Ordered Conformational Changes in Damaged DNA Induced by Nucleotide Excision Repair Factors

Abstract

In response to genotoxic attacks, cells activate sophisticated DNA repair pathways such as nucleotide excision repair (NER), which consists of damage removal via dual incision and DNA resynthesis. Using permanganate footprinting as well as highly purified factors, we show that NER is a dynamic process that takes place in a number of successive steps during which the DNA is remodeled around the lesion in response to the various NER factors. XPC/HR23B first recognizes the damaged structure and initiates the opening of the helix from position ؊3 to ؉6. TFIIH is then recruited and, in the presence of ATP, extends the opening from position ؊6 to ؉6; it also displaces XPC downstream from the lesion, thereby providing the topological structure for recruiting XPA and RPA, which will enlarge the opening. Once targeted by XPG, the damaged DNA is further melted from position ؊19 to ؉8. XPG and XPF/ERCC1 endonucleases then cut the damaged DNA at the limit of the opened structure that was previously "labeled" by the positioning of XPC/HR23B and TFIIH.To counteract the detrimental effect of genotoxic attacks, cells activate sophisticated and specific DNA repair pathways. Damage induced by UV radiation, environmental agents, and anticancer drugs are removed by two distinct nucleotide excision repair (NER) 1 subpathways, namely global genome repair (GGR), which eliminates lesions from the entire genome, and transcription-coupled repair (TCR), a specialized pathway that repairs damages on a transcribed strand of active genes (1-3). Human NER involves the ordered action of factors in dual incision and DNA repair resynthesis steps (4). Any mutation that affects either the enzymatic activity or the ordered assembly of the dual incision complex leads to genetic disorders such as xeroderma pigmentosum, trichothiodystrophy, or Cockayne syndrome (5, 6).In global genome repair, the dual incision is a multistep process that results from the coordinated action of XPC/ HR23B, TFIIH, XPA, RPA, XPG, and XPF/ERCC1, resulting in the removal of the damaged oligonucleotide (4, 7, 8). After being recognized by the XPC/HR23B complex, the damaged DNA structure is targeted by TFIIH, which recruits the other factors upon the addition of ATP (9 -11). The unwound DNA is then incised by the two endonucleases XPG and XPF/ERCC1 on the 3Ј and 5Ј side of the lesion, respectively (12-15), leaving a gap structure that is filled up by the DNA polymerase ⑀ or ␦ and the accompanying factors PCNA, RF-C, RPA, and DNA ligase I (16). Whether or not the NER reaction occurs by sequential arrival of the various factors or by a pre-assembled complex referred to as the repairosome or the holoenzyme is still under debate (17)(18)(19). Although the hypothesis of the sequential assembly, which has gained a lot of support from recent biological studies, seems to be more accepted, the order of assembly of the NER factors on the damaged DNA and their contribution to the DNA remodeling to allow the repair are not fully understood (10,20,21). As an example, to further learn abou...

Xeroderma pigmentosum group C gene expression is predominantly regulated by promoter hypermethylation and contributes to p53 mutation in lung cancers

“…XPC is a subunit of the NER multi-protein machinery that facilitates DNA damage recognition (Friedberg, 2001;Riedl et al, 2003). An in vitro study suggested that binding of XPC to damaged DNA is the rate-limiting step for NER (Janicijevic et al, 2003;Tapias et al, 2004). Accumulated evidence also indicates that genetic polymorphisms of DNA repair genes such as XPA, XPC and ERCC1 are associated with increased risk of lung cancer (Mellon et al, 2002;Park et al, 2002;Marin et al, 2004;Hu et al, 2005;Suk et al, 2005).…”

Dynamics of a Benzo[a]pyrene-derived Guanine DNA Lesion in TGT and CGC Sequence Contexts: Enhanced Mobility in TGT Explains Conformational Heterogeneity, Flexible Bending, and Greater Susceptibility to Nucleotide Excision Repair

“…The basic hypothesis in the field of NER is that the mammalian DNA repair apparatus recognizes structural distortions and thermodynamic destabilization in the DNA duplex caused by the lesions, followed by a verification step that detects the presence of a chemically modified nucleobase and the excision of a fragment 24-32 nucleotides long that contains the damaged base. [3][4][5][6][7][8][9][10][11] It is now established that the first and rate-determining step in NER is the recognition of the bulky lesions by the XPC/HR23B protein heterodimer complex. 12 Since the NER machinery processes a diverse array of bulky lesions with different efficiencies, the conformational features that invoke NER are of great interest.…”

Structural insights into the recognition of cisplatin and AAF-dG lesion by Rad14 (XPA)

“…Several lines of evidence lead to the currently accepted hypothesis that the lesions are initially recognized by XPC with the help of UV-DDB (UV DNA damage binding protein) for cyclobutane pyrimidine dimer (CPD) lesions (17)(18)(19)(20) followed by damage verification by XPD/TFIIH (21)(22)(23)(24), XPA, and XPG (3) to finally assemble to the preincision complex (3,(25)(26)(27). This step seems to involve recruitment of the XPA protein, which is one of the proteins essential for both GG-NER and TC-NER (28).…”