Elongating RNA Polymerase II Is Disassembled through Specific Degradation of Its Largest but Not Other Subunits in Response to DNA Damage in Vivo

Shivani Malik,Shruti Bagla,Priyasri Chaurasia,Zhen Duan,Sukesh R Bhaumik

doi:10.1074/jbc.m707649200

Shivani Malik, Shruti Bagla + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m707649200

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Abstract

Although previous biochemical studies have demonstrated global degradation of the largest subunit, Rpb1p, of RNA polymerase II in response to DNA damage, it is still not clear whether the initiating or elongating form of Rpb1p is targeted for degradation in vivo. Further, whether other components of RNA polymerase II are degraded in response to DNA damage remains unknown. Here, we show that the Rpb1p subunit of the elongating, but not initiating, form of RNA polymerase II is degraded at the active genes in response to 4-nitroquinoline-1-oxide-induced DNA damage in Saccharomyces cerevisiae. However, other subunits of RNA polymerase II are not degraded in response to DNA damage. Further, we show that Rpb1p is essential for RNA polymerase II assembly at the active gene, and thus, the degradation of Rpb1p following DNA damage disassembles elongating RNA polymerase II. Taken together, our data demonstrate that Rpb1p but not other subunits of elongating RNA polymerase II is specifically degraded in response to DNA damage, and such a degradation of Rpb1p is critical for the disassembly of elongating RNA polymerase II at the DNA lesion in vivo.

Highlights

A severe ramification of DNA damage is the formation of lesions in the transcribing strands of the active genes
Degradation of Rpb1p but Not Other Subunits of RNA Polymerase II in 4NQO-treated Yeast Cells—To analyze the fate of RNA polymerase II at the DNA lesions in the transcriptionally active genes in vivo, we developed a protocol to cause and analyze 4NQO-induced DNA damage in living yeast cells. 4NQO forms bulky adducts primarily with guanine bases and to a lesser extent with adenine bases in DNA
Several studies have demonstrated that the Rpb1p subunit of RNA polymerase II is globally degraded in response to DNA damage [16, 17, 32, 33, 37]

Summary

EXPERIMENTAL PROCEDURES

Plasmids—The plasmid pFA6a-13Myc-KanMX6 [34] was used for genomic Myc epitope tagging of the proteins of interest. The 4NQO-treated cells were grown in YPG for different time points at 30 °C prior to formaldehyde cross-linking. For ts mutant and its wild-type equivalent, yeast cells were grown in YPG up to an A600 of 0.85 at 23 °C and transferred to 37 °C for 1 h before cross-linking. For analysis of the global levels of the Rpb1p, Rpb2p, Rpb3p, Rpb4p, Rpb6p, Rpb7p, Rpb8p, Rpb9p, Rpb10p, and Rpb11p subunits of RNA polymerase II, the yeast strains carrying Myc epitope-tagged Rpb1p, Rpb2p, Rpb3p, Rpb4p, Rpb6p, Rpb7p, Rpb8p, Rpb9p, Rpb10p, and Rpb11p were grown in YPG up to an A600 of 0.8 prior to 4NQO treatment. Genomic DNA Preparation—Five ml of 4NQO-treated yeast cells was harvested at different time points. Analysis of Global Levels of RNA Polymerase II Subunits— The yeast culture (5 ml) was harvested following 4NQO treatment.

WCE following the protocol as described previously for the

RESULTS

DISCUSSION