Chemical-Oxidation Cleavage Triggered Isothermal Exponential Amplification Reaction for Attomole Gene-Specific Methylation Analysis

Genomic 5-methylcytosine (5-mC) modification is known to extensively regulate gene expression. The sensitive and convenient analysis of gene-specific methylation is wishful but challenging due to the lack of means that can sensitively and sequence-selectively discriminate 5-mC from cytosine without the need for polymerase chain reaction. Here we report a chemical-oxidation cleavage triggered exponential amplification reaction (EXPAR) method named COEXPAR for gene-specific methylation analysis. EXPAR was proved to not only have rapid amplification kinetics under isothermal condition but also show excellent sequence-selectivity and linear-dependence on EXPAR trigger. Further initiation of EXPAR by chemical-cleavage of DNA at 5-mC, the COEXPAR showed high specificity for methylated and nonmethylated DNA, and ∼10⁷ copies of triggers were replicated in 20 min, which were used to quantify the methylation level at the methylation loci. As a result, the gene-specific methylation level of a p53 gene fragment, as a target model, was analyzed in two linear ranges of 10 fM–1 pM and 1 pM–10 nM, and limits of detection of 411 aM (<i>S</i>/<i>N</i> = 3) by fluorescence, and 576 aM (<i>S</i>/<i>N</i> = 3) by electrochemistry. The method fulfilled the assay in an isothermal way in ∼5 h without the need for tedious sample preparation and accurate thermocycling equipment, which is likely to be a facile and ultrasensitive way for gene-specific methylation analysis

Hepatitis B virus infection disrupts homologous recombination in hepatocellular carcinoma by stabilizing resection inhibitor ADRM1

Many cancers harbor homologous recombination defects (HRDs). A HRD is a therapeutic target that is being successfully utilized in treatment of breast/ovarian cancer via synthetic lethality. However, canonical HRD caused by BRCAness mutations do not prevail in liver cancer. Here we report a subtype of HRD caused by the perturbation of a proteasome variant (CDW19S) in hepatitis B virus–bearing (HBV-bearing) cells. This amalgamate protein complex contained the 19S proteasome decorated with CRL4WDR70 ubiquitin ligase, and assembled at broken chromatin in a PSMD4Rpn10- and ATM-MDC1-RNF8–dependent manner. CDW19S promoted DNA end processing via segregated modules that promote nuclease activities of MRE11 and EXO1. Contrarily, a proteasomal component, ADRM1Rpn13, inhibited resection and was removed by CRL4WDR70-catalyzed ubiquitination upon commitment of extensive resection. HBx interfered with ADRM1Rpn13 degradation, leading to the imposition of ADRM1Rpn13-dependent resection barrier and consequent viral HRD subtype distinguishable from that caused by BRCA1 defect. Finally, we demonstrated that viral HRD in HBV-associated hepatocellular carcinoma can be exploited to restrict tumor progression. Our work clarifies the underlying mechanism of a virus-induced HRD subtype.</p