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The XPG protein is a DNA endonuclease with a variety of structure specific properties. It cleaves near junction in between the double and single stranded DNA of specific polarity i.e. the N-terminal region (N-region) and internal region (I-region). The sequence of XPG has similarities with a variety of other nucleases. It includes T4 RNase H, T5 D15 proteins and 5'ïƒ 3' exonuclease domain of eubacterial DNA polymerases. The XPF cleaves on the 3' end of the unwinded DNA strand which is recognized with damage which is a process of the NER pathway . It also plays as cofactor for a DNA glycosylase that removes oxidized pyrimidines from DNA [i].
XPG/RAD2 endonuclease family
The ERCC5/XPG gene contains 17 exons, a molecular weight of 32 kb and located on 13q32.3 -q33.1 of the chromosome .
Gene sequence obtained from: http://www.ncbi.nlm.nih.gov/gene/2073
Amino acid sequence obtained from: http://www.uniprot.org/uniprot/P28715.fasta
The DNA endonuclease activity of XPG protein performs 3' incision in nucleotide excision pathway (NER). At the site of lesion, NER protein undergoes a DNA bubble structure formation over a length of 25 nucleotides. Later the XPG protein incises the damage region of the DNA strand prior to 0-2 nucleotides at 3' of the ss-DNA of the ds-DNA junction. It has been found that XPG mostly incise at the 3' end and another protein XPF/ERCC1 incise at the 5' end of the same DNA strand. Moreover the XPG protein is also used non-enzymatically for subsequent 5' incision by XPF heterodimer [ii]. It also cleaves various types of artificial DNA substrates like, bubbles, splayed arms and stem-loops. Moreover it also has a structural function independent of its cleavage activity in the assembly of the NER DNA protein complex .
DNA repair pathways are important for removal of lesions from the DNA strands for further transcription processes. There are 3 types of repair process; Base Excision Repair, Nucleotide Excision Repair and Mis-Match Repair.
Fig1. Above diagram shows nucleotide excision repair pathway of damaged DNA by radiation, oxygen radicals, hydrocarbons and chemicals used during chemotherapy.
NER process undergoes damage recognition, local opening of the DNA duplex around the lesion, dual incision of the damaged DNA strand, gap repair synthesis and strand ligation. It has got 2 forms; GG-NER (Global Genomic-NER) and TC-NER (Transcription Coupled NER). Global Genomic NER are responsible for correcting damages in the DNA those are transcriptionally silent. The damages are recognised by XPC-hHR23D* (Xeroderma Pigmentosum Complementation Group-C), (Rad23 homolog B) and XPAÂ and heterotrimericÂ RPAÂ (Replication Protein-A) also bind to the site that further helps in damage recognition. Alternately in transcription coupled NER the transcription mechanism by DNA polymerase II gets stalled at the lesion site of the strand. Then polymerase II is replaced by CSA (Cockayne Syndrome A), CSB (Cockayne Syndrome B) and XAB2 (XPA Binding Protein 2). The above 2 process create a signal for the complex TFIIH for unwinding of the damaged strand. The GG-NER and TC-NER have the same identical process for the repair. TFIIH has 2 helicases, XPD and XPB that unwind the DNA duplex in the region of the lesion. Later incision is done by XPG (Xeroderma Pigmentosum complementation Group G) and ERCC1/XPF (Excision Repair Cross Complementing Group 1) that cleaves at one strand of the DNA from 3'ïƒ 5' creating a 30 base oligonucleotide containing the lesion. Hence a gap is created in the stand which is later filled up by DNA Polymerase Delta** or DNA Polymerase Epsilon**. And finally sealed by DNA ligase I .
* This complex is combination of XPC and hHR23B complexes. It is similar to one of the two human homologs of the yeast NER pathway protein. A minor fraction of the complex is associated with XPC. The main function of this complex is repair and DNA/chromatin metabolism. They are mainly situated at the nucleus .
** DNA polymerase delta is a molecule of the DNA polymerase that binds to one strand of the DNA and begins moving along 3'ïƒ 5' direction. It is used as template for assembling a leading strand in the damaged DNA double helix [iii].
** DNA polymerase epsilon is used in DNA replication, repair and cell cycle control. It is conserved in sequence and subunit in eukaryotes .
Fig2. Above diagram shows the base excision pathway of damaged DNA in single nucleotide defect or 2-8 nucleotides defect.
This process is mainly used for removal of small, non-helix distorting base lesions from the genome. It removes the bases that cause mutation by mispairing with the other nucleotide or breakage in the DNA during replication . They are first recognised by DNA glycosylase and later flip the damaged base out from the DNA double helix, cleaving the N-glucosidic bond and leaving an AP site . They are later cleaving by AP endonuclease to form a 3' hydroxyl adjacent to a 5, deoxy-ribosephosphate . The resulting strand breaks then proceeds for short patch or long patch. In short patch process a single nucleotide is being replaced in the damaged strand and in long patch 2-10 nucleotides are being replaced in the damaged strand .
5.1 Carcinogen identification
For lungs: Benzo-a-pyrene diol epoxide (PAH's).
XPG protein plays an important role in NER repair pathway. They repair DNA lesions induced by tobacco carcinogens. NER function requires interaction between many DNA repair proteins including XPG. His1104Asp polymorphism is located in the XPG C-terminus, which is required for interactions with the XPB, XPD, p62 and p44 subunits of TFIIH in the incision complex of NER. The C-terminal members are conserved in character rather than sequence, being very rich in basic amino acids. Hence, any amino acid change from basic His to acidic Asp could alter the binding to other proteins in the incision complex and the NER capacity
6.1 Lung cancer
Two genetic polymorphisms T335C (His46His, dbSNP no. rs1047768) and G3507C (Asp1104His, dbSNP no. rs17655), had been investigated. Functional effects of these two SNPs are still unknown. Probably the SNPs in the coding DNA sequences may result in a subtle structural alteration of the ERCC5/XPG activity and modulation of lung cancer susceptibility. Asp/Asp genotype of the Asp1104His polymorphism was associated with a significantly increased risk of lung cancer in white's population. T335C of the His46His polymorphism C/C genotype was associated with a significantly increased risk of lung cancer in Norwegians . Studies done on cases and controls for XPG His1104Asp polymorphism associated with the risk of lung cancer. Findings from stratified analyses show small numbers in the subgroups. Hence, the functional relevance of this XPG polymorphism and its role in cancer susceptibility remain to be determined in larger epidemiological studies .
XPC with the replacement of Ala499Val, XPC which includes the substitution of Lys939Gln, XPD with the replacement of Asp312Asn and Lys751Gln and the substitution of XPG with His1104Asp are some of the trivial allele frequencies found in non-Hispanic whites. The SNPs of the gene ERCC1 with the substitution of C8092A and XPA which includes the replacement of G23A are found to increase the risk of SCCHN . Individuals with at least one variant allele of ERCC5 His1104Asp exhibited an increased risk for the larynx cancer .
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