RESUMO
DNA polymerase δ (pol δ) holoenzymes, comprised of pol δ and the processivity sliding clamp, PCNA, carry out DNA synthesis during lagging strand replication, initiation of leading strand replication, and the major DNA damage repair and tolerance pathways. Pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a stepwise process involving the major single strand DNA (ssDNA)-binding protein complex, RPA, the processivity sliding clamp loader, RFC, PCNA and pol δ. During this process, the interactions of RPA, RFC and pol δ with a P/T junction all significantly overlap. A burning issue that has yet to be resolved is how these overlapping interactions are accommodated during this process. To address this, we design and utilize novel, ensemble FRET assays that continuously monitor the interactions of RPA, RFC, PCNA and pol δ with DNA as pol δ holoenzymes are assembled and initiate DNA synthesis. Results from the present study reveal that RPA remains engaged with P/T junctions throughout this process and the RPAâ¢DNA complexes dynamically re-organize to allow successive binding of RFC and pol δ. These results have broad implications as they highlight and distinguish the functional consequences of dynamic RPAâ¢DNA interactions in RPA-dependent DNA metabolic processes.
Assuntos
DNA Polimerase III , Replicação do DNA , DNA , Antígeno Nuclear de Célula em Proliferação , Proteína de Replicação A , Proteína de Replicação C , Moldes Genéticos , Proteína de Replicação A/metabolismo , DNA Polimerase III/metabolismo , DNA Polimerase III/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Holoenzimas/metabolismo , DNA/metabolismo , DNA/biossíntese , Proteína de Replicação C/metabolismo , Proteína de Replicação C/genética , Primers do DNA/genética , Transferência Ressonante de Energia de Fluorescência , HumanosRESUMO
PARP2 is a DNA-dependent ADP-ribosyl transferase (ARTs) enzyme with Poly(ADP-ribosyl)ation activity that is triggered by DNA breaks. It plays a role in the Base Excision Repair pathway, where it has overlapping functions with PARP1. However, additional roles for PARP2 have emerged in the response of cells to replication stress. In this study, we demonstrate that PARP2 promotes replication stress-induced telomere fragility and prevents telomere loss following chronic induction of oxidative DNA lesions and BLM helicase depletion. Telomere fragility results from the activity of the break-induced replication pathway (BIR). During this process, PARP2 promotes DNA end resection, strand invasion and BIR-dependent mitotic DNA synthesis by orchestrating POLD3 recruitment and activity. Our study has identified a role for PARP2 in the response to replication stress. This finding may lead to the development of therapeutic approaches that target DNA-dependent ART enzymes, particularly in cancer cells with high levels of replication stress.
Assuntos
Reparo do DNA , DNA , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , DNA/metabolismo , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Telômero/genética , Telômero/metabolismoRESUMO
Fragile sites are unstable genomic regions that are prone to breakage during stressed DNA replication. Several common fragile sites (CFS) contain A+T-rich regions including perfect [AT/TA] microsatellite repeats that may collapse into hairpins when in single-stranded DNA (ssDNA) form and coincide with chromosomal hotspots for breakage and rearrangements. While many factors contribute to CFS instability, evidence exists for replication stalling within [AT/TA] microsatellite repeats. Currently, it is unknown how stress causes replication stalling within [AT/TA] microsatellite repeats. To investigate this, we utilized FRET to characterize the structures of [AT/TA]25 sequences and also reconstituted lagging strand replication to characterize the progression of pol δ holoenzymes through A+T-rich sequences. The results indicate that [AT/TA]25 sequences adopt hairpins that are unwound by the major ssDNA-binding complex, RPA, and the progression of pol δ holoenzymes through A+T-rich sequences saturated with RPA is dependent on the template sequence and dNTP concentration. Importantly, the effects of RPA on the replication of [AT/TA]25 sequences are dependent on dNTP concentration, whereas the effects of RPA on the replication of A+T-rich, nonstructure-forming sequences are independent of dNTP concentration. Collectively, these results reveal complexities in lagging strand replication and provide novel insights into how [AT/TA] microsatellite repeats contribute to genome instability.