UCSC Engineering
(Last Update: 04/18/00 )
Cyclobutane pyrimidine dimers (CPDs) and [6-4]pyrimidine-pyrimidinone photoproducts ([6-4]PPs) produced in DNA by UVC or UVB radiation are repaired by a complex multistep process involving many more interacting gene products in eukaryotes than prokaryotes. The system is essentially the same in single cell organisms such as S. cerevisiae and large multicellular organisms such a H. sapiens.
The repair process, in principle, involves removal of a 27-29 nt oligonucleotide containing the photoproduct by precisely positioned cleavages 5 nt on the 3' side of the photoproduct, and 24 nt on the 5' side. Once this oligonucleotide is removed, the resulting gap is filled in by DNA polymerase delta, proliferating cell nuclear antigen (PCNA), single strand binding protein and ligase. These processes can be considered as involving sequential steps of photoproduct recognition, assembly of the excision complex, displacement of the excised fragment, and polymerization of the replacement patch. NER operates by assembly of individual factors at sites of DNA damage rather than by preassembly of holocomplexes. The core protein factors required for excision of damage include the XPA protein, the heterotrimeric replication protein (RPA), the 6 to 9 subunit transcription factor TFIIH, the XPC-hHR23B complex, the XPG nuclease, and the ERCC1-XPF nuclease.
Photoproduct recognition is achieved by the specific binding of several proteins: XPA, XPC, and XPE. The XPA gene product was the first damage-recognition protein to be identified and appears to occupy a central role in repair of all regions of DNA, and is rate-limiting for repair in human cells. Recognition may occur due to distortions and single strandedness in DNA from photoproducts, or the photoproducts could swing out of the DNA helix into a pocket in the protein, as occurs in some other DNA repair enzymes. XPA is located on chromosome 9q34.1 and encodes a 273 amino acid Zn2+ finger protein (XPA) that participates in photoproduct recognition and DNA binding. The earliest reaction step that has been reported involves XPC by itself. This binding may be followed by the formation of a quasi-stable complex consisting of XPA, XPC, RPA, and TFIIH, which then acts as a nucleation site for binding of the incision/excision enzymes.
Of the two major photoproducts, [6-4] photoproducts and cyclobutane pyrimidine dimers, XPA alone shows only weak binding to pyrimidine dimers. The XPA-RPA complex appears to bind to damaged sites in DNA once they have been recognized and bound by either XPC/HHR23B in nontranscribed regions of DNA or by stalled RNA polymerase II transcription machinery in transcribed regions. XPA is also required for repair of oxidative damage in mitochondrial DNA , indicating potential overlap between nucleotide and base excision repair also found with XPG.
Mutations have been found in XPA patients throughout the gene with the exception of exon I. The first exon is essential for nuclear localization but not for DNA repair when the protein is expressed at high levels. Exon II encodes a domain for binding to ERCC1, a component of the heterodimeric 5' endonuclease composed of ERCC1 and XPF (ERCC4) , and deletion of the ERCC1 binding region in vitro generates a dominant negative phenotype. Exon III encodes the Zn2+ finger, which is part of the DNA binding domain encoded by exons III - V. The TFIIH complex interacts with exon VI of XPA.
The XPC-hHR23B complex is the earliest damage detector to initiate NER in nontranscribed DNA, acting before the XPA protein, and serves to stabilize XPA binding to the damaged site with a high affinity for the (6-4)PP. The XPC protein may be required for transient nucleosome unfolding during NER. This complex is specifically involved in global genome repair (GGR) but not transcription coupled repair (TCR), where the arrest of RNA polymerase II at a damaged base may function in its place. Stable association of TFIIH with DNA lesions is dependent on the integrity of XPA and XPC proteins.
The XPE protein is composed of two subunits that copurify. One, p48 is found to carry mutations from several XPE patients, and the other p125 does not, and these may be involved in the repair of less accessible lesions in nontranscribed DNA. The p48 subunit is inducible in human cells and is not expressed in hamster cells that fail to repair CPDs in nontranscribed DNA, although this is not due to an absence of the genes for either subunit. There is a strong dependence of p48 mRNA levels on basal p53 expression which is one of several links between p53 and NER.
After assembly the XPC-hHR23B complex dissociates and the XPG protein cuts 3' to the lesion and the ERCC1-XPF heterodimer cuts 5' to the CPD. The nuclease complex plus the 29-30 nt single strand fragment is released by the action of transcription factor TFIIH which contains both 3'-5' (XPB) and 5'-3' (XPD) helicases. The XPG protein is also required for TCR of oxidative damage. At least one component of TFIIH, XPB, interacts with p53 and initiates a signal cascade leading to apoptosis in damaged cells. The whole NER process requires about 100 nt of DNA along which to operate. PCNA, which is required for repair synthesis, also interacts with GADD45, a damage inducible protein, which stimulates excision repair in vitro, though its in vivo function is not known.
Many of the components of the excision repair machinery are the products of genes that give rise to a variety of sun-sensitive and developmental disorders, including xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). Eight genes have been identified among XP patients : seven are involved in nucleotide excision repair (XPA-XPG) and one, the XP variant (XPV), is a damage-specific polymerase that is required for accurate replication of damaged DNA. The complexity of these diseases comes not only from the specific steps that gene products play in repair but also from secondary roles in transcription factors and signaling cascades. Several components of excision repair, especially the genes ERCC1 and hHR23B have not been found among excision repair defective complementation groups. Inactivation of the ERCC1 gene produces UV sensitive cells and causes lethal liver failure in mice.
The following tables of genes were modified from Joel Huberman's DNA Repair Lectures, which explain the color coding and organization of the tables. Eventually each of the proteins should have a link to a page of detailed information---for now, just XPA and XPD have pages.
| S. cerevisiae protein | Human protein | Probable function |
|---|---|---|
| Rad14 | XPA | Binds damaged DNA after XPC or RNA pol II |
| Rpa1,2,3 | RPAp70,p32, p14 |
Stabilizes open complex (with Rad14/XPA); positions nucleases |
| Rad4 | XPC | Works with hHR23B; binds damaged DNA; recruits other NER proteins |
| Rad23 | hHR23B | Cooperates with XPC (see above); contains ubiquitin domain; interacts with proteasome and XPC |
| Ssl2 (Rad25) | XPB | 3' to 5' helicase |
| Tfb1 | p62 | ? |
| Tfb2 | p52 | ? |
| Ssl1 | p44 | DNA binding? |
| Tfb4 | p34 | DNA binding? |
Rad3 |
5' to 3' helicase | |
| Tfb3/Rig2 | MAT1 | CDK assembly factor |
| Kin28 | Cdk7 | CDK; C-terminal domain kinase; CAK |
| Ccl1 | CycH | Cyclin |
| Rad2 | XPG | Endonuclease (3' incision); stabilizes full open complex |
| Rad1 | XPF | Part of endonuclease (5' incision) |
| Rad10 | ERCC1 | Part of endonuclease (5' incision) |
| (icon needed) UCSC Center for Biomolecular Engineering |
UCSC Engineering
|
(icon needed) DNA repair main page |
The Bioinformatics group at UCSC is supported in part by NSF grant BIR-9408579, NSF grant MIP-9488395, DOE grant DE-FG03-95ER62112, LACOR grant 4158U0015-3A-01, and by GANN and NSF graduate fellowships.
The DNA-repair pages are not currently funded, so development of them is very slow.
Questions about page content should be directed to
Kevin Karplus