A distinct role for recombination repair factors in an immediate cellular response to transcription-replication conflicts

TY - BOOK

T1 - A distinct role for recombination repair factors in an immediate cellular response to transcription-replication conflicts

AU - Shao, Xin

PY - 2018

Y1 - 2018

N2 - Various perturbations, originating from endogenous or exogenous sources, can challenge DNA replication to compromise its integrity, and pose a serious threat to chromosomal stability. The cell has evolved various cellular pathways and different checkpoint activities in order to cope with DNA damage. Emerging evidence suggests that transcription-replication conflicts (TRCs) are a potent source of mutagenesis and carcinogenesis (Wilson et al. 2015; Hamperl et al. 2017). The replication forks compete with the transcription machinery on the same DNA template, and this may lead to replication fork arrest. Normally, transcription and replication are coordinated to minimize TRCs (Wei et al.1998). However, deregulated replication, rapid cell division and high transcription activity may contribute to frequent TRCs, particularly in cancer cells (Hanahan and Weinberg 2011; Techer et al. 2017). Moreover, TRCs are inevitable at the largest genes because it takes more than one cell cycle to complete transcription of these genes (Helmrich et al. 2011). In this study, we have taken advantage of the Cdk9 inhibitors, flavopiridol (flavo) and 5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB) to characterize the immediate cellular response to TRCs (hereafter referred to as the TRi response). These inhibitors prevent phosphorylation of Ser2 in the RNAPII CTD repeats, thereby blocking the transition from transcription initiation to transcription elongation (Hazelbaker et al. 2013). Treatment with these drugs trap RNAPII at the promoter proximal regions (Chen et al. 2017), and the trapped complex may block a trespassing replication fork. We find that FANCD2, BLM and BRCA2 colocalizing foci rapidly form in response to Cdk9 inhibitor treatment in chicken DT40 cells. Furthermore, these factors are required for cellular survival after transient drug treatment. FANCD2 focus formation induced by Cdk9 inhibitors are dependent on R-loop accumulation and basal level of ATR activity. Interestingly, monoubiquitylation of FANCD2 is not triggered by flavo treatment. Finally, abrogating the TRi response results in DNA damage at mitosis, promoting chromosome instability and cell death. Collectively, our data show that FANCD2, BRCA2 and BLM play an important role in the TRi response to ensure genome integrity. This may have clinical implications for cancer therapy.

AB - Various perturbations, originating from endogenous or exogenous sources, can challenge DNA replication to compromise its integrity, and pose a serious threat to chromosomal stability. The cell has evolved various cellular pathways and different checkpoint activities in order to cope with DNA damage. Emerging evidence suggests that transcription-replication conflicts (TRCs) are a potent source of mutagenesis and carcinogenesis (Wilson et al. 2015; Hamperl et al. 2017). The replication forks compete with the transcription machinery on the same DNA template, and this may lead to replication fork arrest. Normally, transcription and replication are coordinated to minimize TRCs (Wei et al.1998). However, deregulated replication, rapid cell division and high transcription activity may contribute to frequent TRCs, particularly in cancer cells (Hanahan and Weinberg 2011; Techer et al. 2017). Moreover, TRCs are inevitable at the largest genes because it takes more than one cell cycle to complete transcription of these genes (Helmrich et al. 2011). In this study, we have taken advantage of the Cdk9 inhibitors, flavopiridol (flavo) and 5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB) to characterize the immediate cellular response to TRCs (hereafter referred to as the TRi response). These inhibitors prevent phosphorylation of Ser2 in the RNAPII CTD repeats, thereby blocking the transition from transcription initiation to transcription elongation (Hazelbaker et al. 2013). Treatment with these drugs trap RNAPII at the promoter proximal regions (Chen et al. 2017), and the trapped complex may block a trespassing replication fork. We find that FANCD2, BLM and BRCA2 colocalizing foci rapidly form in response to Cdk9 inhibitor treatment in chicken DT40 cells. Furthermore, these factors are required for cellular survival after transient drug treatment. FANCD2 focus formation induced by Cdk9 inhibitors are dependent on R-loop accumulation and basal level of ATR activity. Interestingly, monoubiquitylation of FANCD2 is not triggered by flavo treatment. Finally, abrogating the TRi response results in DNA damage at mitosis, promoting chromosome instability and cell death. Collectively, our data show that FANCD2, BRCA2 and BLM play an important role in the TRi response to ensure genome integrity. This may have clinical implications for cancer therapy.

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/fbp0ps/alma99122205886405763

M3 - Ph.D. thesis

BT - A distinct role for recombination repair factors in an immediate cellular response to transcription-replication conflicts

PB - Department of Biology, Faculty of Science, University of Copenhagen

ER -