The coexistence of DNA replication and transcription on the same DNA template requires the coordination of the machineries involved in both processes to prevent harmful collisions. Such events, if not properly controlled, can create barriers to DNA replication, leading to replication stress (RS) and genome instability. Inactivation of several RS and DNA damage repair genes results in embryonic lethality, suggesting that rapidly dividing embryonic cells require efficient RS counteracting mechanisms for survival. Cancer cells, similar to embryos, divide fast and require repair activities to tolerate high rates of DNA transcription and replication. We developed transcription and replication competent Xenopus cell-free extracts, which, in contrast to the egg extracts, are made from embryos isolated at mid-blastula transition (MBT), a crucial stage in which maternal mRNAs are degraded and zygotic transcription is activated. We observed many aberrant RIs in embryo-derived extracts, absent in transcription-incompetent eggs. These structures could be the consequence of a transcription intermediate (TI) containing a paused RNA polymerase, expanded by nascent RNA invading DNA ahead or behind the transcription bubble to generate a DNA-RNA hybrid (R-loop). Alternatively, they could be due to stalled RIs cut and processed by DNA repair factors. Paused TIs could interfere with the progression of a nearby replication fork, which appears to be stalled as shown by un-replicated ssDNA at the fork junction, a feature common to RIs isolated from transcribing-replicating embryo extracts. Also frequent are spontaneous reversed forks, formed by the annealing of complementary nascent strands at stalled forks. These aberrant RIs are increased when transcription is perturbed. Future study will use imaging and biochemical approaches to identify novel factors required to prevent, tolerate, or resolve collisions in biological contexts where RS usually occurs as a result of active transcription.
Transcription-replication interference: implications for genome integrity / L. Falbo, H. Chmes, Y. Wijesekarahanthi, V. Costanzo.. ((Intervento presentato al 17th. convegno SIBBM 2022 - Frontiers in Molecular Biology - The RNA world 3.0 tenutosi a Roma : 20-22 Giugno nel 2022.
Transcription-replication interference: implications for genome integrity
L. Falbo;
2022
Abstract
The coexistence of DNA replication and transcription on the same DNA template requires the coordination of the machineries involved in both processes to prevent harmful collisions. Such events, if not properly controlled, can create barriers to DNA replication, leading to replication stress (RS) and genome instability. Inactivation of several RS and DNA damage repair genes results in embryonic lethality, suggesting that rapidly dividing embryonic cells require efficient RS counteracting mechanisms for survival. Cancer cells, similar to embryos, divide fast and require repair activities to tolerate high rates of DNA transcription and replication. We developed transcription and replication competent Xenopus cell-free extracts, which, in contrast to the egg extracts, are made from embryos isolated at mid-blastula transition (MBT), a crucial stage in which maternal mRNAs are degraded and zygotic transcription is activated. We observed many aberrant RIs in embryo-derived extracts, absent in transcription-incompetent eggs. These structures could be the consequence of a transcription intermediate (TI) containing a paused RNA polymerase, expanded by nascent RNA invading DNA ahead or behind the transcription bubble to generate a DNA-RNA hybrid (R-loop). Alternatively, they could be due to stalled RIs cut and processed by DNA repair factors. Paused TIs could interfere with the progression of a nearby replication fork, which appears to be stalled as shown by un-replicated ssDNA at the fork junction, a feature common to RIs isolated from transcribing-replicating embryo extracts. Also frequent are spontaneous reversed forks, formed by the annealing of complementary nascent strands at stalled forks. These aberrant RIs are increased when transcription is perturbed. Future study will use imaging and biochemical approaches to identify novel factors required to prevent, tolerate, or resolve collisions in biological contexts where RS usually occurs as a result of active transcription.
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