Direct Reversal Repair in Mammalian Cells

Ahmad, Alya; Nay, Stephanie L.; O’Connor, Timothy

doi:10.5772/60037

Cited by 7 publications

(8 citation statements)

References 146 publications

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“…We confirmed that the DNA with the O6mG modification generated a much lower fluorescence signal compared to the unmodified DNA (Figure 4D ). We chose to use single-stranded DNA for our screening, as AlkB is known to prefer ssDNA substrates and O6mG can be found in single-stranded regions of DNA associated with replication forks ( 18 , 23 ).…”

Section: Resultsmentioning

confidence: 99%

A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities

Wang

Katanski

Pan

et al. 2020

Nucleic Acids Research

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AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

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Section: Resultsmentioning

confidence: 99%

A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities

Wang

Katanski

Pan

et al. 2020

Nucleic Acids Research

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“…This repair process does not require excision, synthesis, and ligation. In mammalian cells, DR is performed by O 6 -methylguanine DNA methyltransferase (MGMT) or ALKBH (AlkB homolog) proteins in the nucleus ( Ragg et al, 2000 ; Ahmad et al, 2015 ). MGMT repairs the O 6 -methylguanine lesions by removing methyl groups, whereas ALKBH repairs certain N-alkyl lesions via dealkylation.…”

Section: Other Mtdna Damage Repair Mechanismsmentioning

confidence: 99%

The Mitochondrial Response to DNA Damage

Rong

et al. 2021

Front. Cell Dev. Biol.

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Mitochondria are double membrane organelles in eukaryotic cells that provide energy by generating adenosine triphosphate (ATP) through oxidative phosphorylation. They are crucial to many aspects of cellular metabolism. Mitochondria contain their own DNA that encodes for essential proteins involved in the execution of normal mitochondrial functions. Compared with nuclear DNA, the mitochondrial DNA (mtDNA) is more prone to be affected by DNA damaging agents, and accumulated DNA damages may cause mitochondrial dysfunction and drive the pathogenesis of a variety of human diseases, including neurodegenerative disorders and cancer. Therefore, understanding better how mtDNA damages are repaired will facilitate developing therapeutic strategies. In this review, we focus on our current understanding of the mtDNA repair system. We also discuss other mitochondrial events promoted by excessive DNA damages and inefficient DNA repair, such as mitochondrial fusion, fission, and mitophagy, which serve as quality control events for clearing damaged mtDNA.

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“…Direct reversal repair (DRR) removes certain DNA and RNA modifications, without excision, resynthesis, or ligation ( Ahmad et al, 2015 ). It is an error-free repair pathway that retains the original genetic information because it does not involve the breaking of the phosphodiester backbone.…”

Section: Dna Repair Pathwaysmentioning

confidence: 99%

“…MGMT binds in the minor groove of DNA, scans the DNA, repairs the alkylated bases, and therefore provides a quick repair for such DNA lesion. The MGMT protein, whose bacterial analog is called Ogt, specifically reverses guanine base methylation by removing methyl groups from the guanine ( Pegg, 1990 ; Esteller et al, 2000 ; Ahmad et al, 2015 ). As each MGMT molecule can be used only once, the procedure is costly; the reaction is stoichiometric rather than catalytic ( Ibrahim Al-Obaide et al, 2021 ).…”

Section: Dna Repair Pathwaysmentioning

confidence: 99%

Mechanisms of Genome Maintenance in Plants: Playing It Safe With Breaks and Bumps

Raina

Sahu

Laskar³

et al. 2021

Front. Genet.

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Maintenance of genomic integrity is critical for the perpetuation of all forms of life including humans. Living organisms are constantly exposed to stress from internal metabolic processes and external environmental sources causing damage to the DNA, thereby promoting genomic instability. To counter the deleterious effects of genomic instability, organisms have evolved general and specific DNA damage repair (DDR) pathways that act either independently or mutually to repair the DNA damage. The mechanisms by which various DNA repair pathways are activated have been fairly investigated in model organisms including bacteria, fungi, and mammals; however, very little is known regarding how plants sense and repair DNA damage. Plants being sessile are innately exposed to a wide range of DNA-damaging agents both from biotic and abiotic sources such as ultraviolet rays or metabolic by-products. To escape their harmful effects, plants also harbor highly conserved DDR pathways that share several components with the DDR machinery of other organisms. Maintenance of genomic integrity is key for plant survival due to lack of reserve germline as the derivation of the new plant occurs from the meristem. Untowardly, the accumulation of mutations in the meristem will result in a wide range of genetic abnormalities in new plants affecting plant growth development and crop yield. In this review, we will discuss various DNA repair pathways in plants and describe how the deficiency of each repair pathway affects plant growth and development.

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Direct Reversal Repair in Mammalian Cells

Cited by 7 publications

References 146 publications

A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities

A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities

The Mitochondrial Response to DNA Damage

Mechanisms of Genome Maintenance in Plants: Playing It Safe With Breaks and Bumps

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