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Replication stress at microsatellites causes DNA double-strand breaks and break-induced replication.

ABSTRACT: Short tandemly repeated DNA sequences, termed microsatellites, are abundant in the human genome. These microsatellites exhibit length instability and susceptibility to DNA double-strand breaks (DSBs) due to their tendency to form stable non-B DNA structures. Replication-dependent microsatellite DSBs are linked to genome instability signatures in human developmental diseases and cancers. To probe the causes and consequences of microsatellite DSBs, we designed a dual-fluorescence reporter system to detect DSBs at expanded (CTG/CAG) _n and polypurine/polypyrimidine (Pu/Py) mirror repeat structures alongside the c-myc replication origin integrated at a single ectopic chromosomal site. Restriction cleavage near the (CTG/CAG)₁₀₀ microsatellite leads to homology-directed single-strand annealing between flanking AluY elements and reporter gene deletion that can be detected by flow cytometry. However, in the absence of restriction cleavage, endogenous and exogenous replication stressors induce DSBs at the (CTG/CAG)₁₀₀ and Pu/Py microsatellites. DSBs map to a narrow region at the downstream edge of the (CTG)₁₀₀ lagging-strand template. (CTG/CAG) _n chromosome fragility is repeat length-dependent, whereas instability at the (Pu/Py) microsatellites depends on replication polarity. Strikingly, restriction-generated DSBs and replication-dependent DSBs are not repaired by the same mechanism. Knockdown of DNA damage response proteins increases (Rad18, polymerase (Pol) η, Pol κ) or decreases (Mus81) the sensitivity of the (CTG/CAG)₁₀₀ microsatellites to replication stress. Replication stress and DSBs at the ectopic (CTG/CAG)₁₀₀ microsatellite lead to break-induced replication and high-frequency mutagenesis at a flanking thymidine kinase gene. Our results show that non-B structure-prone microsatellites are susceptible to replication-dependent DSBs that cause genome instability.

SUBMITTER: Gadgil RY

PROVIDER: S-EPMC7650239 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Common fragile sites (CFSs) are specific regions of all individuals' genome that are predisposed to DNA double strand breaks (DSBs) and undergo subsequent rearrangements. CFS formation can be induced in vitro by mild level of DNA replication stress, such as DNA polymerase inhibition or nucleotide pool disturbance. The mechanisms of CFS formation have been linked to DNA replication timing control, transcription activities, as well as chromatin organization. However, it is unclear what specific cis- or trans-factors regulate the interplay between replication and transcription that determine CFS formation. We recently reported genome-wide mapping of DNA DSBs under replication stress induced by aphidicolin in human lymphoblastoids for the first time. Here, we systematically compared these DSBs with regards to nearby epigenomic features mapped in the same cell line from published studies. We demonstrate that aphidicolin-induced DSBs are strongly correlated with histone 3 lysine 36 trimethylation, a marker for active transcription. We further demonstrate that this DSB signature is a composite effect by the dual treatment of aphidicolin and its solvent, dimethylsulfoxide, the latter of which potently induces transcription on its own. We also present complementing evidence for the association between DSBs and 3D chromosome architectural domains with high density gene cluster and active transcription. Additionally, we show that while DSBs were detected at all but one of the fourteen finely mapped CFSs, they were not enriched in the CFS core sequences and rather demarcated the CFS core region. Related to this point, DSB density was not higher in large genes of greater than 300 kb, contrary to reported enrichment of CFS sites at these large genes. Finally, replication timing analyses demonstrate that the CFS core region contain initiation events, suggesting that altered replication dynamics are responsible for CFS formation in relatively higher level of replication stress.

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Replication stress at microsatellites causes DNA double-strand breaks and break-induced replication.

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