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Constitutive fusion of ubiquitin to PCNA provides DNA damage tolerance independent of translesion polymerase activities.

Pastushok L, Hanna M, Xiao W - Nucleic Acids Res. (2010)

Bottom Line: Several reports suggest a model whereby monoubiquitylated PCNA recruits TLS polymerases through an enhanced physical association.The creation of viable PCNA.Ub strains lacking endogenous PCNA enabled a thorough analysis of roles for PCNA mono-Ub in DDT.Taken together, our data suggest that either the monoubiquitylation of PCNA does not promote TLS activity in all cases or PCNA.Ub reveals a currently undiscovered role for monoubiquitylated PCNA in DNA damage tolerance.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK, Canada.

ABSTRACT
In response to replication-blocking DNA lesions, proliferating cell nuclear antigen (PCNA) can be conjugated with a single ubiquitin (Ub) or Lys63-linked Ub chains at the Lys164 residue, leading to two modes of DNA damage tolerance (DDT), namely translesion synthesis (TLS) and error-free DDT, respectively. Several reports suggest a model whereby monoubiquitylated PCNA recruits TLS polymerases through an enhanced physical association. We sought to examine this model in Saccharomyces cerevisiae through artificial fusions of Ub to PCNA in vivo. We created N- and C- terminal gene fusions of Ub to PCNA-K164R (collectively called PCNA.Ub) and found that both conferred tolerance to DNA damage. The creation of viable PCNA.Ub strains lacking endogenous PCNA enabled a thorough analysis of roles for PCNA mono-Ub in DDT. As expected, the DNA damage resistance provided by PCNA.Ub is not dependent on RAD18 or UBC13. Surprisingly, inactivation of TLS polymerases did not abolish PCNA.Ub resistance to DNA damage, nor did PCNA.Ub cause elevated spontaneous mutagenesis, which is a defining characteristic of REV3-dependent TLS activity. Taken together, our data suggest that either the monoubiquitylation of PCNA does not promote TLS activity in all cases or PCNA.Ub reveals a currently undiscovered role for monoubiquitylated PCNA in DNA damage tolerance.

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MMS gradient plate assays to assess the effects of deleting DDT pathway genes on HK578-10D pol30Δ::HIS3 cells harboring pol30·UB gene fusions. (A) In the pol30Δ rad18Δ strain background; (B) in the pol30Δ ubc13Δ background; (C) in the pol30Δ rev3Δ background; and (D) in the pol30Δ ubc13Δ rev3Δ background. Experimental conditions were as described in Figure 2.
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Figure 5: MMS gradient plate assays to assess the effects of deleting DDT pathway genes on HK578-10D pol30Δ::HIS3 cells harboring pol30·UB gene fusions. (A) In the pol30Δ rad18Δ strain background; (B) in the pol30Δ ubc13Δ background; (C) in the pol30Δ rev3Δ background; and (D) in the pol30Δ ubc13Δ rev3Δ background. Experimental conditions were as described in Figure 2.

Mentions: We wanted to determine the nature of the MMS resistance provided by PCNA·Ub fusions within the DDT pathway. To help confirm that the PCNA·Ub fusions are indeed functional within DDT, we first tested their function in a pol30Δ rad18Δ double-deletion strain. As discussed above, RAD18 and RAD6 govern the entire DDT pathway and as such, deletion of either gene causes a complete loss in DNA damage resistance via DDT. As seen in Figure 5A, each of the fusions provided MMS resistance in a pol30Δ rad18Δ strain, suggesting that Ub-PCNA and PCNA-Ub can at least partially replace Rad18′s role for modifying PCNA with mono-Ub.Figure 5.


Constitutive fusion of ubiquitin to PCNA provides DNA damage tolerance independent of translesion polymerase activities.

Pastushok L, Hanna M, Xiao W - Nucleic Acids Res. (2010)

MMS gradient plate assays to assess the effects of deleting DDT pathway genes on HK578-10D pol30Δ::HIS3 cells harboring pol30·UB gene fusions. (A) In the pol30Δ rad18Δ strain background; (B) in the pol30Δ ubc13Δ background; (C) in the pol30Δ rev3Δ background; and (D) in the pol30Δ ubc13Δ rev3Δ background. Experimental conditions were as described in Figure 2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2926605&req=5

Figure 5: MMS gradient plate assays to assess the effects of deleting DDT pathway genes on HK578-10D pol30Δ::HIS3 cells harboring pol30·UB gene fusions. (A) In the pol30Δ rad18Δ strain background; (B) in the pol30Δ ubc13Δ background; (C) in the pol30Δ rev3Δ background; and (D) in the pol30Δ ubc13Δ rev3Δ background. Experimental conditions were as described in Figure 2.
Mentions: We wanted to determine the nature of the MMS resistance provided by PCNA·Ub fusions within the DDT pathway. To help confirm that the PCNA·Ub fusions are indeed functional within DDT, we first tested their function in a pol30Δ rad18Δ double-deletion strain. As discussed above, RAD18 and RAD6 govern the entire DDT pathway and as such, deletion of either gene causes a complete loss in DNA damage resistance via DDT. As seen in Figure 5A, each of the fusions provided MMS resistance in a pol30Δ rad18Δ strain, suggesting that Ub-PCNA and PCNA-Ub can at least partially replace Rad18′s role for modifying PCNA with mono-Ub.Figure 5.

Bottom Line: Several reports suggest a model whereby monoubiquitylated PCNA recruits TLS polymerases through an enhanced physical association.The creation of viable PCNA.Ub strains lacking endogenous PCNA enabled a thorough analysis of roles for PCNA mono-Ub in DDT.Taken together, our data suggest that either the monoubiquitylation of PCNA does not promote TLS activity in all cases or PCNA.Ub reveals a currently undiscovered role for monoubiquitylated PCNA in DNA damage tolerance.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK, Canada.

ABSTRACT
In response to replication-blocking DNA lesions, proliferating cell nuclear antigen (PCNA) can be conjugated with a single ubiquitin (Ub) or Lys63-linked Ub chains at the Lys164 residue, leading to two modes of DNA damage tolerance (DDT), namely translesion synthesis (TLS) and error-free DDT, respectively. Several reports suggest a model whereby monoubiquitylated PCNA recruits TLS polymerases through an enhanced physical association. We sought to examine this model in Saccharomyces cerevisiae through artificial fusions of Ub to PCNA in vivo. We created N- and C- terminal gene fusions of Ub to PCNA-K164R (collectively called PCNA.Ub) and found that both conferred tolerance to DNA damage. The creation of viable PCNA.Ub strains lacking endogenous PCNA enabled a thorough analysis of roles for PCNA mono-Ub in DDT. As expected, the DNA damage resistance provided by PCNA.Ub is not dependent on RAD18 or UBC13. Surprisingly, inactivation of TLS polymerases did not abolish PCNA.Ub resistance to DNA damage, nor did PCNA.Ub cause elevated spontaneous mutagenesis, which is a defining characteristic of REV3-dependent TLS activity. Taken together, our data suggest that either the monoubiquitylation of PCNA does not promote TLS activity in all cases or PCNA.Ub reveals a currently undiscovered role for monoubiquitylated PCNA in DNA damage tolerance.

Show MeSH
Related in: MedlinePlus