Biomedical subjects

Stuart G Clarkson

Publications and source records attributed to Stuart G Clarkson.

7 recordsLinked to original sources

PubMed2005

Polymorphisms in the human XPD (ERCC2) gene, DNA repair capacity and cancer susceptibility: an appraisal.

Using the human XPD (ERCC2) gene as an example, we evaluate the suggestion that polymorphisms in DNA repair genes lead to decreased DNA repair capacity and to increased cancer susceptibility. This intuitively appealing idea provides the rationale for a large number of studies that have attracted much attention from scientists, clinicians and the general public. Unfortunately, most of this work presupposes that a functional effect has been established for the DNA repair gene polymorphisms under study. For XPD, there is no credible evidence for any effect on DNA repair of the two common polymorphisms leading to p.D312N and p.K751Q amino acid variations, and evolutionary analyses strongly predict that both polymorphisms are benign. Current evidence suggests no causal relationship between XPD polymorphisms, reduced DNA repair and increased cancer risk. We do not believe that more studies of the same kind will be useful. Instead, we suggest a combination of several other approaches, which up to now have been used in only a sporadic way, to examine more rigorously the possibility that phenotypic differences are associated with polymorphisms in other DNA repair genes.

Amino Acid Sequence↗

PubMed2005

Retraction.

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Retraction Notice↗

PubMed2004

The spacer region of XPG mediates recruitment to nucleotide excision repair complexes and determines substrate specificity.

XPG has structural and catalytic roles in nucleotide excision repair (NER) and belongs to the FEN-1 family of structure-specific nucleases. XPG contains a stretch of over 600 amino acids termed the "spacer region" between the conserved N- and I-nuclease regions. Its role is unknown, and it is not similar to any known protein. To investigate its possible functions, we generated and analyzed several deletion mutants of XPG. The spacer region is not required for endonuclease activity, but amino acids 111-550 contribute to the substrate specificity of XPG, and they are required for interaction with TFIIH and for NER activity in vitro and in vivo. Deletion of residues 184-210 and 554-730 leads only to a partial defect in NER activity and a weakened interaction with TFIIH. XPGDelta184-210 and XPGDelta554-730 are not observed at sites of local UV damage in living cells by immunofluorescence, suggesting that the weakened interaction between XPG and TFIIH results in an NER reaction with altered kinetics. This study demonstrates that the N-terminal portion of the spacer region is particularly important for NER progression by mediating the XPG-TFIIH interaction and XPG substrate specificity.

Amino Acid Sequence↗

PubMed2004

Definition of a short region of XPG necessary for TFIIH interaction and stable recruitment to sites of UV damage.

XPG is the human endonuclease that cuts 3' to DNA lesions during nucleotide excision repair. Missense mutations in XPG can lead to xeroderma pigmentosum (XP), whereas truncated or unstable XPG proteins cause Cockayne syndrome (CS), normally yielding life spans of <7 years. One XP-G individual who had advanced XP/CS symptoms at 28 years has been identified. The genetic, biochemical, and cellular defects in this remarkable case provide insight into the onset of XP and CS, and they reveal a previously unrecognized property of XPG. Both of this individual's XPG alleles produce a severely truncated protein, but an infrequent alternative splice generates an XPG protein lacking seven internal amino acids, which can account for his very slight cellular UV resistance. Deletion of XPG amino acids 225 to 231 does not abolish structure-specific endonuclease activity. Instead, this region is essential for interaction with TFIIH and for the stable recruitment of XPG to sites of local UV damage after the prior recruitment of TFIIH. These results define a new functional domain of XPG, and they demonstrate that recruitment of DNA repair proteins to sites of damage does not necessarily lead to productive repair reactions. This observation has potential implications that extend beyond nucleotide excision repair.

Alternative Splicing↗

PubMed2003

Structural determinants for substrate binding and catalysis by the structure-specific endonuclease XPG.

XPG belongs to the Fen1 family of structure-specific nucleases and is responsible for the 3' endonucleolytic incision during mammalian nucleotide excision repair. In addition, it has ill-defined roles in the transcription-coupled repair of oxidative DNA damage and likely also in transcription that are independent of its nuclease activity. We have used DNA binding and footprinting assays with various substrates to gain insight into how XPG interacts with DNA. Ethylation interference footprinting revealed that XPG binds to its substrates through interaction with the phosphate backbone on one face of the helix, mainly to the double-stranded DNA. By comparing DNA binding and cleavage activity using single-/double-stranded DNA junction substrates differing in the length of the single-stranded regions, we have found that the 3' but not the 5' single-stranded arm was necessary for DNA binding and incision activity. Furthermore, we show that although a 5' overhang is not required for XPG activity, an overhang containing double-stranded DNA near the junction inhibits the nuclease but not substrate binding activity. Apparently, junction accessibility or flexibility is important for catalysis but not binding of XPG. These results show that XPG has distinct requirements for binding and cleaving DNA substrates.

Animals↗

PubMed2002

No benefits of ultrafractionation in two head-and-neck cancer cell lines with different inherent radiosensitivity.

PURPOSE: To assess if ultrafractionation is applicable in the context of an unknown hyperradiosensitivity (HRS) status, we studied the survival and repair capacity of two tumor cell lines after irradiation with two different dose/fractionation schedules that can be used in a clinical setting. MATERIALS AND METHODS: Squamous cell carcinoma cell lines SCC-3 (radioresistant) and SCC-6 (radiosensitive) were used. Survival was studied by clonogenic assay after multiple fractions of 0.5 Gy (2 fractions/day, 6-h interval) and 2 Gy (1 fraction/day) for a total dose of 8 Gy of gamma-rays. The capacity to repair single-strand and double-strand breaks (SSB, DSB) was assessed by comet assay. The messenger RNA (mRNA) levels of DNA-dependent protein kinase (PK) components were analyzed by RNase protection and real-time polymerase chain reaction (PCR). RESULTS: In both cell lines, no apparent difference was noted between the two fractionation protocols. In particular for SCC-3, the mean surviving fraction tended to be lower after 2 Gy than after 0.5 Gy fractions. In SCC-3 and SCC-6 no significant difference was observed in the repair capacity of SSB and DSB after exposure to single doses of 0.5 Gy or 2 Gy. After exposure to the same single doses, the mRNA levels of DNA-PK catalytic subunit (PKcs), Ku 70, and Ku 80 were similar. CONCLUSIONS: Our data do not support the concept of ultrafractionation, at least when using fractions of 0.5 Gy in the cell lines studied. This suggests that methods for testing HRS status in individual tumors need to be developed before the relevance of ultrafractionation can be investigated in the clinic.

Biomarkers↗

PubMed2002

The founding members of xeroderma pigmentosum group G produce XPG protein with severely impaired endonuclease activity.

Of the eight human genes implicated in xeroderma pigmentosum, defects in XPG produce some of the most clinically diverse symptoms. These range from mild freckling to severe skeletal and neurologic abnormalities characteristic of Cockayne syndrome. Mildly affected xeroderma pigmentosum group G patients have diminished XPG endonuclease activity in nucleotide excision repair, whereas severely affected xeroderma pigmentosum group G/Cockayne syndrome patients produce truncated XPG proteins that are unable to function in either nucleotide excision repair or the transcription-coupled repair of oxidative lesions. The first two xeroderma pigmentosum group G patients, XP2BI and XP3BR, were reported before the relationship between xeroderma pigmentosum group G and Cockayne syndrome was appreciated. Here we provide evidence that both patients produce truncated proteins from one XPG allele. From the second allele, XP2BI generates full-length XPG of 1186 amino acids containing a single L858P substitution that has reduced stability and greatly impaired endonuclease activity. In XP3BR, a single base deletion and alternative splicing at a rare noncanonical AT-AC intron produces a 1185 amino acid protein containing 44 internal non-XPG residues. This protein is stably expressed but it also has greatly impaired endonuclease activity. These four XPG products can thus account for the severe ultraviolet sensitivity of XP2BI and XP3BR fibroblasts. These cells, unlike those from xeroderma pigmentosum group G/Cockayne syndrome patients, are capable of limited transcription-coupled repair of oxidative lesions. Our results suggest that the L858P protein in XP2BI and the almost full-length XPG protein in XP3BR are responsible for this activity and for the absence of severe early onset Cockayne syndrome symptoms in these patients.

Amino Acid Sequence↗