TY - JOUR
T1 - Replication protein A dynamically re-organizes on primer/template junctions to permit DNA polymerase δ holoenzyme assembly and initiation of DNA synthesis
AU - Norris, Jessica L.
AU - Rogers, Lindsey O.
AU - Pytko, Kara G.
AU - Dannenberg, Rachel L.
AU - Perreault, Samuel
AU - Kaushik, Vikas
AU - Kuppa, Sahiti
AU - Antony, Edwin
AU - Hedglin, Mark
N1 - Publisher Copyright:
© 2024 The Author(s). Published by Oxford University Press on behalf of Nucleic Acids Research.
PY - 2024/7/22
Y1 - 2024/7/22
N2 - DNA polymerase δ(pol δ) holoenzymes, comprised of pol δand 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 δholoenzymes 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 δwith 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 δwith DNA as pol δholoenzymes 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 RPA•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 RPA•DNA interactions in RPA-dependent DNA metabolic processes.
AB - DNA polymerase δ(pol δ) holoenzymes, comprised of pol δand 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 δholoenzymes 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 δwith 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 δwith DNA as pol δholoenzymes 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 RPA•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 RPA•DNA interactions in RPA-dependent DNA metabolic processes.
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U2 - 10.1093/nar/gkae475
DO - 10.1093/nar/gkae475
M3 - Article
C2 - 38842913
AN - SCOPUS:85199269507
SN - 0305-1048
VL - 52
SP - 7650
EP - 7664
JO - Nucleic acids research
JF - Nucleic acids research
IS - 13
ER -