The Molecular Basis of Double-Strand DNA Break Repair: Crystal Structure of the RAD52/RPA Complex

2002 ◽  
Author(s):  
Doba D. Jackson ◽  
Gloria Borgstahl
Keyword(s):  
Crystal Structure ◽  
Molecular Basis ◽  
Dna Break ◽  
Dna Break Repair ◽  
Break Repair ◽  
Double Strand Dna

scholarly journals Molecular Basis for K63-Linked Ubiquitination Processes in Double-Strand DNA Break Repair: A Focus on Kinetics and Dynamics

2017 ◽  
Vol 429 (22) ◽  
pp. 3409-3429 ◽  
Author(s):  
Brian L. Lee ◽  
Anamika Singh ◽  
J.N. Mark Glover ◽  
Michael J. Hendzel ◽  
Leo Spyracopoulos
Keyword(s):  
Molecular Basis ◽  
Kinetics And Dynamics ◽  
Dna Break ◽  
Dna Break Repair ◽  
Break Repair ◽  
Double Strand Dna

Radiosensitization of cervical cancer cells via double-strand DNA break repair inhibition

2008 ◽  
Vol 110 (1) ◽  
pp. 93-98 ◽  
Author(s):  
Christa B. Fuhrman ◽  
Josh Kilgore ◽  
Yvette D. LaCoursiere ◽  
Christopher M. Lee ◽  
Brett A. Milash ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cancer Cells ◽  
Cervical Cancer Cells ◽  
Dna Break ◽  
Dna Break Repair ◽  
Break Repair ◽  
Double Strand Dna

scholarly journals Optimized CRISPR-Cas9 Genome Editing for Leishmania and Its Use To Target a Multigene Family, Induce Chromosomal Translocation, and Study DNA Break Repair Mechanisms

mSphere ◽  
2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Wen-Wei Zhang ◽  
Patrick Lypaczewski ◽  
Greg Matlashewski
Keyword(s):  
Genome Editing ◽  
Chromosomal Translocations ◽  
Content Type ◽  
Crispr System ◽  
Repair Mechanisms ◽  
Human Leishmaniasis ◽  
Dna Break ◽  
Dna Break Repair ◽  
Break Repair ◽  
Double Strand Dna

ABSTRACT Leishmania parasites cause human leishmaniasis. To accelerate characterization of Leishmania genes for new drug and vaccine development, we optimized and simplified the CRISPR-Cas9 genome-editing tool for Leishmania. We show that co-CRISPR targeting of the miltefosine transporter gene and serial transfections of an oligonucleotide donor significantly eased isolation of edited mutants. This cotargeting strategy was efficiently used to delete all 11 members of the A2 virulence gene family. This technical advancement is valuable, since there are many gene clusters and supernumerary chromosomes in the various Leishmania species and isolates. We simplified this CRISPR system by developing a gRNA and Cas9 coexpression vector which could be used to delete genes in various Leishmania species. This CRISPR system could also be used to generate specific chromosomal translocations, which will help in the study of Leishmania gene expression and transcription control. This study also provides new information about double-strand DNA break repair mechanisms in Leishmania. CRISPR-Cas9-mediated genome editing has recently been adapted for Leishmania spp. parasites, the causative agents of human leishmaniasis. We have optimized this genome-editing tool by selecting for cells with CRISPR-Cas9 activity through cotargeting the miltefosine transporter gene; mutation of this gene leads to miltefosine resistance. This cotargeting strategy integrated into a triple guide RNA (gRNA) expression vector was used to delete all 11 copies of the A2 multigene family; this was not previously possible with the traditional gene-targeting method. We found that the Leishmania donovani rRNA promoter is more efficient than the U6 promoter in driving gRNA expression, and sequential transfections of the oligonucleotide donor significantly eased the isolation of edited mutants. A gRNA and Cas9 coexpression vector was developed that was functional in all tested Leishmania species, including L. donovani, L. major, and L. mexicana. By simultaneously targeting sites from two different chromosomes, all four types of targeted chromosomal translocations were generated, regardless of the polycistronic transcription direction from the parent chromosomes. It was possible to use this CRISPR system to create a single conserved amino acid substitution (A189G) mutation for both alleles of RAD51, a DNA recombinase involved in homology-directed repair. We found that RAD51 is essential for L. donovani survival based on direct observation of the death of mutants with both RAD51 alleles disrupted, further confirming that this CRISPR system can reveal gene essentiality. Evidence is also provided that microhomology-mediated end joining (MMEJ) plays a major role in double-strand DNA break repair in L. donovani. IMPORTANCE Leishmania parasites cause human leishmaniasis. To accelerate characterization of Leishmania genes for new drug and vaccine development, we optimized and simplified the CRISPR-Cas9 genome-editing tool for Leishmania. We show that co-CRISPR targeting of the miltefosine transporter gene and serial transfections of an oligonucleotide donor significantly eased isolation of edited mutants. This cotargeting strategy was efficiently used to delete all 11 members of the A2 virulence gene family. This technical advancement is valuable, since there are many gene clusters and supernumerary chromosomes in the various Leishmania species and isolates. We simplified this CRISPR system by developing a gRNA and Cas9 coexpression vector which could be used to delete genes in various Leishmania species. This CRISPR system could also be used to generate specific chromosomal translocations, which will help in the study of Leishmania gene expression and transcription control. This study also provides new information about double-strand DNA break repair mechanisms in Leishmania.

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