Mechanism of open complex and dual incision formation by human nucleotide excision repair factors

Area of Science:

• Molecular Biology
• DNA Repair Mechanisms
• Human Cellular Processes

Background:

• Nucleotide excision repair (NER) involves dual endonuclease cleavage of damaged DNA strands.
• XPG and ERCC1-XPF enzymes perform 3' and 5' incisions, respectively, at DNA-protein adducts.
• ATP-dependent formation of an open DNA structure precedes dual incision in NER.

Purpose of the Study:

• To investigate the mechanism of open complex formation during nucleotide excision repair.
• To elucidate the roles of specific proteins, including TFIIH subunits and XPC, in NER initiation.
• To understand the sequential steps leading to dual incision and DNA repair.

Main Methods:

• Utilized cell-free extracts from human cells with specific protein deficiencies (XPB, XPD, XPC, RPA, XPA, XPG, ERCC1-XPF).
• Assessed the ability of these extracts to form open DNA structures and perform dual incisions.
• Analyzed the impact of mutations in TFIIH (XPB/XPD) and other NER factors on DNA opening and cleavage.

Main Results:

• Mutations in XPB or XPD (TFIIH subunits) or deficiency in XPC protein completely inhibited DNA opening and dual incision.
• Absence of RPA, XPA, or XPG resulted in intermediate levels of DNA strand separation.
• ERCC1-XPF-defective extracts formed the 3' incision but lacked the 5' incision, indicating a role for XPB in facilitating the 5' cleavage.

Conclusions:

• TFIIH-associated helicase activity and XPC protein are crucial for initiating the formation of the open DNA intermediate in NER.
• Full extension of the open structure to cleavage sites is promoted by other core NER factors.
• The 3' incision is made before the 5' incision, and XPB plays a role in facilitating the ERCC1-XPF-mediated 5' incision.