Inserting Site-Specific DNA Lesions into Whole Genomes

2017 ◽  
pp. 107-118 ◽  
Author(s):  
Vincent Pagès ◽  
Robert P. Fuchs
Keyword(s):  
Dna Lesions ◽  
Site Specific ◽  
Whole Genomes

scholarly journals Site-specific targeting of a light activated dCas9-KIllerRed fusion protein generates transient, localized regions of oxidative DNA damage

2020 ◽  
Author(s):  
Nealia C.M. House ◽  
Jacob V. Layer ◽  
Brendan D. Price
Keyword(s):  
Reactive Oxygen Species ◽  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Light Exposure ◽  
Green Light ◽  
Reactive Oxygen ◽  
Dna Lesions ◽  
Oxygen Species ◽  
Dna Breaks ◽  
Site Specific

AbstractDNA repair requires reorganization of the local chromatin structure to facilitate access to and repair of the DNA. Studying DNA double-strand break (DSB) repair in specific chromatin domains has been aided by the use of sequence-specific endonucleases to generate targeted breaks. Here, we describe a new approach that combines KillerRed, a photosensitizer that generates reactive oxygen species (ROS) when exposed to light, and the genome-targeting properties of the CRISPR/Cas9 system. Fusing KillerRed to catalytically inactive Cas9 (dCas9) generates dCas9-KR, which can then be targeted to any desired genomic region with an appropriate guide RNA. Activation of dCas9-KR with green light generates a local increase in reactive oxygen species, resulting in “clustered” oxidative damage, including both DNA breaks and base damage. Activation of dCas9-KR rapidly (within minutes) increases both γH2AX and recruitment of the KU70/80 complex. Importantly, this damage is repaired within 10 minutes of termination of light exposure, indicating that the DNA damage generated by dCas9-KR is both rapid and transient. Further, repair is carried out exclusively through NHEJ, with no detectable contribution from HR-based mechanisms. Surprisingly, sequencing of repaired DNA damage regions did not reveal any increase in either mutations or INDELs in the targeted region, implying that NHEJ has high fidelity under the conditions of low level, limited damage. The dCas9-KR approach for creating targeted damage has significant advantages over the use of endonucleases, since the duration and intensity of DNA damage can be controlled in “real time” by controlling light exposure. In addition, unlike endonucleases that carry out multiple cut-repair cycles, dCas9-KR produces a single burst of damage, more closely resembling the type of damage experienced during acute exposure to reactive oxygen species or environmental toxins. dCas9-KR is a promising system to induce DNA damage and measure site-specific repair kinetics at clustered DNA lesions.

scholarly journals SOS and UVM Pathways Have Lesion-Specific Additive and Competing Effects on Mutation Fixation at Replication-Blocking DNA Lesions

1999 ◽  
Vol 181 (5) ◽  
pp. 1515-1523 ◽  
Author(s):  
M. Sayeedur Rahman ◽  
M. Zafri Humayun
Keyword(s):  
Dna Damage ◽  
Transfection Efficiency ◽  
Sos Response ◽  
Genetic Network ◽  
Dna Lesions ◽  
Site Specific ◽  
E Coli ◽  
Mutational Specificity ◽  
Class 1 ◽  
Ap Sites

ABSTRACT Escherichia coli cells have multiple mutagenic pathways that are induced in response to environmental and physiological stimuli. Unlike the well-investigated classical SOS response, little is known about newly recognized pathways such as the UVM (UV modulation of mutagenesis) response. In this study, we compared the contributions of the SOS and UVM pathways on mutation fixation at two representative noninstructive DNA lesions: 3, N4-ethenocytosine (ɛC) and abasic (AP) sites. Because both SOS and UVM responses are induced by DNA damage, and defined UVM-defective E. colistrains are not yet available, we first constructed strains in which expression of the SOS mutagenesis proteins UmuD′ and UmuC (and also RecA in some cases) is uncoupled from DNA damage by being placed under the control of a heterologous lac-derived promoter. M13 single-stranded viral DNA bearing site-specific lesions was transfected into cells induced for the SOS or UVM pathway. Survival effects were determined from transfection efficiency, and mutation fixation at the lesion was analyzed by a quantitative multiplex sequence analysis procedure. Our results suggest that induction of the SOS pathway can independently elevate mutagenesis at both lesions, whereas the UVM pathway significantly elevates mutagenesis at ɛC in an SOS-independent fashion and at AP sites in an SOS-dependent fashion. Although mutagenesis at ɛC appears to be elevated by the induction of either the SOS or the UVM pathway, the mutational specificity profiles for ɛC under SOS and UVM pathways are distinct. Interestingly, when both pathways are active, the UVM effect appears to predominate over the SOS effect on mutagenesis at ɛC, but the total mutation frequency is significantly increased over that observed when each pathway is individually induced. These observations suggest that the UVM response affects mutagenesis not only at class 2 noninstructive lesions (ɛC) but also at classical SOS-dependent (class 1) lesions such as AP sites. Our results add new layers of complexity to inducible mutagenic phenomena: DNA damage activates multiple pathways that have lesion-specific additive as well as suppressive effects on mutation fixation, and some of these pathways are not directly regulated by the SOS genetic network.

Hypersensitivity of DNA polymerase β null mouse fibroblasts reflects accumulation of cytotoxic repair intermediates from site-specific alkyl DNA lesions

DNA Repair ◽  
2003 ◽  
Vol 2 (1) ◽  
pp. 27-48 ◽  
Author(s):  
Julie K. Horton ◽  
Donna F. Joyce-Gray ◽  
Brian F. Pachkowski ◽  
James A. Swenberg ◽  
Samuel H. Wilson
Keyword(s):  
Dna Polymerase ◽  
Dna Lesions ◽  
Null Mouse ◽  
Dna Polymerase Β ◽  
Site Specific ◽  
Mouse Fibroblasts

scholarly journals A novel method for site specific introduction of single model oxidative DNA lesions into oligodeoxyribonucleotides

1993 ◽  
Vol 21 (7) ◽  
pp. 1563-1568 ◽  
Author(s):  
Zafer Hatahet ◽  
Andrei A. Purmal ◽  
Susan S. Wallace
Keyword(s):  
Dna Lesions ◽  
Single Model ◽  
Site Specific ◽  
Novel Method

scholarly journals Nucleotide Excision Repair and Impact of Site-Specific 5′,8-Cyclopurine and Bulky DNA Lesions on the Physical Properties of Nucleosomes

Biochemistry ◽  
2018 ◽  
Vol 58 (6) ◽  
pp. 561-574 ◽  
Author(s):  
Vladimir Shafirovich ◽  
Marina Kolbanovskiy ◽  
Konstantin Kropachev ◽  
Zhi Liu ◽  
Yuquin Cai ◽  
...  
Keyword(s):  
Physical Properties ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Dna Lesions ◽  
Site Specific ◽  
Nucleotide Excision ◽  
Bulky Dna Lesions

scholarly journals Site-specific proteolytic cleavage prevents ubiquitination and degradation of human REV3L, the catalytic subunit of DNA polymerase ζ

2020 ◽  
Vol 48 (7) ◽  
pp. 3619-3637 ◽  
Author(s):  
Fengting Wang ◽  
Pan Li ◽  
Yuan Shao ◽  
Yanyan Li ◽  
Kai Zhang ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Genome Stability ◽  
Catalytic Domain ◽  
Hct116 Cell ◽  
Point Mutations ◽  
Dna Lesions ◽  
Human Cells ◽  
Catalytic Subunit ◽  
New Paradigm ◽  
Site Specific

Abstract REV3L, the catalytic subunit of DNA polymerase ζ (Pol ζ), is indispensable for translesion DNA synthesis, which protects cells from deleterious DNA lesions resulting from various intrinsic and environmental sources. However, REV3L lacks a proofreading exonuclease activity and consequently bypasses DNA lesions at the expense of increased mutations, which poses a severe threat to genome stability. Here we report a site-specific proteolytic event of human REV3L. We show that REV3L is cleaved by a threonine aspartase, Taspase1 (TASP1), to generate an N-terminal 70-kDa fragment (N70) and a polypeptide carrying the C-terminal polymerase catalytic domain in human cells. Strikingly, such a post-translational cleavage event plays a vital role in controlling REV3L stability by preventing ubiquitination and proteasome-mediated degradation of REV3L. Indicative of the biological importance of the above REV3L post-translational processing, cellular responses to UV and cisplatin-induced DNA lesions are markedly impaired in human HCT116 cell derivatives bearing defined point mutations in the endogenous REV3L gene that compromise REV3L cleavage. These findings establish a new paradigm in modulating the abundance of REV3L through site-specific proteolysis in human cells.

scholarly journals Site-specific targeting of a light activated dCas9-KillerRed fusion protein generates transient, localized regions of oxidative DNA damage

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0237759
Author(s):  
Nealia C. M. House ◽  
Ramya Parasuram ◽  
Jacob V. Layer ◽  
Brendan D. Price
Keyword(s):  
Reactive Oxygen Species ◽  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Light Exposure ◽  
Green Light ◽  
Reactive Oxygen ◽  
Dna Lesions ◽  
Oxygen Species ◽  
Dna Breaks ◽  
Site Specific

DNA repair requires reorganization of the local chromatin structure to facilitate access to and repair of the DNA. Studying DNA double-strand break (DSB) repair in specific chromatin domains has been aided by the use of sequence-specific endonucleases to generate targeted breaks. Here, we describe a new approach that combines KillerRed, a photosensitizer that generates reactive oxygen species (ROS) when exposed to light, and the genome-targeting properties of the CRISPR/Cas9 system. Fusing KillerRed to catalytically inactive Cas9 (dCas9) generates dCas9-KR, which can then be targeted to any desired genomic region with an appropriate guide RNA. Activation of dCas9-KR with green light generates a local increase in reactive oxygen species, resulting in “clustered” oxidative damage, including both DNA breaks and base damage. Activation of dCas9-KR rapidly (within minutes) increases both γH2AX and recruitment of the KU70/80 complex. Importantly, this damage is repaired within 10 minutes of termination of light exposure, indicating that the DNA damage generated by dCas9-KR is both rapid and transient. Further, repair is carried out exclusively through NHEJ, with no detectable contribution from HR-based mechanisms. Surprisingly, sequencing of repaired DNA damage regions did not reveal any increase in either mutations or INDELs in the targeted region, implying that NHEJ has high fidelity under the conditions of low level, limited damage. The dCas9-KR approach for creating targeted damage has significant advantages over the use of endonucleases, since the duration and intensity of DNA damage can be controlled in “real time” by controlling light exposure. In addition, unlike endonucleases that carry out multiple cut-repair cycles, dCas9-KR produces a single burst of damage, more closely resembling the type of damage experienced during acute exposure to reactive oxygen species or environmental toxins. dCas9-KR is a promising system to induce DNA damage and measure site-specific repair kinetics at clustered DNA lesions.

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