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Rpb1 sumoylation in response to UV radiation or transcriptional impairment in yeast.

Chen X, Ding B, LeJeune D, Ruggiero C, Li S - PLoS ONE (2009)

Bottom Line: K1487, which is located in the acidic linker region between the C-terminal domain and the globular domain of Rpb1, is the major sumoylation site.Rpb1 sumoylation is not affected by its ubiquitylation, and vice versa, indicating that the two processes do not crosstalk.However, deficiency in TCR enhances UV-induced Rpb1 sumoylation, presumably due to the persistence of transcription-blocking DNA lesions in the transcribed strand of a gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, United States of America.

ABSTRACT
Covalent modifications of proteins by ubiquitin and the Small Ubiquitin-like MOdifier (SUMO) have been revealed to be involved in a plethora of cellular processes, including transcription, DNA repair and DNA damage responses. It has been well known that in response to DNA damage that blocks transcription elongation, Rpb1, the largest subunit of RNA polymerase II (Pol II), is ubiquitylated and subsequently degraded in mammalian and yeast cells. However, it is still an enigma regarding how Pol II responds to damaged DNA and conveys signal(s) for DNA damage-related cellular processes. We found that Rpb1 is also sumoylated in yeast cells upon UV radiation or impairment of transcription elongation, and this modification is independent of DNA damage checkpoint activation. Ubc9, an E2 SUMO conjugase, and Siz1, an E3 SUMO ligase, play important roles in Rpb1 sumoylation. K1487, which is located in the acidic linker region between the C-terminal domain and the globular domain of Rpb1, is the major sumoylation site. Rpb1 sumoylation is not affected by its ubiquitylation, and vice versa, indicating that the two processes do not crosstalk. Abolishment of Rpb1 sumoylation at K1487 does not affect transcription elongation or transcription coupled repair (TCR) of UV-induced DNA damage. However, deficiency in TCR enhances UV-induced Rpb1 sumoylation, presumably due to the persistence of transcription-blocking DNA lesions in the transcribed strand of a gene. Remarkably, abolishment of Rpb1 sumoylation at K1487 causes enhanced and prolonged UV-induced phosphorylation of Rad53, especially in TCR-deficient cells, suggesting that the sumoylation plays a role in restraining the DNA damage checkpoint response caused by transcription-blocking lesions. Our results demonstrate a novel covalent modification of Rpb1 in response to UV induced DNA damage or transcriptional impairment, and unravel an important link between the modification and the DNA damage checkpoint response.

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Abolishment of Rpb1 sumoylation at K1487 does not affect overall TCR or Rad26-independent TCR.(A) DNA sequencing gels showing repair of UV-induced cyclobutane pyrimidine dimers (CPDs) in the transcribed strand of the RPB2 gene in rad16 cells expressing wild type (CX85) or K1487R mutant (CX87) Rpb1. (B) DNA sequencing gels showing repair of CPDs in the transcribed strand of the RPB2 gene in rad16 rad26 cells expressing wild type (CX112) or K1487R mutant (CX113) Rpb1. Lanes U are unirradiated controls. Other lanes are samples from cells incubated for different times (min) following UV irradiation. The arrow on the left of the gels indicates the transcription start site of RPB2.
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pone-0005267-g006: Abolishment of Rpb1 sumoylation at K1487 does not affect overall TCR or Rad26-independent TCR.(A) DNA sequencing gels showing repair of UV-induced cyclobutane pyrimidine dimers (CPDs) in the transcribed strand of the RPB2 gene in rad16 cells expressing wild type (CX85) or K1487R mutant (CX87) Rpb1. (B) DNA sequencing gels showing repair of CPDs in the transcribed strand of the RPB2 gene in rad16 rad26 cells expressing wild type (CX112) or K1487R mutant (CX113) Rpb1. Lanes U are unirradiated controls. Other lanes are samples from cells incubated for different times (min) following UV irradiation. The arrow on the left of the gels indicates the transcription start site of RPB2.

Mentions: The observation that deficiency in TCR enhances UV-induced Rpb1 sumoylation can be explained by the persistence (or slower removal) of transcription-blocking lesions in the transcribed strand of the genes, as the mere impairment of transcription elongation by MPA treatment also induces Rpb1 sumoylation (Fig. 1D). Alternatively, Rpb1 sumoylation may serve as a TCR signal, which may be removed (Rpb1 de-sumoylated) during or after the TCR process in TCR-proficient cells. To test the second possibility, we examined the effect of K1487R mutation of Rpb1 on repair of CPDs in the constitutively transcribed RPB2 gene in rad16 cells where GGR is abolished [5], [57] and TCR can be unambiguously analyzed. Yeast cells were cultured to late log phase, UV irradiated, and incubated in a repair medium for various lengths of time. Total DNA was isolated, digested with a restriction enzyme to excise the fragment of interest, and incised at the UV-induced CPDs with an excess amount of T4 endonuclease V [58]. The incised fragments were strand-specifically end-labeled, resolved on a DNA sequencing gel, and exposed against a Phosphoimager screen. The band intensities in the gel lane of “0” time repair indicate the yields of CPDs at different sites. A decrease in band intensities with time at respective sites indicates CPD repair at these sites. In rad16 cells expressing the wild type Rpb1, fast repair can be seen in the transcribed strand of the RPB2 gene, initiating at ∼40 nucleotides upstream of the transcription start site (Fig. 6A), in agreement with our previous results [10]. The TCR rate in rad16 cells expressing K1487R Rpb1 was similar to those expressing the wild-type Rpb1 (Fig. 6A). In agreement with previous results [9], [10], [11], [57], deletion of RAD26 dramatically diminishes TCR in the RPB2 gene (Fig. 6B). The TCR rate in rad16 rad26 cells expressing K1487R Rpb1 was also similar to those expressing the wild-type Rpb1 (Fig. 6B). These results indicate that the K1487R mutation affects neither the overall TCR nor the Rad26-independent TCR.


Rpb1 sumoylation in response to UV radiation or transcriptional impairment in yeast.

Chen X, Ding B, LeJeune D, Ruggiero C, Li S - PLoS ONE (2009)

Abolishment of Rpb1 sumoylation at K1487 does not affect overall TCR or Rad26-independent TCR.(A) DNA sequencing gels showing repair of UV-induced cyclobutane pyrimidine dimers (CPDs) in the transcribed strand of the RPB2 gene in rad16 cells expressing wild type (CX85) or K1487R mutant (CX87) Rpb1. (B) DNA sequencing gels showing repair of CPDs in the transcribed strand of the RPB2 gene in rad16 rad26 cells expressing wild type (CX112) or K1487R mutant (CX113) Rpb1. Lanes U are unirradiated controls. Other lanes are samples from cells incubated for different times (min) following UV irradiation. The arrow on the left of the gels indicates the transcription start site of RPB2.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2668072&req=5

pone-0005267-g006: Abolishment of Rpb1 sumoylation at K1487 does not affect overall TCR or Rad26-independent TCR.(A) DNA sequencing gels showing repair of UV-induced cyclobutane pyrimidine dimers (CPDs) in the transcribed strand of the RPB2 gene in rad16 cells expressing wild type (CX85) or K1487R mutant (CX87) Rpb1. (B) DNA sequencing gels showing repair of CPDs in the transcribed strand of the RPB2 gene in rad16 rad26 cells expressing wild type (CX112) or K1487R mutant (CX113) Rpb1. Lanes U are unirradiated controls. Other lanes are samples from cells incubated for different times (min) following UV irradiation. The arrow on the left of the gels indicates the transcription start site of RPB2.
Mentions: The observation that deficiency in TCR enhances UV-induced Rpb1 sumoylation can be explained by the persistence (or slower removal) of transcription-blocking lesions in the transcribed strand of the genes, as the mere impairment of transcription elongation by MPA treatment also induces Rpb1 sumoylation (Fig. 1D). Alternatively, Rpb1 sumoylation may serve as a TCR signal, which may be removed (Rpb1 de-sumoylated) during or after the TCR process in TCR-proficient cells. To test the second possibility, we examined the effect of K1487R mutation of Rpb1 on repair of CPDs in the constitutively transcribed RPB2 gene in rad16 cells where GGR is abolished [5], [57] and TCR can be unambiguously analyzed. Yeast cells were cultured to late log phase, UV irradiated, and incubated in a repair medium for various lengths of time. Total DNA was isolated, digested with a restriction enzyme to excise the fragment of interest, and incised at the UV-induced CPDs with an excess amount of T4 endonuclease V [58]. The incised fragments were strand-specifically end-labeled, resolved on a DNA sequencing gel, and exposed against a Phosphoimager screen. The band intensities in the gel lane of “0” time repair indicate the yields of CPDs at different sites. A decrease in band intensities with time at respective sites indicates CPD repair at these sites. In rad16 cells expressing the wild type Rpb1, fast repair can be seen in the transcribed strand of the RPB2 gene, initiating at ∼40 nucleotides upstream of the transcription start site (Fig. 6A), in agreement with our previous results [10]. The TCR rate in rad16 cells expressing K1487R Rpb1 was similar to those expressing the wild-type Rpb1 (Fig. 6A). In agreement with previous results [9], [10], [11], [57], deletion of RAD26 dramatically diminishes TCR in the RPB2 gene (Fig. 6B). The TCR rate in rad16 rad26 cells expressing K1487R Rpb1 was also similar to those expressing the wild-type Rpb1 (Fig. 6B). These results indicate that the K1487R mutation affects neither the overall TCR nor the Rad26-independent TCR.

Bottom Line: K1487, which is located in the acidic linker region between the C-terminal domain and the globular domain of Rpb1, is the major sumoylation site.Rpb1 sumoylation is not affected by its ubiquitylation, and vice versa, indicating that the two processes do not crosstalk.However, deficiency in TCR enhances UV-induced Rpb1 sumoylation, presumably due to the persistence of transcription-blocking DNA lesions in the transcribed strand of a gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, United States of America.

ABSTRACT
Covalent modifications of proteins by ubiquitin and the Small Ubiquitin-like MOdifier (SUMO) have been revealed to be involved in a plethora of cellular processes, including transcription, DNA repair and DNA damage responses. It has been well known that in response to DNA damage that blocks transcription elongation, Rpb1, the largest subunit of RNA polymerase II (Pol II), is ubiquitylated and subsequently degraded in mammalian and yeast cells. However, it is still an enigma regarding how Pol II responds to damaged DNA and conveys signal(s) for DNA damage-related cellular processes. We found that Rpb1 is also sumoylated in yeast cells upon UV radiation or impairment of transcription elongation, and this modification is independent of DNA damage checkpoint activation. Ubc9, an E2 SUMO conjugase, and Siz1, an E3 SUMO ligase, play important roles in Rpb1 sumoylation. K1487, which is located in the acidic linker region between the C-terminal domain and the globular domain of Rpb1, is the major sumoylation site. Rpb1 sumoylation is not affected by its ubiquitylation, and vice versa, indicating that the two processes do not crosstalk. Abolishment of Rpb1 sumoylation at K1487 does not affect transcription elongation or transcription coupled repair (TCR) of UV-induced DNA damage. However, deficiency in TCR enhances UV-induced Rpb1 sumoylation, presumably due to the persistence of transcription-blocking DNA lesions in the transcribed strand of a gene. Remarkably, abolishment of Rpb1 sumoylation at K1487 causes enhanced and prolonged UV-induced phosphorylation of Rad53, especially in TCR-deficient cells, suggesting that the sumoylation plays a role in restraining the DNA damage checkpoint response caused by transcription-blocking lesions. Our results demonstrate a novel covalent modification of Rpb1 in response to UV induced DNA damage or transcriptional impairment, and unravel an important link between the modification and the DNA damage checkpoint response.

Show MeSH
Related in: MedlinePlus