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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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Related in: MedlinePlus

Western blots showing Rpb1 sumoylation in response to UV radiation or impairment of transcription elongation.(A) Rpb1 was immunoprecipitated from the unirradiated and UV irradiated cells using antibody 8WG16 (anti-Rpb1) and probed with anti-SUMO and 8WG16 antibodies. (B) Sumoylated proteins were immunoprecipitated from the unirradiated and UV irradiated cells and probed with 8WG16 and anti-SUMO antibodies. (C) UV-induced Rpb1 sumoylation in wild type (JKM179), sml1 (YFD756) and sml1 mec1 (YAA25) cells. (D) Sumoylation of Rpb1 in response to UV or treatments of transcription inhibitors. (E) UV-induced Rpb1 sumoylation in cells expressing wild type (CX84) or K1487R mutant (CX79) Rpb1. Bars on the left of the blot indicate distinct bands formed by wild type Rpb1. Arrow heads on the right of the blot mark bands abolished by the K1487R mutation. (F) UV-induced Rpb1 sumoylation in cells expressing wild type (CX84) or K to R mutant (CX79, CX105, CX106, CX108, CX110 and CX110) Rpb1. Bars on the left of the blot indicate distinct bands formed by wild type Rpb1. Arrow heads on the right of the blot mark bands not shown by the mutant Rpb1. WT, wild type.
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pone-0005267-g001: Western blots showing Rpb1 sumoylation in response to UV radiation or impairment of transcription elongation.(A) Rpb1 was immunoprecipitated from the unirradiated and UV irradiated cells using antibody 8WG16 (anti-Rpb1) and probed with anti-SUMO and 8WG16 antibodies. (B) Sumoylated proteins were immunoprecipitated from the unirradiated and UV irradiated cells and probed with 8WG16 and anti-SUMO antibodies. (C) UV-induced Rpb1 sumoylation in wild type (JKM179), sml1 (YFD756) and sml1 mec1 (YAA25) cells. (D) Sumoylation of Rpb1 in response to UV or treatments of transcription inhibitors. (E) UV-induced Rpb1 sumoylation in cells expressing wild type (CX84) or K1487R mutant (CX79) Rpb1. Bars on the left of the blot indicate distinct bands formed by wild type Rpb1. Arrow heads on the right of the blot mark bands abolished by the K1487R mutation. (F) UV-induced Rpb1 sumoylation in cells expressing wild type (CX84) or K to R mutant (CX79, CX105, CX106, CX108, CX110 and CX110) Rpb1. Bars on the left of the blot indicate distinct bands formed by wild type Rpb1. Arrow heads on the right of the blot mark bands not shown by the mutant Rpb1. WT, wild type.

Mentions: TCR is believed to be triggered by the stalling of RNA polymerase II (Pol II) [3]. In response to UV-induced DNA damage, Rpb1, the largest subunit of Pol II, is ubiquitylated and subsequently degraded in both human and yeast cells [13], [14]. The early studies proposed that Pol II ubiquitylation and subsequent degradation may be required for TCR to take place. However, it was later found that these events are not related to TCR in either human [17] or yeast [15], [16], [19] cells. To explore potential Pol II-related signal(s) for TCR and/or other DNA damage responses, we examined other possible modifications of Rpb1 following UV irradiation. Rpb1 was immunoprecipited from yeast cells using antibody 8WG16 which specifically recognizes the C-terminal heptapeptide repeats of Rpb1 [25]. The immunoprecipitates were subject to Western blot and probed with antibodies that were known to recognize potential covalent modifications. Interestingly, when the immunoprecipitated Rpb1 was probed with an anti-SUMO antibody, several bands could be seen in the UV irradiated samples, but not in the unirradiated ones (Fig. 1 A), indicating that Rpb1 was sumoylated in response to UV-induced DNA damage. To confirm this finding, a reciprocal immunoprecipitation was carried out. Sumoylated proteins were immunoprecipitated from normally cultured and UV-irradiated cells using an anti-SUMO antibody, and the immunoprecipitates were probed with 8WG16 on a Western blot. Several bands could be detected in the UV irradiated sample, but not in the unirradiated one (Fig. 1B), indicating that Rpb1 is sumoylated in response to UV-induced DNA damage. The different bands may reflect different forms of sumoylated Rpb1 (e.g., mono-, poly- or multi-sumoylated).


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)

Western blots showing Rpb1 sumoylation in response to UV radiation or impairment of transcription elongation.(A) Rpb1 was immunoprecipitated from the unirradiated and UV irradiated cells using antibody 8WG16 (anti-Rpb1) and probed with anti-SUMO and 8WG16 antibodies. (B) Sumoylated proteins were immunoprecipitated from the unirradiated and UV irradiated cells and probed with 8WG16 and anti-SUMO antibodies. (C) UV-induced Rpb1 sumoylation in wild type (JKM179), sml1 (YFD756) and sml1 mec1 (YAA25) cells. (D) Sumoylation of Rpb1 in response to UV or treatments of transcription inhibitors. (E) UV-induced Rpb1 sumoylation in cells expressing wild type (CX84) or K1487R mutant (CX79) Rpb1. Bars on the left of the blot indicate distinct bands formed by wild type Rpb1. Arrow heads on the right of the blot mark bands abolished by the K1487R mutation. (F) UV-induced Rpb1 sumoylation in cells expressing wild type (CX84) or K to R mutant (CX79, CX105, CX106, CX108, CX110 and CX110) Rpb1. Bars on the left of the blot indicate distinct bands formed by wild type Rpb1. Arrow heads on the right of the blot mark bands not shown by the mutant Rpb1. WT, wild type.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2668072&req=5

pone-0005267-g001: Western blots showing Rpb1 sumoylation in response to UV radiation or impairment of transcription elongation.(A) Rpb1 was immunoprecipitated from the unirradiated and UV irradiated cells using antibody 8WG16 (anti-Rpb1) and probed with anti-SUMO and 8WG16 antibodies. (B) Sumoylated proteins were immunoprecipitated from the unirradiated and UV irradiated cells and probed with 8WG16 and anti-SUMO antibodies. (C) UV-induced Rpb1 sumoylation in wild type (JKM179), sml1 (YFD756) and sml1 mec1 (YAA25) cells. (D) Sumoylation of Rpb1 in response to UV or treatments of transcription inhibitors. (E) UV-induced Rpb1 sumoylation in cells expressing wild type (CX84) or K1487R mutant (CX79) Rpb1. Bars on the left of the blot indicate distinct bands formed by wild type Rpb1. Arrow heads on the right of the blot mark bands abolished by the K1487R mutation. (F) UV-induced Rpb1 sumoylation in cells expressing wild type (CX84) or K to R mutant (CX79, CX105, CX106, CX108, CX110 and CX110) Rpb1. Bars on the left of the blot indicate distinct bands formed by wild type Rpb1. Arrow heads on the right of the blot mark bands not shown by the mutant Rpb1. WT, wild type.
Mentions: TCR is believed to be triggered by the stalling of RNA polymerase II (Pol II) [3]. In response to UV-induced DNA damage, Rpb1, the largest subunit of Pol II, is ubiquitylated and subsequently degraded in both human and yeast cells [13], [14]. The early studies proposed that Pol II ubiquitylation and subsequent degradation may be required for TCR to take place. However, it was later found that these events are not related to TCR in either human [17] or yeast [15], [16], [19] cells. To explore potential Pol II-related signal(s) for TCR and/or other DNA damage responses, we examined other possible modifications of Rpb1 following UV irradiation. Rpb1 was immunoprecipited from yeast cells using antibody 8WG16 which specifically recognizes the C-terminal heptapeptide repeats of Rpb1 [25]. The immunoprecipitates were subject to Western blot and probed with antibodies that were known to recognize potential covalent modifications. Interestingly, when the immunoprecipitated Rpb1 was probed with an anti-SUMO antibody, several bands could be seen in the UV irradiated samples, but not in the unirradiated ones (Fig. 1 A), indicating that Rpb1 was sumoylated in response to UV-induced DNA damage. To confirm this finding, a reciprocal immunoprecipitation was carried out. Sumoylated proteins were immunoprecipitated from normally cultured and UV-irradiated cells using an anti-SUMO antibody, and the immunoprecipitates were probed with 8WG16 on a Western blot. Several bands could be detected in the UV irradiated sample, but not in the unirradiated one (Fig. 1B), indicating that Rpb1 is sumoylated in response to UV-induced DNA damage. The different bands may reflect different forms of sumoylated Rpb1 (e.g., mono-, poly- or multi-sumoylated).

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