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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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Effects of Rpb1 sumoylation at K1487 on UV-induced Rad53 phosphorylation.(A–C) UV-induced Rad53 phosphorylation in log phase wild type (for NER genes) cells expressing wild type (CX84) or K1487R mutant (CX79) Rpb1. (D–F) UV-induced Rad53 phosphorylation in log phase rad16 cells expressing wild type (CX85) or K1487R mutant (CX87) Rpb1. (G–I) UV-induced Rad53 phosphorylation in log phase rad16 rad26 cells expressing wild type (CX112) or K1487R mutant (CX113) Rpb1. The cells were irradiated with UV and incubated in a rich medium at 30°C. Whole cell extracts were prepared from the cells at different times of the post-UV incubation. Rad53 in the whole cell extracts was probed with an anti-Rad53 antibody on Western blots. p and u on the left of the blots indicate bands of phosphorylated and unphosphorylated Rad53, respectively. Plots C, F and I show ratios of phosphorylated Rad53 (Rad53p) to unphosphorylated Rad53 (Rad53u) in the wild type, rad16 and rad16 rad26 cells, respectively. Error bars represents standard deviations.
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pone-0005267-g007: Effects of Rpb1 sumoylation at K1487 on UV-induced Rad53 phosphorylation.(A–C) UV-induced Rad53 phosphorylation in log phase wild type (for NER genes) cells expressing wild type (CX84) or K1487R mutant (CX79) Rpb1. (D–F) UV-induced Rad53 phosphorylation in log phase rad16 cells expressing wild type (CX85) or K1487R mutant (CX87) Rpb1. (G–I) UV-induced Rad53 phosphorylation in log phase rad16 rad26 cells expressing wild type (CX112) or K1487R mutant (CX113) Rpb1. The cells were irradiated with UV and incubated in a rich medium at 30°C. Whole cell extracts were prepared from the cells at different times of the post-UV incubation. Rad53 in the whole cell extracts was probed with an anti-Rad53 antibody on Western blots. p and u on the left of the blots indicate bands of phosphorylated and unphosphorylated Rad53, respectively. Plots C, F and I show ratios of phosphorylated Rad53 (Rad53p) to unphosphorylated Rad53 (Rad53u) in the wild type, rad16 and rad16 rad26 cells, respectively. Error bars represents standard deviations.

Mentions: To examine if Rpb1 sumoylation plays a role in DNA damage checkpoint response, we analyzed UV-induced Rad53 phosphorylation in log phase yeast cells. In wild type cells, UV irradiation caused rapid phosphorylation of Rad53, which is reflected by the slower migrating Rad53 bands on a Western blot (Fig. 7A, see Rad53 bands marked with ‘p’). The level of Rad53 phosphorylation, as indicated by the ratio of the phosphorylated to unphophorylated Rad53, peaked ∼30 minutes after UV irradiation and gradually decreased afterwards (Fig. 7A and C). UV-induced Rad53 phosphorylation was somewhat weakened in rad16 cells, and dramatically impaired and delayed in rad16 rad26 cells (Fig. 7, compare panels A, D and G, and panels C, F and I). These results agree well with previous reports showing that both GGR mediated by Rad16 and TCR mediated by Rad26 contribute to DNA damage checkpoint response [62]. In fact, all yeast mutants deficient in incision during NER have been shown to be deficient in the rapid phosphorylation of Rad53 in response to UV radiation [3].


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)

Effects of Rpb1 sumoylation at K1487 on UV-induced Rad53 phosphorylation.(A–C) UV-induced Rad53 phosphorylation in log phase wild type (for NER genes) cells expressing wild type (CX84) or K1487R mutant (CX79) Rpb1. (D–F) UV-induced Rad53 phosphorylation in log phase rad16 cells expressing wild type (CX85) or K1487R mutant (CX87) Rpb1. (G–I) UV-induced Rad53 phosphorylation in log phase rad16 rad26 cells expressing wild type (CX112) or K1487R mutant (CX113) Rpb1. The cells were irradiated with UV and incubated in a rich medium at 30°C. Whole cell extracts were prepared from the cells at different times of the post-UV incubation. Rad53 in the whole cell extracts was probed with an anti-Rad53 antibody on Western blots. p and u on the left of the blots indicate bands of phosphorylated and unphosphorylated Rad53, respectively. Plots C, F and I show ratios of phosphorylated Rad53 (Rad53p) to unphosphorylated Rad53 (Rad53u) in the wild type, rad16 and rad16 rad26 cells, respectively. Error bars represents standard deviations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2668072&req=5

pone-0005267-g007: Effects of Rpb1 sumoylation at K1487 on UV-induced Rad53 phosphorylation.(A–C) UV-induced Rad53 phosphorylation in log phase wild type (for NER genes) cells expressing wild type (CX84) or K1487R mutant (CX79) Rpb1. (D–F) UV-induced Rad53 phosphorylation in log phase rad16 cells expressing wild type (CX85) or K1487R mutant (CX87) Rpb1. (G–I) UV-induced Rad53 phosphorylation in log phase rad16 rad26 cells expressing wild type (CX112) or K1487R mutant (CX113) Rpb1. The cells were irradiated with UV and incubated in a rich medium at 30°C. Whole cell extracts were prepared from the cells at different times of the post-UV incubation. Rad53 in the whole cell extracts was probed with an anti-Rad53 antibody on Western blots. p and u on the left of the blots indicate bands of phosphorylated and unphosphorylated Rad53, respectively. Plots C, F and I show ratios of phosphorylated Rad53 (Rad53p) to unphosphorylated Rad53 (Rad53u) in the wild type, rad16 and rad16 rad26 cells, respectively. Error bars represents standard deviations.
Mentions: To examine if Rpb1 sumoylation plays a role in DNA damage checkpoint response, we analyzed UV-induced Rad53 phosphorylation in log phase yeast cells. In wild type cells, UV irradiation caused rapid phosphorylation of Rad53, which is reflected by the slower migrating Rad53 bands on a Western blot (Fig. 7A, see Rad53 bands marked with ‘p’). The level of Rad53 phosphorylation, as indicated by the ratio of the phosphorylated to unphophorylated Rad53, peaked ∼30 minutes after UV irradiation and gradually decreased afterwards (Fig. 7A and C). UV-induced Rad53 phosphorylation was somewhat weakened in rad16 cells, and dramatically impaired and delayed in rad16 rad26 cells (Fig. 7, compare panels A, D and G, and panels C, F and I). These results agree well with previous reports showing that both GGR mediated by Rad16 and TCR mediated by Rad26 contribute to DNA damage checkpoint response [62]. In fact, all yeast mutants deficient in incision during NER have been shown to be deficient in the rapid phosphorylation of Rad53 in response to UV radiation [3].

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