Mechanistic basis for microhomology identification and genome scarring by polymerase theta
| Autor: | Wanjuan Feng, Pablo Carvajal-Garcia, Jeff Sekelsky, Steven A. Roberts, Jang Eun Cho, Richard D. Wood, Juan Carvajal-Garcia, Dale A. Ramsden, Gaorav P. Gupta |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Genome instability
DNA End-Joining Repair BRCA chromosome break repair DNA Polymerase Theta mutational signatures Breast Neoplasms Pol theta DNA-Directed DNA Polymerase Computational biology Biology Biochemistry Genome Genomic Instability Germline Resection Mice chemistry.chemical_compound Animals Humans DNA Breaks Double-Stranded Nucleotide Germ-Line Mutation Polymerase BRCA2 Protein Mice Knockout chemistry.chemical_classification Multidisciplinary BRCA1 Protein Genome Human Chromosome Breakage Biological Sciences Fibroblasts microhomology-mediated end joining Mice Inbred C57BL Microhomology-mediated end joining chemistry biology.protein Female DNA |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.1921791117 |
| Popis: | Significance Repair of chromosome breaks by polymerase theta-mediated end joining (TMEJ) requires short sequence identities in flanking DNA (microhomologies)—a sequence-context constraint that is unique among DNA repair pathways. Though microhomologies have a central role in TMEJ, it has been uncertain whether an organized mechanism to identify them even exists. Using a combination of chromosomal and extrachromosomal substrates, we describe how polymerase theta efficiently locates microhomologies when present, and creates them de novo when absent. We show how this generates a pattern of microhomology-mediated end joining products that is sufficiently distinct from other end joining pathways and that it can be used as a biomarker for TMEJ activity in cancer genomes. DNA polymerase theta mediates an end joining pathway (TMEJ) that repairs chromosome breaks. It requires resection of broken ends to generate long, 3′ single-stranded DNA tails, annealing of complementary sequence segments (microhomologies) in these tails, followed by microhomology-primed synthesis sufficient to resolve broken ends. The means by which microhomologies are identified is thus a critical step in this pathway, but is not understood. Here we show microhomologies are identified by a scanning mechanism initiated from the 3′ terminus and favoring bidirectional progression into flanking DNA, typically to a maximum of 15 nucleotides into each flank. Polymerase theta is frequently insufficiently processive to complete repair of breaks in microhomology-poor, AT-rich regions. Aborted synthesis leads to one or more additional rounds of microhomology search, annealing, and synthesis; this promotes complete repair in part because earlier rounds of synthesis generate microhomologies de novo that are sufficiently long that synthesis is more processive. Aborted rounds of synthesis are evident in characteristic genomic scars as insertions of 3 to 30 bp of sequence that is identical to flanking DNA (“templated” insertions). Templated insertions are present at higher levels in breast cancer genomes from patients with germline BRCA1/2 mutations, consistent with an addiction to TMEJ in these cancers. Our work thus describes the mechanism for microhomology identification and shows how it both mitigates limitations implicit in the microhomology requirement and generates distinctive genomic scars associated with pathogenic genome instability. |
| Databáze: | OpenAIRE |
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