The cause of on-target point mutations generated by CRISPR-Cas9 treatment in the yeast Xanthophyllomyces dendrorhous

ABSTRACTRecognizing outcomes of DNA repair induced by CRISPR-Cas9 cutting is vital for precise genome editing. Reported DNA repair outcomes after Cas9 cutting include deletions/insertions and low frequency of genomic rearrangements and nucleotide substitutions. Thus far, substitution mutations caused by CRISPR-Cas9 has not attracted much attention. Here, we identified on-target point mutations induced by CRISPR-Cas9 treatment in the yeast Xanthophyllomyces dendrorhous by Sanger and Illumina sequencing. Different from previous studies, our findings suggested that the on-target mutations are not random and they cannot render the gRNA effective. Moreover, these point mutations showed strong sequence dependence that is not consistent with the observations in Hela cells, in which CRISPR-mediated substitutions were considered lacking sequence dependence and conversion preferences. Furthermore, this study demonstrated that the NHEJ components Ku70, Ku80, Mre11, or RAD50, and the overlapping roles of non-essential DNA polymerases were necessary for the emergence of point mutations, increasing the knowledge on CRISPR-Cas9 mediated DNA repair.

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Contribution of Large Genomic Rearrangements in Italian Lynch Syndrome Patients: Characterization of a Novel Alu-Mediated Deletion

BioMed Research International ◽

10.1155/2013/219897 ◽

2013 ◽

Vol 2013 ◽

pp. 1-7 ◽

Cited By ~ 15

Author(s):

Francesca Duraturo ◽

Angela Cavallo ◽

Raffaella Liccardo ◽

Bianca Cudia ◽

Marina De Rosa ◽

...

Keyword(s):

Lynch Syndrome ◽

Germ Line ◽

Point Mutations ◽

Low Frequency ◽

Genomic Rearrangements ◽

Dna Mismatch ◽

Mmr Genes ◽

Large Genomic Rearrangements ◽

Large Deletions ◽

Long Range Pcr

Lynch syndrome is associated with germ-line mutations in the DNA mismatch repair (MMR) genes, mainlyMLH1andMSH2. Most of the mutations reported in these genes to date are point mutations, small deletions, and insertions. Large genomic rearrangements in the MMR genes predisposing to Lynch syndrome also occur, but the frequency varies depending on the population studied on average from 5 to 20%. The aim of this study was to examine the contribution of large rearrangements in theMLH1andMSH2genes in a well-characterised series of 63 unrelated Southern Italian Lynch syndrome patients who were negative for pathogenic point mutations in theMLH1,MSH2, andMSH6genes. We identified a large novel deletion in theMSH2gene, including exon 6 in one of the patients analysed (1.6% frequency). This deletion was confirmed and localised by long-range PCR. The breakpoints of this rearrangement were characterised by sequencing. Further analysis of the breakpoints revealed that this rearrangement was a product of Alu-mediated recombination. Our findings identified a novel Alu-mediated rearrangement withinMSH2gene and showed that large deletions or duplications inMLH1andMSH2genes are low-frequency mutational events in Southern Italian patients with an inherited predisposition to colon cancer.

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CRISPR-Associated Primase-Polymerases are implicated in prokaryotic CRISPR-Cas adaptation

Nature Communications ◽

10.1038/s41467-021-23535-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Katerina Zabrady ◽

Matej Zabrady ◽

Peter Kolesar ◽

Arthur W. H. Li ◽

Aidan J. Doherty

Keyword(s):

Dna Repair ◽

Dna Synthesis ◽

Genome Stability ◽

Dna Polymerases ◽

Acquired Immunity ◽

Strand Displacement ◽

Genetic Studies ◽

Type Iiia ◽

Maintain Genome Stability ◽

Cas Proteins

AbstractCRISPR-Cas pathways provide prokaryotes with acquired “immunity” against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.

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DNA-repair reactions by purified HeLa DNA polymerases and exonucleases.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)37744-5 ◽

1988 ◽

Vol 263 (25) ◽

pp. 12228-12234 ◽

Cited By ~ 1

Author(s):

H Randahl ◽

G C Elliott ◽

S Linn

Keyword(s):

Dna Repair ◽

Dna Polymerases

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Low frequency vibrational modes in proteins: Changes induced by point-mutations in the protein-cofactor matrix of bacterial reaction centers

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.95.21.12306 ◽

1998 ◽

Vol 95 (21) ◽

pp. 12306-12311 ◽

Cited By ~ 42

Author(s):

C. Rischel ◽

D. Spiedel ◽

J. P. Ridge ◽

M. R. Jones ◽

J. Breton ◽

...

Keyword(s):

Point Mutations ◽

Low Frequency ◽

Reaction Centers ◽

Vibrational Modes ◽

Bacterial Reaction Centers ◽

Cofactor Matrix

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KGF facilitates repair of radiation-induced DNA damage in alveolar epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.272.6.l1174 ◽

1997 ◽

Vol 272 (6) ◽

pp. L1174-L1180 ◽

Cited By ~ 25

Author(s):

M. Takeoka ◽

W. F. Ward ◽

H. Pollack ◽

D. W. Kamp ◽

R. J. Panos

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Epithelial Cells ◽

Dna Polymerase ◽

Dna Polymerases ◽

Dna Strand Breaks ◽

Strand Breaks ◽

Alveolar Epithelial ◽

Radiation Induced ◽

Dna Strand

Administration of exogenous keratinocyte growth factor (KGF) prevents or attenuates several forms of oxidant-mediated lung injury. Because DNA damage in epithelial cells is a component of radiation pneumotoxicity, we determined whether KGF ameliorated DNA strand breaks in irradiated A549 cells. Cells were exposed to 137Cs gamma rays, and DNA damage was measured by alkaline unwinding and ethidium bromide fluorescence after a 30-min recovery period. Radiation induced a dose-dependent increase in DNA strand breaks. The percentage of double-stranded DNA after exposure to 30 Gy increased from 44.6 +/- 3.5% in untreated control cells to 61.6 +/- 5.0% in cells cultured with 100 ng/ml KGF for 24 h (P < 0.05). No reduction in DNA damage occurred when the cells were cultured with KGF but maintained at 0 degree C during and after irradiation. The sparing effect of KGF on radiation-induced DNA damage was blocked by aphidicolin, an inhibitor of DNA polymerases-alpha, -delta, and -epsilon and by butylphenyl dGTP, which blocks DNA polymerase-alpha strongly and polymerases-delta and -epsilon less effectively. However, dideoxythymidine triphosphate, a specific inhibitor of DNA polymerase-beta, did not abrogate the KGF effect. Thus KGF increases DNA repair capacity in irradiated pulmonary epithelial cells, an effect mediated at least in part by DNA polymerases-alpha, -delta, and -epsilon. Enhancement of DNA repair capability after cell damage may be one mechanism by which KGF is able to ameliorate oxidant-mediated alveolar epithelial injury.

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DNA Repair after γ Irradiation in Lymphocytes Exposed to Low-Frequency Pulsed Electromagnetic Fields

Radiation Research ◽

10.2307/3577431 ◽

1989 ◽

Vol 118 (1) ◽

pp. 161 ◽

Cited By ~ 33

Author(s):

A. Cossarizza ◽

D. Monti ◽

P. Sola ◽

G. Moschini ◽

R. Cadossi ◽

...

Keyword(s):

Dna Repair ◽

Electromagnetic Fields ◽

Low Frequency ◽

Pulsed Electromagnetic Fields ◽

Γ Irradiation ◽

Pulsed Electromagnetic

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Mechanistic Comparison of High-Fidelity and Error-Prone DNA Polymerases and Ligases Involved in DNA Repair

ChemInform ◽

10.1002/chin.200621259 ◽

2006 ◽

Vol 37 (21) ◽

Author(s):

Alexander K. Showalter ◽

Brandon J. Lamarche ◽

Marina Bakhtina ◽

Mei-I Su ◽

Kuo-Hsiang Tang ◽

...

Keyword(s):

Dna Repair ◽

Dna Polymerases ◽

High Fidelity

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The essential DNA polymerases delta and epsilon are involved in repair of UV-damaged DNA in the yeast Saccharomyces cerevisiae.

Acta Biochimica Polonica ◽

10.18388/abp.1999_4162 ◽

1999 ◽

Vol 46 (2) ◽

pp. 289-298 ◽

Cited By ~ 1

Author(s):

A Hałas ◽

Z Policińska ◽

H Baranowska ◽

W J Jachymczyk

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Repair ◽

Uv Irradiation ◽

Dna Polymerases ◽

Relative Molecular Mass ◽

Yeast Saccharomyces Cerevisiae ◽

Strand Breaks ◽

Single Strand Breaks ◽

Mutant Strains ◽

Cellular Dna

We have studied the ability of yeast DNA polymerases to carry out repair of lesions caused by UV irradiation in Saccharomyces cerevisiae. By the analysis of postirradiation relative molecular mass changes in cellular DNA of different DNA polymerases mutant strains, it was established that mutations in DNA polymerases delta and epsilon showed accumulation of single-strand breaks indicating defective repair. Mutations in other DNA polymerase genes exhibited no defects in DNA repair. Thus, the data obtained suggest that DNA polymerases delta and epsilon are both necessary for DNA replication and for repair of lesions caused by UV irradiation. The results are discussed in the light of current concepts concerning the specificity of DNA polymerases in DNA repair.

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