Biology subjects

Piberger, A. L.

Publications and source records attributed to Piberger, A. L..

3 recordsLinked to original sources

bioRxiv2019

PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts

Obstacles on the DNA template can lead to DNA replication fork stalling and genomic rearrangements. RAD51-mediated homologous recombination (HR) can promote restart and repair of stalled forks, but also post-replicative repair once the obstacle has been bypassed. Bulky DNA adducts are important replication-blocking lesions induced by environmental carcinogens, but it is not known whether they activate HR directly at stalled forks, or at gaps left behind ongoing forks. Here we show that in mammalian cells, bulky adducts predominantly induce HR at post-replicative gaps formed by the DNA/RNA primase PrimPol. Using BPDE and other bulky model lesions, we report that RAD51 is not recruited to stalled or collapsed forks, but instead to long gaps formed by PrimPol re-priming activity and resection by MRE11 and EXO1. In contrast, RAD51 loading at DSBs does not require PrimPol. At bulky adducts, PrimPol is required for the induction of sister chromatid exchanges and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in DNA damage tolerance by homologous recombination.

molecular biology↗

bioRxiv2018

BET inhibition induces HEXIM1- and RAD51-dependent conflicts between transcription and replication

BET bromodomain proteins are epigenetic readers required for oncogenic transcription activities, and BET inhibitors have been rapidly advanced into clinical trials. Understanding the effects of BET inhibition on other nuclear processes such as DNA replication will be important for future clinical applications. Here we show that BET inhibition causes replication stress in cancer and non-cancer cells due to a rapid burst in global RNA synthesis and interference of transcription with replication. We identify BRD4 as the main BET inhibitor target in this process and provide evidence that BRD4 inhibition causes transcription-replication interference through release of P-TEFb from its inhibitor HEXIM1, promoting RNA Polymerase II phosphorylation. Unusually, BET inhibitor-induced transcription-replication interference does not activate the classic ATM/ATR-dependent DNA damage response. We show however that they promote foci formation of the homologous recombination factor RAD51. Both HEXIM1 and RAD51 are required for BET inhibitor-induced fork slowing, but rescuing fork slowing by HEXIM1 or RAD51 depletion activate a DNA damage response. Our data support a new mechanism where BRD4 inhibition slows replication and suppresses DNA damage through concerted action of transcription and homologous recombination machineries. They shed new light on the roles of DNA replication and recombination in the action of this new class of cancer drugs.

molecular biology↗

bioRxiv2018

PARP1 and PARP2 stabilise replication forks at base excision repair intermediates through Fbh1-dependent Rad51 regulation

PARP1 regulates the repair of DNA single strand breaks (SSBs) generated directly, or during base excision repair (BER). However, the role of PARP2 in these and other repair mechanisms is unknown. Here, we report a requirement for PARP2 in stabilising replication forks that encounter BER intermediates through Fbh1-dependent regulation of Rad51. Whilst PARP2 is dispensable for tolerance of cells to SSBs or homologous recombination dysfunction, it is redundant with PARP1 in BER. Therefore, combined disruption of PARP1 and PARP2 leads to defective BER, resulting in elevated levels of replication associated DNA damage due to an inability to stabilise Rad51 at damaged replication forks and prevent uncontrolled DNA resection. Together, our results demonstrate how PARP1 and PARP2 regulate two independent, but intrinsically linked aspects of DNA base damage tolerance by promoting BER directly, and through stabilising replication forks that encounter BER intermediates.

cell biology↗