CPD damage recognition by transcribing RNA polymerase II.

Area of Science:

• Molecular Biology
• DNA Repair Mechanisms
• Biochemistry

Background:

• Transcription-coupled repair (TCR) is crucial for removing DNA lesions, such as ultraviolet light-induced cyclobutane pyrimidine dimers (CPDs).
• RNA polymerase II (Pol II) plays a central role in TCR by stalling at DNA damage sites.

Purpose of the Study:

• To elucidate the structure-based mechanism of the initial step in eukaryotic TCR: CPD-induced stalling of Pol II.
• To understand how DNA lesions like CPDs impede polymerase progression.

Main Methods:

• Structural analysis of RNA polymerase II stalled at cyclobutane pyrimidine dimers.
• Investigating the role of nucleotide misincorporation in polymerase stalling.

Main Results:

• A CPD in the transcribed DNA strand enters the Pol II active site, directing uridine misincorporation into the nascent RNA.
• This uridine misincorporation blocks polymerase translocation, causing stalling.
• Replacing uridine with adenosine allows CPD bypass, confirming CPD-directed misincorporation is key to stalling.
• The stalled polymerase conformation remains unchanged, with the lesion inaccessible.

Conclusions:

• Pol II stalling at CPDs is mediated by lesion-directed uridine misincorporation, not allosteric changes in the polymerase.
• The stalled complex facilitates nonallosteric recruitment of repair factors for lesion excision.