The recombinant retroviral vector pLCDSN containing E. coli cytosine deaminase gene was constructed. After packaging with PA317 cell line, the infectious particles were used to infect human colon carcinoma cell line LoVo. A single clone harbouring EC-CD gene was picked after G418 selection. There was no significant difference in cell growth curve or morphology between the LoVo/LCDSN and LoVo cells. Both of them were very sensitive to 5-FU in vitro (IC(50), approximately 0.5 &mgr;mol/L). However, the expression of the CD gene did increase the sensitivity of these cells to the nontoxic prodrug, 5-FC, decreasing the IC(50) for 5-FC from 22 000 &mgr;mol/L for parental LoVo cells to 13 &mgr;mol/L for LoVo/LCDSN cells. Obvious by side effect was also observed. When cells transduced with CD gene were mixed with wild type cells at a ratio of 30:70, above 80% of the cancer cells could be killed after treatment with a nontoxic concentration of 5-FC.
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ACS Synth Biol
January 2025
Laboratory of Synthetic Microbiology, School of Chemical Engineering & Technology, Tianjin University, Tianjin 300072, P. R. China.
The fusion expression of deoxyribonucleic acid (DNA) replication-related proteins with nucleotide deaminase enzymes promotes random mutations in bacterial genomes, thereby increasing genetic diversity among the population. Most previous studies have focused on cytosine deaminase, which produces only C → T mutations, significantly limiting the variety of mutation types. In this study, we developed a fusion expression system by combining DnaG (RNA primase) with adenine deaminase TadA-8e (DnaG-TadA) in , which is capable of rapidly introducing A → G mutations into the genome, resulting in a 664-fold increase in terms of mutation rate.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
January 2025
Program in Genetics, Molecular, and Cellular Biology, Tufts University Graduate School of Biomedical Sciences, Boston, MA 02111.
CAG/CTG repeats are prone to expansion, causing several inherited human diseases. The initiating sources of DNA damage which lead to inaccurate repair of the repeat tract to cause expansions are not fully understood. Expansion-prone CAG/CTG repeats are actively transcribed and prone to forming stable R-loops with hairpin structures forming on the displaced single-stranded DNA (S-loops).
View Article and Find Full Text PDFInt J Mol Sci
December 2024
Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
DNA polymerases frequently misincorporate ribonucleoside 5'-triphosphates into nascent DNA strands. This study examined the effects of an incorporated ribonucleoside on untargeted mutations in human cells. Riboguanosine (rG) was introduced into the downstream region of the gene to preferentially detect the untargeted mutations.
View Article and Find Full Text PDFBinding of transcription factors (TFs) at gene regulatory elements controls cellular epigenetic state and gene expression. Current genome-wide chromatin profiling approaches have inherently limited resolution, complicating assessment of TF occupancy and co-occupancy, especially at individual alleles. In this work, we introduce Accessible Chromatin by Cytosine Editing Site Sequencing with ATAC-seq (ACCESS-ATAC), which harnesses a double-stranded DNA cytosine deaminase (Ddd) enzyme to stencil TF binding locations within accessible chromatin regions.
View Article and Find Full Text PDFACS Synth Biol
January 2025
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
targeted mutagenesis technologies are the basis for the continuous directed evolution of specific proteins. Here, an efficient mutagenesis system (CgMutaT7) for continuous evolution of the targeted gene in was developed. First, cytosine deaminase and uracil-DNA glycosylase inhibitor were sequentially fused to T7 RNA polymerase using flexible linkers to build the CgMutaT7 system, which introduces mutations in targeted regions controlled by the T7 promoter.
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