Abstract
Replication-Factor-C (RFC) and RFC-like complexes (RLCs) mediate chromatin engagement of the proliferating cell nuclear antigen (PCNA). It remains controversial how RFC and RLCs cooperate to regulate PCNA loading and unloading. Here, we show the distinct PCNA loading or unloading activity of each clamp loader. ATAD5-RLC possesses the potent PCNA unloading activity. ATPase motif and collar domain of ATAD5 are crucial for the unloading activity. DNA structures did not affect PCNA unloading activity of ATAD5-RLC. ATAD5-RLC could unload ubiquitinated PCNA. Through single molecule measurements, we reveal that ATAD5-RLC unloaded PCNA through one intermediate state before ATP hydrolysis. RFC loaded PCNA through two intermediate states on DNA, separated by ATP hydrolysis. Replication proteins such as Fen1 could inhibit the PCNA unloading activity of Elg1-RLC, a yeast homolog of ATAD5-RLC in vitro. Our findings provide molecular insights into how PCNA is released from chromatin to finalize DNA replication/repair.
Highlights
Replication-Factor-C (RFC) and RFC-like complexes (RLCs) mediate chromatin engagement of the proliferating cell nuclear antigen (PCNA)
Deletion of N-terminus of ATAD5 up to 692 amino acids (ΔN692) did not affect PCNA unloading activity. These results suggest that the PCNA unloading activity of ATAD5 is conferred by its C-terminal domain and can be functionally separated from its de-ubiquitination function
We examined whether ubiquitination or SUMOylation of PCNA could affect unloading by Elg1-RLC
Summary
Replication-Factor-C (RFC) and RFC-like complexes (RLCs) mediate chromatin engagement of the proliferating cell nuclear antigen (PCNA). It remains controversial how RFC and RLCs cooperate to regulate PCNA loading and unloading. RFC loaded PCNA through two intermediate states on DNA, separated by ATP hydrolysis Replication proteins such as Fen[1] could inhibit the PCNA unloading activity of Elg1-RLC, a yeast homolog of ATAD5-RLC in vitro. Based on various experimental results, a model for RFC-mediated PCNA loading on the primedtemplate DNA has been suggested[12,13,14] In this model, a spiralshaped RFC complex assembles with PCNA in the presence of ATP, opening a gap in the trimeric ring. There have been no studies on the mechanism of PCNA unloading, which, due to the irreversible step of ATP hydrolysis, cannot be the reversal of the PCNA loading reaction
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