Replication protein A dynamically regulates monoubiquitination of proliferating cell nuclear antigen

Mark Hedglin, Mahesh Aitha, Anthony Pedley, Stephen J. Benkovic

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)5157-5168
Number of pages12
JournalJournal of Biological Chemistry
Volume294
Issue number13
DOIs
StatePublished - Jan 1 2019

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Replication Protein A
Proliferating Cell Nuclear Antigen
DNA
Post Translational Protein Processing
DNA Replication
Damage tolerance
Clamping devices
S Phase
DNA Damage
Chromatin
Assays
Carrier Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Hedglin, Mark ; Aitha, Mahesh ; Pedley, Anthony ; Benkovic, Stephen J. / Replication protein A dynamically regulates monoubiquitination of proliferating cell nuclear antigen. In: Journal of Biological Chemistry. 2019 ; Vol. 294, No. 13. pp. 5157-5168.
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Replication protein A dynamically regulates monoubiquitination of proliferating cell nuclear antigen. / Hedglin, Mark; Aitha, Mahesh; Pedley, Anthony; Benkovic, Stephen J.

In: Journal of Biological Chemistry, Vol. 294, No. 13, 01.01.2019, p. 5157-5168.

Research output: Contribution to journalArticle

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