBV genotype from the liver tissue of each chronic HBV infected patient in clinical practice to understand the disease prognosis and also to select therapeutic strategy

Targeting MCL-1 triggers DNA damage and an anti-proliferative response independent from apoptosis induction

Summary: MCL-1 is a high-priority target due to its dominant role in the pathogenesis and chemoresistance of cancer, yet clinical trials of MCL-1 inhibitors are revealing toxic side effects. MCL-1 biology is complex, extending beyond apoptotic regulation and confounded by its multiple isoforms, its domains of unresolved structure and function, and challenges in distinguishing noncanonical activities from the apoptotic response. We find that, in the presence or absence of an intact mitochondrial apoptotic pathway, genetic deletion or pharmacologic targeting of MCL-1 induces DNA damage and retards cell proliferation. Indeed, the cancer cell susceptibility profile of MCL-1 inhibitors better matches that of anti-proliferative than pro-apoptotic drugs, expanding their potential therapeutic applications, including synergistic combinations, but heightening therapeutic window concerns. Proteomic profiling provides a resource for mechanistic dissection and reveals the minichromosome maintenance DNA helicase as an interacting nuclear protein complex that links MCL-1 to the regulation of DNA integrity and cell-cycle progression

Novel Point and Combo-Mutations in the Genome of Hepatitis B Virus-Genotype D: Characterization and Impact on Liver Disease Progression to Hepatocellular Carcinoma

<div><p>Background</p><p>The contribution of chronic hepatitis B virus (HBV) infection in the pathogenesis of hepatocellular carcinoma (HCC) through progressive stages of liver fibrosis is exacerbated by the acquisition of naturally occurring mutations in its genome. This study has investigated the prevalence of single and combo mutations in the genome of HBV-genotype D from treatment naïve Indian patients of progressive liver disease stages and assessed their impact on the disease progression to HCC.</p><p>Methods</p><p>The mutation profile was determined from the sequence analysis of the full-length HBV genome and compared with the reference HBV sequences. SPSS 16.0 and R software were used to delineate their statistical significance in predicting HCC occurrence.</p><p>Results</p><p>Age was identified as associated risk factor for HCC development in chronic hepatitis B (CHB) patients (p≤0.01). Beyond the classical mutations in basal core promoter (BCP) (A1762T/G1764A) and precore (G1862T), persistence of progressively accumulated mutations in enhancer-I, surface, HBx and core were showed significant association to liver disease progression. BCP_T1753C, core_T147C, surface_L213I had contributed significantly in the disease progression to HCC (p<0.05) in HBeAg positive patients whereas precore_T1858C, core_I116L, core_P130Q and preS1_S98T in HBeAg negative patients. Furthermore, the effect of individual mutation was magnified by the combination with A1762T/G1764A in HCC pathogenesis. Multivariate risk analysis had confirmed that core_P130Q [OR 20.71, 95% CI (1.64–261.77), p = 0.019] in B cell epitope and core_T147C [OR 14.58, 95% CI (1.17–181.76), p = 0.037] in CTL epitope were two independent predictors of HCC in HBeAg positive and negative patients respectively.</p><p>Conclusions</p><p>Thus distinct pattern of mutations distributed across the entire HBV genome may be useful in predicting HCC in high-risk CHB patients and pattern of mutational combinations may exert greater impact on HCC risk prediction more accurately than point mutations and hence these predictors may support the existing surveillance strategies in proper management of the patients.</p></div

Frequencies of naturally occurring single and combination of mutations accumulated in the HBV genome were found significantly higher in hepatocellular carcinoma (HCC) than non-HCC patients.

<p>p<0.05 was considered as significant. ** indicates p>0.05.</p

Frequencies of mutations showing escalating trend with the progression of the liver diseases from no liver fibrosis (nLF) or liver fibrosis (LF) to liver cirrhosis (LC) and hepatocellular carcinoma (HCC).

<p>Significant mutations were indicated in bold (p≤0.05) and marginal significant values were represented in italics.</p><p>Frequencies of mutations showing escalating trend with the progression of the liver diseases from no liver fibrosis (nLF) or liver fibrosis (LF) to liver cirrhosis (LC) and hepatocellular carcinoma (HCC).</p

Multivariate analysis to identify independent risk factors for HCC development in HBeAg positive and negative patients.

<p>p value <0.05 was considered as significant.</p><p>Multivariate analysis to identify independent risk factors for HCC development in HBeAg positive and negative patients.</p

Clinical, biochemical and demographic profile of sixty-eight patients included in the study.

<p>SD =  Standard deviation; NS =  Not significant; ALT =  Alanine aminotransferase; AST = Aspartate aminotransferase; INR =  International normalized ratio; ALP = Alkaline phosphatase; IU =  International unit; Δ Parameters presented as Mean±SD; β parameters presented as Median [Range]; δ Mutations with statistically significant difference between the groups are presented.</p><p>Clinical, biochemical and demographic profile of sixty-eight patients included in the study.</p

List of primers used to generate PCR products and promoter construct.

<p>List of primers used to generate PCR products and promoter construct.</p