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A Self-Replicating Linear DNA

Liew PS, Tan TH, Wong YC, Sim EUH, Lee CW, Narayanan K

ACS Synth Biol, 2020 04 17;9(4):804-813.
PMID: 32196315 DOI: 10.1021/acssynbio.9b00478

TelN and tos are a unique DNA linearization unit isolated from bacteriophage N15. While being transferable, the TelN cleaving-rejoining activities remained stable to function on tos in both bacterial and mammalian environments. However, TelN contribution in linear plasmid replication in mammalian cells remains unknown. Herein, we investigated the association of TelN in linear tos-containing DNA (tos-DNA) replication in mammalian cells. Additionally, the mammalian origin of replication (ori) that is well-known to initiate the replication event of plasmid vectors was also studied. In doing so, we identified that both TelN and mammalian initiation sites were essential for the replication of linear tos-DNA, determined by using methylation sensitive DpnI/MboI digestion and polymerase chain reaction (PCR) amplification approaches. Furthermore, we engineered the linear tos-DNA to be able to retain in mammalian cells using S/MAR technology. The resulting S/MAR containing tos-DNA was robust for at least 15 days, with (1) continuous tos-DNA replication, (2) correct splicing of gene transcripts, and (3) stable exogenous gene expression that was statistically comparable to the endogenous gene expression level. Understanding the activities of TelN and tos in mammalian cells can potentially provide insights for adapting this simple DNA linearization unit in developing novel genetic engineering tools, especially to the eukaryotic telomere/telomerase study.

Matched MeSH terms: DNA Replication/genetics*
Fulltext Somatic mitochondrial DNA D-loop mutations in meningioma discovered: A preliminary data

Mohamed Yusoff AA, Mohd Khair SZN, Wan Abdullah WS, Abd Radzak SM, Abdullah JM

J Cancer Res Ther, 2020 12 22;16(6):1517-1521.
PMID: 33342822 DOI: 10.4103/jcrt.JCRT_1132_16

Background and Objective: Meningiomas are among the most common intracranial tumors of the central nervous system. It is widely accepted that the initiation and progression of meningiomas involve the accumulation of nucleus genetic alterations, but little is known about the implication of mitochondrial genomic alterations during development of these tumors. The human mitochondrial DNA (mtDNA) contains a short hypervariable, noncoding displacement loop control region known as the D-Loop. Alterations in the mtDNA D-loop have been reported to occur in most types of human cancers. The purpose of this study was to assess the mtDNA D-loop mutations in Malaysian meningioma patients.
Materials and Methods: Genomic DNA was extracted from 21 fresh-frozen tumor tissues and blood samples of the same meningioma patients. The entire mtDNA D-loop region (positions 16024-576) was polymerase chain reaction amplified using designed primers, and then amplification products were purified before the direct DNA sequencing proceeds.
Results: Overall, 10 (47.6%) patients were detected to harbor a total of 27 somatic mtDNA D-loop mutations. Most of these mtDNA mutations were identified in the hypervariable segment II (40.7%), with 33.3% being located mainly in the conserved sequence block II of the D310 sequence. Furthermore, 58 different germline variations were observed at 21 nucleotide positions.
Conclusion: Our results suggest that mtDNA alterations in the D-loop region may be an important and early event in developing meningioma. Further studies are needed, including validation in a larger patient cohort, to verify the clinicopathological outcomes of mtDNA mutation biomarkers in meningiomas.

Matched MeSH terms: DNA Replication/genetics
Fulltext Suppression of cell division-associated genes by Helicobacter pylori attenuates proliferation of RAW264.7 monocytic macrophage cells

Tan GM, Looi CY, Fernandez KC, Vadivelu J, Loke MF, Wong WF

Sci Rep, 2015;5:11046.
PMID: 26078204 DOI: 10.1038/srep11046

Helicobacter pylori at multiplicity of infection (MOI ≥ 50) have been shown to cause apoptosis in RAW264.7 monocytic macrophage cells. Because chronic gastric infection by H. pylori results in the persistence of macrophages in the host's gut, it is likely that H. pylori is present at low to moderate, rather than high numbers in the infected host. At present, the effect of low-MOI H. pylori infection on macrophage has not been fully elucidated. In this study, we investigated the genome-wide transcriptional regulation of H. pylori-infected RAW264.7 cells at MOI 1, 5 and 10 in the absence of cellular apoptosis. Microarray data revealed up- and down-regulation of 1341 and 1591 genes, respectively. The expression of genes encoding for DNA replication and cell cycle-associated molecules, including Aurora-B kinase (AurkB) were down-regulated. Immunoblot analysis verified the decreased expression of AurkB and downstream phosphorylation of Cdk1 caused by H. pylori infection. Consistently, we observed that H. pylori infection inhibited cell proliferation and progression through the G1/S and G2/M checkpoints. In summary, we suggest that H. pylori disrupts expression of cell cycle-associated genes, thereby impeding proliferation of RAW264.7 cells, and such disruption may be an immunoevasive strategy utilized by H. pylori.

Matched MeSH terms: DNA Replication/genetics

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