Abstract
DNA damage tolerance permits bypass of DNA lesions encountered during S-phase and may be carried out by translesion DNA synthesis (TLS). Human TLS requires selective monoubiquitination of proliferating cell nuclear antigen (PCNA) sliding clamps encircling damaged DNA. This posttranslational modification (PTM) is catalyzed by Rad6/Rad18. Recent studies revealed that replication protein A (RPA), the major ssDNA-binding protein, is involved in the regulation of PCNA monoubiquitination and interacts directly with Rad18 on chromatin and in the nucleoplasm. However, it is unclear how RPA regulates this critical PTM and what functional role(s) these interactions serve. Here, we developed an in vitro assay to quantitatively monitor PCNA monoubiquitination under in vivo scenarios. Results from extensive experiments revealed that RPA regulates Rad6/Rad18 activity in an ssDNA-dependent manner. We found that "DNA-free" RPA inhibits monoubiquitination of free PCNA by directly interacting with Rad18. This interaction is promoted under native conditions when there is an overabundance of free RPA in the nucleoplasm where Rad6/Rad18 and a significant fraction of PCNA reside. During DNA replication stress, RPA binds the ssDNA exposed downstream of stalled primer/template (P/T) junctions, releasing Rad6/Rad18. RPA restricted the resident PCNAs to the upstream duplex regions by physically blocking diffusion of PCNA along ssDNA, and this activity was required for efficient monoubiquitination of PCNA on DNA. Furthermore, upon binding ssDNA, RPA underwent a conformational change that increased its affinity for Rad18. Rad6/Rad18 complexed with ssDNA-bound RPA was active, and this interaction may selectively promote monoubiquitination of PCNA on long RPA-coated ssDNA.
Highlights
DNA damage tolerance permits bypass of DNA lesions encountered during S-phase and may be carried out by translesion DNA synthesis (TLS)
replication protein A (RPA) is the major eukaryotic ssDNA-binding complex and protects exposed ssDNA from degradation and prevents formation of alternative DNA structures that are refractory to DNA synthesis [2]. double-stranded DNA (dsDNA) unwinding eventually stalls after an uncoupling event and failure to re-couple DNA synthesis and dsDNA unwinding often results in dsDNA breaks that may lead to gross chromosomal rearrangements, cell-cycle arrest, and cell death
Rad6/Rad18 is active throughout the cell cycle and does not require proliferating cell nuclear antigen (PCNA) to encircle DNA for monoubiquitination of PCNA to occur [4, 6, 7, 11,12,13,14,15,16,17,18]
Summary
DNA damage tolerance permits bypass of DNA lesions encountered during S-phase and may be carried out by translesion DNA synthesis (TLS). Recent studies revealed that replication protein A (RPA), the major ssDNA-binding protein, is involved in the regulation of PCNA monoubiquitination and interacts directly with Rad on chromatin and in the nucleoplasm. It is unclear how RPA regulates this critical PTM and what functional role(s) these interactions serve. Recent cellular studies revealed that RPA is involved in the regulation of PCNA monoubiquitination by Rad6/Rad and interacts with Rad on chromatin and in the nucleoplasm (i.e. nonchromatin associated/soluble fraction) The latter are quite prominent in nonperturbed human cells (8 –10).
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