Untersuchungen zum Einfluss von Sulforaphan auf die DNA-Einzelstrangbruch- und Nukleotidexzisionsreparatur

Eine Risikobewertung von Broccoli-basierten Nahrungsergänzungsmitteln fehlt bis dato. Die vorliegende Arbeit leistet in diesem Kontext nun erstmals einen kritischen Beitrag zur Auswirkung der bioaktiven Substanz Sulforaphan (SFN) auf essentielle DNA-Reparaturprozesse, die anhand der Prozessierung und Reparatur zweier Modellläsionen in Zellkulturen beurteilt wurden. Es zeigte sich, dass SFN beide Reparaturwege inhibiert und Zink-bindende Strukturen dabei potentielle Angriffspunkte darstellen

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

Summary: BET bromodomain proteins are required for oncogenic transcription activities, and BET inhibitors have been rapidly advanced into clinical trials. Understanding the effects of BET inhibition on processes such as DNA replication will be important for future clinical applications. Here, we show that BET inhibition, and specifically inhibition of BRD4, causes replication stress through a rapid overall increase in RNA synthesis. We provide evidence that BET inhibition acts by releasing P-TEFb from its inhibitor HEXIM1, promoting interference between transcription and replication. Unusually, these transcription-replication conflicts do not activate the ATM/ATR-dependent DNA damage response but recruit the homologous recombination factor RAD51. Both HEXIM1 and RAD51 promote BET inhibitor-induced fork slowing but also prevent a DNA damage response. Our data suggest that BET inhibitors slow replication through concerted action of transcription and recombination machineries and shed light on the importance of replication stress in the action of this class of experimental cancer drugs. : Bowry et al. show that BET inhibitors, emerging cancer therapeutics that target transcription programs, cause conflicts between transcription and replication by deregulating P-TEFb. These conflicts recruit the homologous recombination factor RAD51, which slows down replication and suppresses DNA damage. This highlights the importance of replication stress for BET inhibitor treatment. Keywords: JQ1, I-BET151, BRD4, P-TEFb, homologous recombination, replication fork, replication stress, DNA damage, cance

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

Mechanisms of synthetic lethality between BRCA1/2 and 53BP1 deficiencies and DNA polymerase theta targeting

Abstract A synthetic lethal relationship exists between disruption of polymerase theta (Polθ), and loss of either 53BP1 or homologous recombination (HR) proteins, including BRCA1; however, the mechanistic basis of these observations are unclear. Here we reveal two distinct mechanisms of Polθ synthetic lethality, identifying dual influences of 1) whether Polθ is lost or inhibited, and 2) the underlying susceptible genotype. Firstly, we find that the sensitivity of BRCA1/2- and 53BP1-deficient cells to Polθ loss, and 53BP1-deficient cells to Polθ inhibition (ART558) requires RAD52, and appropriate reduction of RAD52 can ameliorate these phenotypes. We show that in the absence of Polθ, RAD52 accumulations suppress ssDNA gap-filling in G2/M and encourage MRE11 nuclease accumulation. In contrast, the survival of BRCA1-deficient cells treated with Polθ inhibitor are not restored by RAD52 suppression, and ssDNA gap-filling is prevented by the chemically inhibited polymerase itself. These data define an additional role for Polθ, reveal the mechanism underlying synthetic lethality between 53BP1, BRCA1/2 and Polθ loss, and indicate genotype-dependent Polθ inhibitor mechanisms