Strong functional interactions of TFIIH with XPC and XPG in human DNA nucleotide excision repair, without a preassembled repairosome

In mammalian cells, the core factors involved in the damage recognition and incision steps of DNA nucleotide excision repair are XPA, TFIIH complex, XPC-HR23B, replication protein A (RPA), XPG, and ERCC1-XPF. Many interactions between these components have been detected, using different physical methods, in human cells and for the homologous factors in Saccharomyces cerevisiae. Several human nucleotide excision repair (NER) complexes, including a high-molecular-mass repairosome complex, have been proposed. However, there have been no measurements of activity of any mammalian NER protein complex isolated under native conditions. In order to assess relative strengths of interactions between NER factors, we captured TFIIH from cell extracts with an anti-cdk7 antibody, retaining TFIIH in active form attached to magnetic beads. Coimmunoprecipitation of other NER proteins was then monitored functionally in a reconstituted repair system with purified proteins. We found that all detectable TFIIH in gently prepared human cell extracts was present in the intact nine-subunit form. There was no evidence for a repair complex that contained all of the NER components. At low ionic strength TFIIH could associate with functional amounts of each NER factor except RPA. At physiological ionic strength, TFIIH associated with significant amounts of XPC-HR23B and XPG but not other repair factors. The strongest interaction was between TFIIH and XPC-HR23B, indicating a coupled role of these proteins in early steps of repair. A panel of antibodies was used to estimate that there are on the order of 10(5) molecules of each core NER factor per HeLa cell.     

DNA damage recognition during nucleotide excision repair in mammalian cells

For the bulk of mammalian DNA, the core protein factors needed for damage recognition and incision during nucleotide excision repair (NER) are the XPA protein, the heterotrimeric RPA protein, the 6 to 9-subunit TFIIH, the XPC-hHR23B complex, the XPG nuclease, and the ERCC1-XPF nuclease. With varying efficiencies, NER can repair a very wide range of DNA adducts, from bulky helical distortions to subtle modifications on sugar residues. Several of the NER factors have an affinity for damaged DNA. The strongest binding factor appears to be XPC-hHR23B but preferential binding to damage is also a property of XPA, RPA, and components of TFIIH. It appears that in order to be repaired by NER, an adduct in DNA must have two features: it must create a helical distortion, and there must be a change in DNA chemistry. Initial recognition of the distortion is the most likely function for XPC-hHR23B and perhaps XPA and RPA, whereas TFIIH is well-suited to locate the damaged DNA strand by locating altered DNA chemistry that blocks translocation of the XPB and XPD components.     

Recruitment of the nucleotide excision repair endonuclease XPG to sites of UV-induced dna damage depends on functional TFIIH

The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3' side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5' incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the DDB2 protein, which is thought to enhance damage recognition by NER factor XPC. Together, our data present a comprehensive view of the in vivo behavior of a protein that is involved in a complex chromatin-associated process.