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

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Nucleotide excision repair (NER) is a crucial DNA repair pathway for bulky adducts.
• Current genome-wide methods for studying DNA repair have limited resolution due to measuring signal loss over time.

Purpose of the Study:

• To develop and present a novel method, excision repair sequencing (XR-seq), for studying DNA adduct repair.
• To enable direct detection of DNA repair events with high resolution.

Main Methods:

• XR-seq isolates excised oligomers from NER reactions via cell fractionation and immunoprecipitation.
• Excised oligomers are processed into sequencing libraries, with options for damage reversal or conversion to double-stranded DNA using translesion synthesis (TLS) polymerases.
• The protocol is adaptable for use in humans, mice, Arabidopsis thaliana, yeast, and Escherichia coli.

Main Results:

• XR-seq directly detects the DNA repair signal, providing a significant advantage over methods relying on the loss of damage signal.
• The XR-seq protocol yields a library within 7-9 days.
• The method is applicable across a wide range of organisms and DNA adduct types, including irreversible ones like benzo[a]pyrene adducts.

Conclusions:

• XR-seq is a versatile and high-resolution technique for studying nucleotide excision repair of DNA adducts.
• This method offers a significant advancement in the study of DNA repair mechanisms across diverse species.