Dataset Information

Molecular basis of microhomology-mediated end-joining by purified full-length Pol?.

ABSTRACT: DNA polymerase ? (Pol?) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Pol? performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Pol? MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Pol? MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Pol? complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Pol? multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Pol?.

SUBMITTER: Black SJ

PROVIDER: S-EPMC6764996 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Publications

Molecular basis of microhomology-mediated end-joining by purified full-length Polθ.

Black Samuel J SJ Ozdemir Ahmet Y AY Kashkina Ekaterina E Kent Tatiana T Rusanov Timur T Ristic Dejan D Shin Yeonoh Y Suma Antonio A Hoang Trung T Chandramouly Gurushankar G Siddique Labiba A LA Borisonnik Nikita N Sullivan-Reed Katherine K Mallon Joseph S JS Skorski Tomasz T Carnevale Vincenzo V Murakami Katsuhiko S KS Wyman Claire C Pomerantz Richard T RT

Nature communications 20190927 1

DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, a ...[more]

PMID: 31562312

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Project description:One key problem in precision genome editing is the unpredictable plurality of sequence outcomes at the site of targeted DNA double stranded breaks (DSBs). This is due to the typical activation of the versatile Non-homologous End Joining (NHEJ) pathway. Such unpredictability limits the utility of somatic gene editing for applications including gene therapy and functional genomics. For germline editing work, the accurate reproduction of the identical alleles using NHEJ is a labor intensive process. In this study, we propose Microhomology-mediated End Joining (MMEJ) as a viable solution for improving somatic sequence homogeneity in vivo, capable of generating a single predictable allele at high rates (56% ~ 86% of the entire mutant allele pool). Using a combined dataset from zebrafish (Danio rerio) in vivo and human HeLa cell in vitro, we identified specific contextual sequence determinants surrounding genomic DSBs for robust MMEJ pathway activation. We then applied our observation to prospectively design MMEJ-inducing sgRNAs against a variety of proof-of-principle genes and demonstrated high levels of mutant allele homogeneity. MMEJ-based DNA repair at these target loci successfully generated F0 mutant zebrafish embryos and larvae that faithfully recapitulated previously reported, recessive, loss-of-function phenotypes. We also tested the generalizability of our approach in cultured human cells. Finally, we provide a novel algorithm, MENTHU (http://genesculpt.org/menthu/), for improved and facile prediction of candidate MMEJ loci. We believe that this MMEJ-centric approach will have a broader impact on genome engineering and its applications. For example, whereas somatic mosaicism hinders efficient recreation of knockout mutant allele at base pair resolution via the standard NHEJ-based approach, we demonstrate that F0 founders transmitted the identical MMEJ allele of interest at high rates. Most importantly, the ability to directly dictate the reading frame of an endogenous target will have important implications for gene therapy applications in human genetic diseases.

Dataset Information

Molecular basis of microhomology-mediated end-joining by purified full-length Pol?.

Publications

Molecular basis of microhomology-mediated end-joining by purified full-length Polθ.

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