A genetic study based on PCNA-ubiquitin fusions reveals no requirement for PCNA polyubiquitylation in DNA damage tolerance

Judit Z. Gervai, Judit Gálicza, Z. Szeltner, Judit Zámborszky, D. Szüts

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Post-translational modifications of Proliferating Cell Nuclear Antigen (PCNA) play a key role in regulating the bypass of DNA lesions during DNA replication. PCNA can be monoubiquitylated at lysine 164 by the RAD6-RAD18 ubiquitin ligase complex. Through this modification, PCNA can interact with low fidelity Y family DNA polymerases to promote translesion synthesis. Monoubiquitylated PCNA can be polyubiquitylated on lysine 63 of ubiquitin by a further ubiquitin-conjugating complex. This modification promotes a template switching bypass process in yeast, while its role in higher eukaryotes is less clear. We investigated the function of PCNA ubiquitylation using a PCNAK164R mutant DT40 chicken B lymphoblastoma cell line, which is hypersensitive to DNA damaging agents such as methyl methanesulfonate (MMS), cisplatin or ultraviolet radiation (UV) due to the loss of PCNA modifications. In the PCNAK164R mutant we also detected cell cycle arrest following UV treatment, a reduced rate of damage bypass through translesion DNA synthesis on synthetic UV photoproducts, and an increased rate of genomic mutagenesis following MMS treatment. PCNA-ubiquitin fusion proteins have been reported to mimic endogenous PCNA ubiquitylation. We found that the stable expression of a PCNAK164R-ubiquitin fusion protein fully or partially rescued the observed defects of the PCNAK164R mutant. The expression of a PCNAK164R-ubiquitinK63R fusion protein, on which the formation of lysine 63-linked polyubiquitin chains is not possible, similarly rescued the cell cycle arrest, DNA damage sensitivity, reduction of translesion synthesis and increase of MMS-induced genomic mutagenesis. Template switching bypass was not affected by the genetic elimination of PCNA polyubiquitylation, but it was reduced in the absence of the recombination proteins BRCA1 or XRCC3. Our study found no requirement for PCNA polyubiquitylation to protect cells from replication-stalling DNA damage.

Original languageEnglish
Pages (from-to)46-54
Number of pages9
JournalDNA Repair
Volume54
DOIs
Publication statusPublished - Jun 1 2017

Fingerprint

Damage tolerance
Proliferating Cell Nuclear Antigen
Ubiquitin
DNA Damage
Fusion reactions
DNA
Methyl Methanesulfonate
Ultraviolet radiation
Lysine
Mutagenesis
Ubiquitination
Cells
Radiation
Cell Cycle Checkpoints
BRCA1 Protein
Polyubiquitin
Proteins
DNA-Directed DNA Polymerase
Post Translational Protein Processing
Ligases

Keywords

  • DNA damage bypass
  • PCNA
  • PCNA monoubiquitylation
  • PCNA polyubiquitylation
  • Template switching
  • Translesion synthesis

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

A genetic study based on PCNA-ubiquitin fusions reveals no requirement for PCNA polyubiquitylation in DNA damage tolerance. / Gervai, Judit Z.; Gálicza, Judit; Szeltner, Z.; Zámborszky, Judit; Szüts, D.

In: DNA Repair, Vol. 54, 01.06.2017, p. 46-54.

Research output: Contribution to journalArticle

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