Ribonucleotide incorporation enables repair of chromosome breaks by nonhomologous end joining

• Published in: Science (American Association for the Advancement of Science) Vol. 361; no. 6407; pp. 1126 - 1129
• Main Authors: Pryor, John M, Conlin, Michael P, Carvajal-Garcia, Juan, Luedeman, Megan E, Luthman, Adam J, Small, George W, Ramsden, Dale A
• Format: Journal Article
• Language: English
• Published: United States The American Association for the Advancement of Science 14-09-2018
• Subjects: Animals; Bacterial Proteins; Cell Line; Chromosome Breakage; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR-Associated Protein 9; Deoxyribonucleic acid; DNA; DNA damage; DNA End-Joining Repair; DNA Repair; DNA-Directed DNA Polymerase - metabolism; Endonucleases; Eosinophil-Derived Neurotoxin - genetics; Eosinophil-Derived Neurotoxin - metabolism; Eukaryotes; Fibroblasts; Genome editing; Genomes; Genomic Instability; Intermediates; Kinetics; Mice; Molecular biology; Non-homologous end joining; Repair; Repair & maintenance; Ribonucleic acid; Ribonucleotides; Ribonucleotides - metabolism; RNA; V(D)J Recombination
• ISSN: 0036-8075; 1095-9203; 1095-9203
• DOI: 10.1126/science.aat2477

The nonhomologous end-joining (NHEJ) pathway preserves genome stability by ligating the ends of broken chromosomes together. It employs end-processing enzymes, including polymerases, to prepare ends for ligation. We show that two such polymerases incorporate primarily ribonucleotides during NHEJ-an exception to the central dogma of molecular biology-both during repair of chromosome breaks made by Cas9 and during V(D)J recombination. Moreover, additions of ribonucleotides but not deoxynucleotides effectively promote ligation. Repair kinetics suggest that ribonucleotide-dependent first-strand ligation is followed by complementary strand repair with deoxynucleotides, then by replacement of ribonucleotides embedded in the first strand with deoxynucleotides. Our results indicate that as much as 65% of cellular NHEJ products have transiently embedded ribonucleotides, which promote flexibility in repair at the cost of more fragile intermediates.