Stringent Primer Termination by an Archaeo-Eukaryotic DNA Primase

Priming of single stranded templates is essential for DNA replication. In recent years, significant progress was made in understanding how DNA primase fulfils this fundamental function, particularly with regard to the initiation. Equally intriguing is the unique property of archeao-eukaryotic primases to terminate primer formation at a well-defined unit length. The apparent ability to “count” the number of bases incorporated prior to primer release is not well understood, different mechanisms having been proposed for different species. We report a mechanistic investigation of primer termination by the pRN1 primase from Sulfolobus islandicus. Using an HPLC-based assay we determined structural features of the primer 5′-end that are required for consistent termination. Mutations within the unstructured linker connecting the catalytic domain to the template binding domain allowed us to assess the effect of altered linker length and flexibility on primer termination.

DNA primase subunit 1 deteriorated progression of hepatocellular carcinoma by activating AKT/mTOR signaling and UBE2C-mediated P53 ubiquitination

Background DNA primase subunit 1 (PRIM1) has been reported as a novel oncogene in several cancer types. However, its roles in hepatocellular carcinoma (HCC) remain unclear. This study aimed to investigate underlying mechanisms of PRIM1 and identify it as a potential molecular target for HCC. Methods Hub genes were screened between HCC tissues and normal liver tissues in 3 gene expression omnibus (GEO) datasets and the cancer genome atlas (TCGA). The expression features and prognostic value of one of the hub genes PRIM1 were analyzed by bioinformatic analyses and immunohistochemistry. Loss-of-function and gain-of-function studies were used to investigate the regulatory role of PRIM1 in HCC cells. Real-time (RT)-qPCR, western blotting, and ubiquitin immunoprecipitation assays were performed to explore the underlying mechanisms. The xenograft model was employed to detect the roles of PRIM1 in tumor growth in vivo. Finally, the 3D spheroid model was conducted to validate the role of PRIM1 in tumor growth and sorafenib resistance. Results The hub genes of HCC were screened in multiple bioinformatic datasets. PRIM1, as one of the hub genes, was significantly overexpressed in HCC tissues in mRNA and protein levels. In addition, high expression of PRIM1 indicated poor prognosis of HCC patients in TCGA, ICGC, and Nantong cohorts. Overexpression of PRIM1 promoted the proliferation, migration/invasion, and sorafenib resistance of HCC cells, with the decrease in apoptosis and cell cycle arrest. Mechanically, PRIM1 facilitated epithelial-mesenchymal transition (EMT) process and the activity of PI3K/AKT/mTOR signaling of HCC cells. Additionally, PRIM1 could cause the ubiquitination and degradation of P53 by upregulating Ubiquitin Conjugating Enzyme E2 C (UBE2C). Furthermore, knockdown of PRIM1 significantly inhibited the growth of xenograft tumors and HCC cells-derived spheroids with enhanced sorafenib resistance. Conclusion This study implies that PRIM1 may play a key role in the progression of HCC and may serve as a potential target for HCC treatment.

Cell-penetrating peptide conjugates of indole-3-acetic acid-based DNA primase/Gyrase inhibitors as potent antitubercular agents against planktonic and biofilm culture of Mycobacterium smegmatis

Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that caused 1.5 million fatalities globally in 2018. New strains of Mtb resistant to all known classes of antibiotics pose a global healthcare problem. In this work we have conjugated novel indole-3-acetic acid-based DNA primase/gyrase inhibitor with cell-penetrating peptide via cleavable and non-cleavable bonds. For non-cleavable linkage, inhibitor was conjugated with peptide via an amide bond to the N-terminus, whereas a cleavable linkage was obtained by conjugating the inhibitor through a disulfide bond. We performed the conjugation of the inhibitor either directly on a solid surface, or by using solution-phase chemistry. M. smegmatis (non-pathogenic model of Mtb) was used to determine the minimal inhibitory concentration (MIC) of the synthetic conjugates. Conjugates were found more active as compared to free inhibitor molecules. Strikingly, the conjugate also impair the development of biofilm, showing a therapeutic potential against infections caused by both planktonic and sessile forms of mycobacterium species.

DNA Primase Subunit 1 Expression in Hepatocellular Carcinoma and Its Clinical Implication

DNA Primase Subunit 1 (PRIM1) is crucial for cancer development and progression. However, there remains a lack of comprehension concerning the clinical implication of PRIM1 in HCC. Here, aberrant expression of PRIM1 was identified in HCC according to available databases. The prognostic value of PRIM1 in patients presenting with HCC was further assessed based on TCGA data. Gene set enrichment analysis (GSEA) was subsequently conducted to investigate the potential function of PRIM1. Additionally, the correlations between tumor-infiltrating immune cells (TIICs) and PRIM1 expression were evaluated. The data from TCGA, GEO, ONCOMINE, and HCCDB databases illustrated that PRIM1 was overexpressed in HCC tissues, compared to normal liver tissues (all p<0.05). Kaplan-Meier analysis revealed that high PRIM1 expression in HCC was closely correlated with worse overall survival (p<0.05). The univariate and multivariate analyses illustrated that PRIM1 expression was an independent novel prognostic indicator in HCC. Additionally, the area under the receiver operating characteristic (AUROC) curve for PRIM1 reached 0.8651, indicating the diagnostic significance of PRIM1 in patients with HCC. GSEA showed that PRIM1 overexpression was significantly enriched in several tumor-related signaling pathways. Besides, TIIC analysis clarified the association between PRIM1 expression and TIICs in HCC. The findings disclose that PRIM1 profoundly implicated in promoting tumorigenesis might work as a desirable biomarker for HCC.