Characterization of FFPE-induced bacterial DNA damage and development of a repair method

Abstract Formalin-fixed, paraffin-embedded (FFPE) specimens have huge potential as source material in the field of human microbiome research. However, the effects of FFPE processing on bacterial DNA remain uncharacterized. Any effects are relevant for microbiome studies, where DNA template is often minimal and sequences studied are not limited to one genome. As such, we aimed to both characterize this FFPE-induced bacterial DNA damage and develop strategies to reduce and repair this damage. Our analyses indicate that bacterial FFPE DNA is highly fragmented, a poor template for PCR, crosslinked and bears sequence artefacts derived predominantly from oxidative DNA damage. Two strategies to reduce this damage were devised – an optimized decrosslinking procedure reducing sequence artefacts generated by high-temperature incubation, and secondly, an in vitro reconstitution of the base excision repair pathway. As evidenced by whole genome sequencing, treatment with these strategies significantly increased fragment length, reduced the appearance of sequence artefacts and improved the sequencing readability of bacterial and mammalian FFPE DNA. This study provides a new understanding of the condition of bacterial DNA in FFPE specimens and how this impacts downstream analyses, in addition to a strategy to improve the sequencing quality of bacterial and possibly mammalian FFPE DNA.

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Oxygen-Dependent Accumulation of Purine DNA Lesions in Cockayne Syndrome Cells

Cells ◽

10.3390/cells9071671 ◽

2020 ◽

Vol 9 (7) ◽

pp. 1671 ◽

Cited By ~ 1

Author(s):

Marios G. Krokidis ◽

Mariarosaria D’Errico ◽

Barbara Pascucci ◽

Eleonora Parlanti ◽

Annalisa Masi ◽

...

Keyword(s):

Dna Damage ◽

Oxidative Dna Damage ◽

Excision Repair ◽

Enzymatic Digestion ◽

Cockayne Syndrome ◽

Dna Lesions ◽

Premature Aging ◽

Neurological Features ◽

Oxygen Tensions ◽

Base Excision

Cockayne Syndrome (CS) is an autosomal recessive neurodegenerative premature aging disorder associated with defects in nucleotide excision repair (NER). Cells from CS patients, with mutations in CSA or CSB genes, present elevated levels of reactive oxygen species (ROS) and are defective in the repair of a variety of oxidatively generated DNA lesions. In this study, six purine lesions were ascertained in wild type (wt) CSA, defective CSA, wtCSB and defective CSB-transformed fibroblasts under different oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%). In particular, the four 5′,8-cyclopurine (cPu) and the two 8-oxo-purine (8-oxo-Pu) lesions were accurately quantified by LC-MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. cPu levels were found comparable to 8-oxo-Pu in all cases (3–6 lesions/106 nucleotides), slightly increasing on going from hyperoxia to physioxia to hypoxia. Moreover, higher levels of four cPu were observed under hypoxia in both CSA and CSB-defective cells as compared to normal counterparts, along with a significant enhancement of 8-oxo-Pu. These findings revealed that exposure to different oxygen tensions induced oxidative DNA damage in CS cells, repairable by NER or base excision repair (BER) pathways. In NER-defective CS patients, these results support the hypothesis that the clinical neurological features might be connected to the accumulation of cPu. Moreover, the elimination of dysfunctional mitochondria in CS cells is associated with a reduction in the oxidative DNA damage.

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Dynamic Compartmentalization of Base Excision Repair Proteins in Response to Nuclear and Mitochondrial Oxidative Stress

Molecular and Cellular Biology ◽

10.1128/mcb.01357-08 ◽

2008 ◽

Vol 29 (3) ◽

pp. 794-807 ◽

Cited By ~ 31

Author(s):

Lyra M. Griffiths ◽

Dan Swartzlander ◽

Kellen L. Meadows ◽

Keith D. Wilkinson ◽

Anita H. Corbett ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Dna Repair ◽

Base Excision Repair ◽

Oxidative Dna Damage ◽

Excision Repair ◽

Intracellular Localization ◽

Genotoxic Stress ◽

Mitochondrial Oxidative Stress ◽

Base Excision

ABSTRACT DNAs harbored in both nuclei and mitochondria of eukaryotic cells are subject to continuous oxidative damage resulting from normal metabolic activities or environmental insults. Oxidative DNA damage is primarily reversed by the base excision repair (BER) pathway, initiated by N-glycosylase apurinic/apyrimidinic (AP) lyase proteins. To execute an appropriate repair response, BER components must be distributed to accommodate levels of genotoxic stress that may vary considerably between nuclei and mitochondria, depending on the growth state and stress environment of the cell. Numerous examples exist where cells respond to signals, resulting in relocalization of proteins involved in key biological transactions. To address whether such dynamic localization contributes to efficient organelle-specific DNA repair, we determined the intracellular localization of the Saccharomyces cerevisiae N-glycosylase/AP lyases, Ntg1 and Ntg2, in response to nuclear and mitochondrial oxidative stress. Fluorescence microscopy revealed that Ntg1 is differentially localized to nuclei and mitochondria, likely in response to the oxidative DNA damage status of the organelle. Sumoylation is associated with targeting of Ntg1 to nuclei containing oxidative DNA damage. These studies demonstrate that trafficking of DNA repair proteins to organelles containing high levels of oxidative DNA damage may be a central point for regulating BER in response to oxidative stress.

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Base excision repair of oxidative DNA damage and association with cancer and aging

Carcinogenesis ◽

10.1093/carcin/bgn250 ◽

2008 ◽

Vol 30 (1) ◽

pp. 2-10 ◽

Cited By ~ 344

Author(s):

S. Maynard ◽

S. H. Schurman ◽

C. Harboe ◽

N. C. de Souza-Pinto ◽

V. A. Bohr

Keyword(s):

Dna Damage ◽

Base Excision Repair ◽

Oxidative Dna Damage ◽

Excision Repair ◽

Base Excision

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The Effect ofMsh2Knockdown on Toxicity Induced bytert-Butyl-hydroperoxide, Potassium Bromate, and Hydrogen Peroxide in Base Excision Repair Proficient and Deficient Cells

BioMed Research International ◽

10.1155/2013/152909 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

N. Cooley ◽

R. H. Elder ◽

A. C. Povey

Keyword(s):

Dna Damage ◽

Base Excision Repair ◽

Oxidative Dna Damage ◽

Excision Repair ◽

Shrna Expression ◽

Cellular Toxicity ◽

Dna Mismatch ◽

Shrna Expression Vector ◽

Repair Systems ◽

Base Excision

The DNA mismatch repair (MMR) and base excision repair (BER) systems are important determinants of cellular toxicity following exposure to agents that cause oxidative DNA damage. To examine the interactions between these different repair systems, we examined whether toxicity, induced byt-BOOH and KBrO3, differs in BER proficient (Mpg+/+,Nth1+/+) and deficient (Mpg−/−,Nth1−/−) mouse embryonic fibroblasts (MEFs) followingMsh2knockdown of between 79 and 88% using an shRNA expression vector.Msh2knockdown inNth1+/+cells had no effect ont-BOOH and KBrO3induced toxicity as assessed by an MTT assay; knockdown inNth1−/−cells resulted in increased resistance tot-BOOH and KBrO3, a result consistent with Nth1 removing oxidised pyrimidines.Msh2knockdown inMpg+/+cells had no effect ont-BOOH toxicity but increased resistance to KBrO3; inMpg−/−cells,Msh2knockdown increased cellular sensitivity to KBrO3but increased resistance to t-BOOH, suggesting a role forMpgin removing DNA damage induced by these agents. MSH2 dependent and independent pathways then determine cellular toxicity induced by oxidising agents. A complex interaction between MMR and BER repair systems, that is, exposure dependent, also exists to determine cellular toxicity.

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Roles of base excision repair enzymes Nth1p and Apn2p from Schizosaccharomyces pombe in processing alkylation and oxidative DNA damage

DNA Repair ◽

10.1016/j.dnarep.2005.06.009 ◽

2005 ◽

Vol 4 (11) ◽

pp. 1270-1280 ◽

Cited By ~ 21

Author(s):

Takanori Sugimoto ◽

Emi Igawa ◽

Haruna Tanihigashi ◽

Mayumi Matsubara ◽

Hiroshi Ide ◽

...

Keyword(s):

Dna Damage ◽

Schizosaccharomyces Pombe ◽

Base Excision Repair ◽

Oxidative Dna Damage ◽

Excision Repair ◽

Repair Enzymes ◽

Base Excision

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Complexities of the DNA base excision repair pathway for repair of oxidative DNA damage

Environmental and Molecular Mutagenesis ◽

10.1002/em.1070 ◽

2001 ◽

Vol 38 (2-3) ◽

pp. 180-190 ◽

Cited By ~ 87

Author(s):

Sankar Mitra ◽

Istvan Boldogh ◽

Tadahide Izumi ◽

Tapas K. Hazra

Keyword(s):

Dna Damage ◽

Base Excision Repair ◽

Oxidative Dna Damage ◽

Excision Repair ◽

Repair Pathway ◽

Base Excision Repair Pathway ◽

Dna Base Excision Repair ◽

Dna Base ◽

Base Excision

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DNA polymerase X from Deinococcus radiodurans implicated in bacterial tolerance to DNA damage is characterized as a short patch base excision repair polymerase

Microbiology ◽

10.1099/mic.0.029223-0 ◽

2009 ◽

Vol 155 (9) ◽

pp. 3005-3014 ◽

Cited By ~ 19

Author(s):

Nivedita P. Khairnar ◽

Hari S. Misra

Keyword(s):

Dna Damage ◽

Dna Polymerase ◽

Base Excision Repair ◽

Deinococcus Radiodurans ◽

Excision Repair ◽

Strand Break ◽

Klenow Fragment ◽

E Coli ◽

Base Excision

The Deinococcus radiodurans R1 genome encodes an X-family DNA repair polymerase homologous to eukaryotic DNA polymerase β. The recombinant deinococcal polymerase X (PolX) purified from transgenic Escherichia coli showed deoxynucleotidyltransferase activity. Unlike the Klenow fragment of E. coli, this enzyme showed short patch DNA synthesis activity on heteropolymeric DNA substrate. The recombinant enzyme showed 5′-deoxyribose phosphate (5′-dRP) lyase activity and base excision repair function in vitro, with the help of externally supplied glycosylase and AP endonuclease functions. A polX disruption mutant of D. radiodurans expressing 5′-dRP lyase and a truncated polymerase domain was comparatively less sensitive to γ-radiation than a polX deletion mutant. Both mutants showed higher sensitivity to hydrogen peroxide. Excision repair mutants of E. coli expressing this polymerase showed functional complementation of UV sensitivity. These results suggest the involvement of deinococcal polymerase X in DNA-damage tolerance of D. radiodurans, possibly by contributing to DNA double-strand break repair and base excision repair.

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