Dataset Information

Genome-wide mapping of nucleotide excision repair with XR-seq.

ABSTRACT: Nucleotide excision repair is a versatile mechanism to repair a variety of bulky DNA adducts. We developed excision repair sequencing (XR-seq) to study nucleotide excision repair of DNA adducts in humans, mice, Arabidopsis thaliana, yeast and Escherichia coli. In this protocol, the excised oligomers, generated in the nucleotide excision repair reaction, are isolated by cell lysis and fractionation, followed by immunoprecipitation with damage- or repair factor-specific antibodies from the non-chromatin fraction. The single-stranded excised oligomers are ligated to adapters and re-immunoprecipitated with damage-specific antibodies. The DNA damage in the excised oligomers is then reversed by enzymatic or chemical reactions before being converted into a sequencing library by PCR amplification. Alternatively, the excised oligomers containing DNA damage, especially those containing irreversible DNA damage such as benzo[a]pyrene-induced DNA adducts, can be converted to a double-stranded DNA (dsDNA) form by using appropriate translesion DNA synthesis (TLS) polymerases and then can be amplified by PCR. The current genome-wide approaches for studying repair measure the loss of damage signal with time, which limits their resolution. By contrast, an advantage of XR-seq is that the repair signal is directly detected above a background of zero. An XR-seq library using the protocol described here can be obtained in 7-9 d.

SUBMITTER: Hu J

PROVIDER: S-EPMC6429938 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Genome-wide mapping of nucleotide excision repair with XR-seq.

Hu Jinchuan J Li Wentao W Adebali Ogun O Yang Yanyan Y Oztas Onur O Selby Christopher P CP Sancar Aziz A

Nature protocols 20190101 1

Nucleotide excision repair is a versatile mechanism to repair a variety of bulky DNA adducts. We developed excision repair sequencing (XR-seq) to study nucleotide excision repair of DNA adducts in humans, mice, Arabidopsis thaliana, yeast and Escherichia coli. In this protocol, the excised oligomers, generated in the nucleotide excision repair reaction, are isolated by cell lysis and fractionation, followed by immunoprecipitation with damage- or repair factor-specific antibodies from the non-chr ...[more]

PMID: 30552409

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Project description:The incorporation of ribonucleotides in DNA has attracted considerable notice in recent years, since the pool of ribonucleotides can exceed that of the deoxyribonucleotides by at least 10-20-fold, and single ribonucleotide incorporation by DNA polymerases appears to be a common event. Moreover ribonucleotides are potentially mutagenic and lead to genome instability. As a consequence, errantly incorporated ribonucleotides are rapidly repaired in a process dependent upon RNase H enzymes. On the other hand, global genomic nucleotide excision repair (NER) in prokaryotes and eukaryotes removes damage caused by covalent modifications that typically distort and destabilize DNA through the production of lesions derived from bulky chemical carcinogens, such as polycyclic aromatic hydrocarbon metabolites, or via crosslinking. However, a recent study challenges this lesion-recognition paradigm. The work of Vaisman et al. (2013) [34] reveals that even a single ribonucleotide embedded in a deoxyribonucleotide duplex is recognized by the bacterial NER machinery in vitro. In their report, the authors show that spontaneous mutagenesis promoted by a steric-gate pol V mutant increases in uvrA, uvrB, or uvrC strains lacking rnhB (encoding RNase HII) and to a greater extent in an NER-deficient strain lacking both RNase HI and RNase HII. Using purified UvrA, UvrB, and UvrC proteins in in vitro assays they show that despite causing little distortion, a single ribonucleotide embedded in a DNA duplex is recognized and doubly-incised by the NER complex. We present the hypothesis to explain the recognition and/or verification of this small lesion, that the critical 2'-OH of the ribonucleotide - with its unique electrostatic and hydrogen bonding properties - may act as a signal through interactions with amino acid residues of the prokaryotic NER complex that are not possible with DNA. Such a mechanism might also be relevant if it were demonstrated that the eukaryotic NER machinery likewise incises an embedded ribonucleotide in DNA.

Dataset Information

Genome-wide mapping of nucleotide excision repair with XR-seq.

Publications

Genome-wide mapping of nucleotide excision repair with XR-seq.

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