Small tandem DNA duplications result from CST-guided Pol \u03b1-primase action at DNA break termini

Joost Schimmel,Núria Muñoz-Subirana,Robin Van Schendel,Marcel Tijsterman,Hanneke Kool

doi:10.1038/s41467-021-25154-w

Joost Schimmel, Núria Muñoz-Subirana + Show 3 more

Open Access

https://doi.org/10.1038/s41467-021-25154-w

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Abstract

Small tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Recent studies have suggested that DNA double-strand breaks are causal to this mutational class, but the underlying mechanism remains elusive. Here, we identify a crucial role for DNA polymerase α (Pol α)-primase in tandem duplication formation at breaks having complementary 3′ ssDNA protrusions. By including so-called primase deserts in CRISPR/Cas9-induced DNA break configurations, we reveal that fill-in synthesis preferentially starts at the 3′ tip, and find this activity to be dependent on 53BP1, and the CTC1-STN1-TEN1 (CST) and Shieldin complexes. This axis generates near-blunt ends specifically at DNA breaks with 3′ overhangs, which are subsequently repaired by non-homologous end-joining. Our study provides a mechanistic explanation for a mutational signature abundantly observed in the genomes of species and cancer cells.

Highlights

Small tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers
While all tested components of the non-homologous end-joining pathway (NHEJ) axis are required for mutagenic NHEJ on double-strand breaks (DSBs) with 3′ protrusions, we found that 53BP1 separates from Shld[2] and CST for the repair of DSBs with 5′ protrusions: whereas the mutational signatures in Shld[2] and CST deficient cells marginally differ from that obtained from wild-type cells (Supplementary Figs. 5f–i, 2b–d), profiles in Tp53bp1−/− cells strongly deviate and are dependent on microhomology as those observed in Ku80 deficient cells (Supplementary Fig. 5h, i compared to Fig. 2c, d)
Our study demonstrates that besides the recently described role in preventing the loss of genetic information flanking DSBs, the 53BP1-Shieldin-CST axis contributes to genome expansion by polymerase α (Pol α)-primase mediated fill-in synthesis of complementary 3′ ssDNA protrusions, resulting in small tandem duplications

Summary

Introduction

Small tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Bioinformatic analysis of naturally occurring DNA insertions in humans and plants has hinted towards a model where TDs arise through error-prone repair of DNA double-strand breaks (DSBs) that may result from nearby single-strand breaks in opposing strands[1,14] This model was recently supported by the observation that TDs abundantly form at DSBs with small overhangs induced by Cas9-nickase enzymes in different species[15,16,17]. We demonstrate that CST-Polα can initiate close to the termini of DSBs with 3′ protruding ends in a 53BP1 and Shieldin dependent manner We show that this activity induces erroneous duplication of genomic sequences at DSBs with complementary ends, resulting in small DNA duplications similar to those observed in human pathologies

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