Svejstrup lab

Mechanisms of Transcription Laboratory

: Ubiquitylation of RNA polymerase II and other proteins

A structural model of elongating RNA polymerase II (cyan) in the act of transcribing a gene. The DNA strands are yellow and green, respectively, while the newly synthesized RNA is red.

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Damage-stalled RNAPII functions as a molecular ‘beacon’ that triggers transcription-coupled nucleotide excision repair (TC-NER).

On the other hand, if that DNA lesion for some reason cannot be removed by TC-NER, a ‘mechanism of last resort’ ensures that RNAPII is ubiquitylated and degraded by the proteasome, enabling repair by other mechanisms, such as global genomic nucleotide excision repair.

Our lab has contributed significantly to the understanding of this last resort pathway over the last two decades. However, the mechanism and factor requirement of RNAPII ubiquitylation and degradation is still not fully understood.

Likewise, it is increasingly clear that not only RNAPII but also a large number of other factors become ubiquitylated in response to DNA damage, and that this is crucial for correct regulation of the DNA damage response. We have developed powerful methods to analyze ubiquitylation on a proteome-wide scale and used them to map hundreds to thousands of damage-induced ubiquitylation sites.

However, it is typically unknown which of the ~1000 ubiquitin ligases found in a human cell is responsible for an individual modification event, and which of these modification events are functionally important. In general, few attempts have thus been made to map ubiquitylation events catalysed by a specific ubiquitin ligase in a proteome-wide manner. We have now successfully achieved this for CSA, a ubiquitylation factor involved in TC-NER, and indirectly in RNAPII ubiquitylation.

In addition to identifying a number of interesting CSA target proteins and their specific ubiquitylation sites that we are presently pursuing, these techniques will be applied to other ubiquitin ligases, which are important in the DNA damage response.