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A genetic study based on PCNA-ubiquitin fusions reveals no requirement for PCNA polyubiquitylation in DNA damage tolerance.
- Source :
-
DNA repair [DNA Repair (Amst)] 2017 Jun; Vol. 54, pp. 46-54. Date of Electronic Publication: 2017 Apr 14. - Publication Year :
- 2017
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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 PCNA <superscript>K164R</superscript> 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 PCNA <superscript>K164R</superscript> 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 PCNA <superscript>K164R</superscript> -ubiquitin fusion protein fully or partially rescued the observed defects of the PCNA <superscript>K164R</superscript> mutant. The expression of a PCNA <superscript>K164R</superscript> -ubiquitin <superscript>K63R</superscript> 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.<br /> (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Subjects :
- Animals
BRCA1 Protein metabolism
Chickens metabolism
DNA drug effects
DNA metabolism
DNA radiation effects
DNA-Binding Proteins metabolism
DNA-Directed DNA Polymerase metabolism
Humans
Methyl Methanesulfonate toxicity
Mutation, Missense
Proliferating Cell Nuclear Antigen chemistry
Proliferating Cell Nuclear Antigen genetics
Ubiquitination
Ultraviolet Rays
Chickens genetics
DNA Damage
DNA Repair
DNA Replication
Proliferating Cell Nuclear Antigen metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1568-7856
- Volume :
- 54
- Database :
- MEDLINE
- Journal :
- DNA repair
- Publication Type :
- Academic Journal
- Accession number :
- 28458162
- Full Text :
- https://doi.org/10.1016/j.dnarep.2017.04.003