Biomedical subjects

Richard D Wood

Publications and source records attributed to Richard D Wood.

14 recordsLinked to original sources

PubMed2006

XPA protein as a limiting factor for nucleotide excision repair and UV sensitivity in human cells.

Nucleotide excision repair (NER) acts on a variety of DNA lesions, including damage induced by many chemotherapeutic drugs. Cancer therapy with such drugs might be improved by reducing the NER capacity of tumors. It is not known, however to what extent any individual NER protein is rate-limiting for any step of the repair reaction. We studied sensitivity to UV radiation and repair of DNA damage with regard to XPA, one of the core factors in the NER incision complex. About 150,000-200,000 molecules of XPA protein are present in NER proficient human cell lines, and no XPA protein in the XP-A cell line XP12RO. Transfected XP12RO cell lines expressing 50,000 or more XPA molecules/cell showed UV resistance similar to normal cells. Suppression of XPA protein to approximately 10,000 molecules/cell in a Tet-regulatable system modestly but significantly increased sensitivity to UV irradiation. No removal of cyclobutane pyrimidine dimers was detected in the SV40 immortalized cell lines tested. Repair proficient WI38-VA fibroblasts and transfected XP-A cells expressing 150,000 molecules of XPA/cell removed (6-4) photoproducts from the genome with a half-life of 1h. Cells in which XPA protein was reduced to about 10,000 molecules/cell removed (6-4) photoproducts more slowly, with a half-life of 3h. A reduced rate of repair of (6-4) photoproducts thus results in increased cellular sensitivity towards UV irradiation. These data indicate that XPA levels must be reduced to <10% of that present in a normal cell to render XPA a limiting factor for NER and consequent cellular sensitivity. To inhibit NER, it may be more effective to interfere with XPA protein function, rather than reducing XPA protein levels.

Antineoplastic Agents↗

PubMed2006

Loss of DNA polymerase zeta causes chromosomal instability in mammalian cells.

Rev3L encodes the catalytic subunit of DNA polymerase zeta (pol zeta) in mammalian cells. In yeast, pol zeta helps cells bypass sites of DNA damage that can block replication enzymes. Targeted disruption of the mouse Rev3L gene causes lethality midway through embryonic gestation, and Rev3L-/- mouse embryonic fibroblasts (MEFs) remain in a quiescent state in culture. This suggests that pol zeta may be necessary for tolerance of endogenous DNA damage during normal cell growth. We report the generation of mitotically active Rev3L-/- MEFs on a p53-/- genetic background. Rev3L null MEFs exhibited striking chromosomal instability, with a large increase in translocation frequency. Many complex genetic aberrations were found only in Rev3L null cells. Rev3L null cells had increased chromosome numbers, most commonly near pentaploid, and double minute chromosomes were frequently found. This chromosomal instability associated with loss of a DNA polymerase activity in mammalian cells is similar to the instability associated with loss of homologous recombination capacity. Rev3L null MEFs were also moderately sensitive to mitomycin C, methyl methanesulfonate, and UV and gamma-radiation, indicating that mammalian pol zeta helps cells tolerate diverse types of DNA damage. The increased occurrence of chromosomal translocations in Rev3L-/- MEFs suggests that loss of Rev3L expression could contribute to genome instability during neoplastic transformation and progression.

Animals↗

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

Human DNA repair genes, 2005.

An updated inventory of about 150 human DNA repair genes is described. The compilation includes genes encoding DNA repair enzymes, some genes associated with cellular responses to DNA damage, and other genes associated with genetic instability or sensitivity to DNA damaging agents. The updated human DNA repair genes table (http://www.cgal.icnet.uk/DNA_Repair_Genes.htmlhttp://www.cgal.icnet.uk/DNA_Repair_Genes.html) is a research and reference tool that directly links to several databases: Gene Cards, Online Mendelian Inheritance in Man, the NCBI MapViewer for chromosome position, and the NCBI Entrez database for the reference nucleotide sequence. This article discusses the approximately 25 genes added, since the original version of the table was first produced in 2001, and some other revisions.

Chromatin↗

PubMed2005

DDB1-DDB2 (xeroderma pigmentosum group E) protein complex recognizes a cyclobutane pyrimidine dimer, mismatches, apurinic/apyrimidinic sites, and compound lesions in DNA.

The DDB protein complex, comprising the subunits DDB1 and DDB2, binds tightly to UV light-irradiated DNA. Mutations in DDB2 are responsible for xeroderma pigmentosum group E, a disorder with defects in nucleotide excision repair of DNA. Both subunits are also components of a complex involved in ubiquitin-mediated proteolysis. Cellular defects in DDB2 disable repair of the major UV radiation photoproduct in DNA, a cyclobutane pyrimidine dimer, but no significant direct binding of DDB to this photoproduct in DNA has ever been demonstrated. Thus, it has been uncertain how DDB could play a specific role in DNA repair of such damage. We investigated DDB function using highly purified proteins. Co-purified DDB1-DDB2 or DDB reconstituted with individual DDB1 and DDB2 subunits binds to damaged DNA as a ternary complex. We found that DDB can indeed recognize a cyclobutane pyrimidine dimer in DNA with an affinity (K(app)a) 6-fold higher than that of nondamaged DNA. The DDB1-DDB2 complex also bound with high specificity to a UV radiation-induced (6-4) photoproduct and to an apurinic site in DNA. Unexpectedly, DDB also bound avidly to DNA containing a 2- or 3-bp mismatch (and does not bind well to DNA containing larger mismatches). These data indicate that DDB does not detect lesions per se. It instead recognizes other structural features of damaged DNA, acting as a sensor that probes DNA for a subset of conformational changes. Lesions recognized may include those arising when translesion polymerases such as POLH incorporate bases across from DNA lesions caused by UV radiation.

Amino Acid Sequence↗

PubMed2005

DNA polymerases and somatic hypermutation of immunoglobulin genes.

Somatic hypermutation of immunoglobulin variable genes, which increases antibody diversity, is initiated by the activation-induced cytosine deaminase (AID) protein. The current DNA-deamination model posits that AID deaminates cytosine to uracil in DNA, and that mutations are generated by DNA polymerases during replication or repair of the uracil residue. Mutations could arise as follows: by DNA replicating past the uracil; by removing the uracil with a uracil glycosylase and replicating past the resulting abasic site with a low-fidelity polymerase; or by repairing the uracil and synthesizing a DNA-repair patch downstream using a low-fidelity polymerase. In this review, we summarize the biochemical properties of specialized DNA polymerases in mammalian cells and discuss their participation in the mechanisms of hypermutation. Many recent studies have examined mice deficient in the genes that encode various DNA polymerases, and have shown that DNA polymerase H (POLH) contributes to hypermutation, whereas POLI, POLK and several other enzymes do not have major roles. The low-fidelity enzyme POLQ has been proposed as another candidate polymerase because it can efficiently bypass abasic sites and recent evidence indicates that it might participate in hypermutation.

Animals↗

PubMed2004

High-efficiency bypass of DNA damage by human DNA polymerase Q.

Endogenous DNA damage arises frequently, particularly apurinic (AP) sites. These must be dealt with by cells in order to avoid genotoxic effects. DNA polymerase theta; is a newly identified enzyme encoded by the human POLQ gene. We find that POLQ has an exceptional ability to bypass an AP site, inserting A with 22% of the efficiency of a normal template, and continuing extension as avidly as with a normally paired base. POLQ preferentially incorporates A opposite an AP site and strongly disfavors C. On nondamaged templates, POLQ makes frequent errors, incorporating G or T opposite T about 1% of the time. This very low fidelity distinguishes POLQ from other A-family polymerases. POLQ has three sequence insertions between conserved motifs in its catalytic site. One insert of approximately 22 residues into the tip of the polymerase thumb subdomain is predicted to confer considerable flexibility and additional DNA contacts to affect enzyme fidelity. POLQ is the only known enzyme that efficiently carries out both the insertion and extension steps for bypass of AP sites, commonly formed as endogenous genomic lesions.

Amino Acid Motifs↗

PubMed2004

Reduced levels of XPA, ERCC1 and XPF DNA repair proteins in testis tumor cell lines.

Over 80% of patients with advanced metastatic testis tumors can be cured using cisplatin-based combination chemotherapy. This is unusual as metastatic cancer in adults is usually incurable. Cell lines derived from testis tumors retain sensitivity to cisplatin in vitro. We previously investigated 2 testis tumor cell lines with a low capacity to remove cisplatin-induced DNA damage and found that they had low levels of the DNA nucleotide excision repair proteins XPA, ERCC1 and XPF. To determine whether low levels of XPA, ERCC1 and XPF proteins are characteristic of testis tumor cell lines, we investigated 35 cell lines derived from cancers to determine whether groups of cell lines from diverse tissue origins differ from one another in constitutive levels of these NER proteins. Quantitative immunoblotting was used to compare groups of cell lines representing prostate, bladder, breast, lung, cervical, ovarian and testis cancers. Only the 6 testis tumor cell lines showed significantly lower mean levels of XPA (p = 0.001), XPF (p = 0.001) and ERCC1 (p = 0.004) proteins from the other groups. Our results encourage further investigation of the possibility that low levels of these nucleotide excision repair proteins could be related to the favorable response of testis tumors to cisplatin-based chemotherapy.

Cell Line, Tumor↗

PubMed2004

Optimisation of the superplastic forming of a dental implant for bone augmentation using finite element simulations.

OBJECTIVES: The phenomenon of superplasticity has made it possible to form complex shapes that require extremely high degrees of ductility in titanium alloy with minimal internal stresses. Combined with the use of an investment casting material as the die material, which makes possible the forming of re-entrant angles, it is possible to produce membranes for ridge augmentation. The aim is to characterise the metal alloy sheet and simulate the superplastic forming process in three dimensions to produce process parameters, namely gas pressure as a function of time, to accurately adapt the titanium sheet to the bone surface. METHOD: The surface of the die was digitised using a 3D laser scanning system (UBM-Keyence LC2450). Ti-6Al-4V sheet of 140 mm diameter was modelled using a grid of triangular membrane elements. This mesh was automatically refined during the simulations. Finite element simulation was carried out using the Superflag software program (University of Wales Swansea) Three different options for gas pressure control were adopted, namely, target flow stress, target strain rate and target energy dissipation. The pressure cycles produced from the simulation were used to form titanium alloy sheet at 900 degrees C using argon gas. The deformed regions of the formed sheet were then examined to determine the regions of contact with the die and to characterise surface damage. RESULTS: Comparison of the simulations with experiment showed that there was good agreement between simulated and experimental thickness distributions in most parts of the sheet that were examined. Interrupted tests showed that in the intermediate positions of the forming sheet the simulations were slightly ahead of the experiment. The target stress option was found to produce the best degree of adaptation and the sheet formed using this cycle showed good surface quality, whereas in highly deformed regions using the other target options, the sheet was found to have formed microcracks. The use of a solid lubricant on the surface of the forming sheet was not found to have a significant influence on the adaptation of the titanium alloy sheet except in areas of high deformation where the sheet perforated. SIGNIFICANCE: The finite element membrane formulation is well adapted to the superplastic forming of a ridge augmentation membrane prosthesis. The simulation accurately describes the evolution of the shape of the prosthesis and its thickness distribution with time, which allows the manufacturer to select an appropriate initial thickness of titanium alloy sheet prior to attempting to form the component. The investment dies are found to have sufficient strength to withstand the forming operation if a suitable orientation of the titanium sheet with respect to the die is adopted. A metal surface of good quality can be produced in the formed prosthesis using the appropriate gas control option.

Alloys↗

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

POLQ (Pol theta), a DNA polymerase and DNA-dependent ATPase in human cells.

The genomes of eukaryotic cells predict the existence of multiple DNA polymerases, which are proposed to serve specialized roles in DNA replication and repair. We report here the isolation of the full-length human DNA POLQ gene, and an initial characterization of its gene product, DNA polymerase theta. POLQ is of particular interest as it is orthologous to Drosophila Mus308, a gene implicated in cellular resistance to interstrand DNA cross-linking agents. The POLQ cDNA encodes a polypeptide of 2592 amino acids with an ATPase-helicase domain in the N-terminal part of the protein, a central spacer domain, and a DNA polymerase domain in the C-terminal portion. This arrangement is conserved with Mus308. Expression of an mRNA of approximately 8.5 kb was detected in human cell lines. In a survey of human and mouse tissues, expression was highest in testis. Immunoblotting with POLQ antibodies detected a protein of >250 kDa in extracts from HeLa cells. Prominent fragments of approximately 100 kDa suggest that POLQ is readily proteolyzed. Full-length human POLQ was expressed from a baculovirus system. Purified POLQ showed DNA polymerase activity on nicked double-stranded DNA and on a singly primed DNA template. The enzyme activity was resistant to aphidicolin, consistent with its membership of the A family of DNA polymerases, and inhibited by dideoxynucleotides. POLQ further exhibited a single-stranded DNA-dependent ATPase activity.

Adenosine Triphosphatases↗

PubMed2003

DDB complexities.

A group of recent publications contribute new insights concerning the role of the DNA damage-binding protein complex (DDB) in DNA repair. Mutations in the 48kDa DDB2 subunit are now found in all confirmed cases of xeroderma pigmentosum complementation group E. Several studies have reported a connection between the 127kDa DDB1 subunit and proteins involved in ubiquitin-mediated proteolysis. One such multiprotein complex containing DDB1 and DDB2 is closely related to a complex containing DDB1 and the Cockayne syndrome group A (CSA) protein. There is accumulating evidence for several levels of cellular regulation of DDB, including translocation to the nucleus, proteolytic degradation of DDB2 protein, and transcriptional induction of DDB2 mRNA. Although the mechanism is not yet known, it appears that DDB assists in nucleotide excision repair in chromatin.

DNA Damage↗

PubMed2003

POLN, a nuclear PolA family DNA polymerase homologous to the DNA cross-link sensitivity protein Mus308.

The Drosophila Mus308 gene is unusual in encoding both a family A DNA polymerase domain and a DNA/RNA helicase domain. A mus308 mutation was shown to result in increased sensitivity to DNA cross-linking agents, leading to the hypothesis that Mus308 functions in the repair of DNA interstrand cross-links. Recently a mammalian ortholog of Mus308, POLQ, has been identified. We report here the identification, cloning, and characterization of POLN and its gene product, a new mammalian DNA polymerase also related to Mus308. The human cDNA encodes a protein of 900 amino acid residues. The region starting from residue 419 shares 33% identity (48% similarity) with the equivalent region of Escherichia coli DNA polymerase I. POLN is expressed in human cell lines with numerous alternatively spliced transcripts, and a full-length human coding region that comprises 24 exons within 160 kilobases of genomic DNA. Expression analysis by northern blotting and in situ hybridization showed highest expression of full-length POLN in human and mouse testis. POLN localized to the nucleus when expressed as a enhanced green fluorescent protein (GFP)-tagged protein in human fibroblasts. GFP-tagged recombinant POLN had DNA polymerase activity on activated calf thymus DNA and on a singly primed template.

Amino Acid Sequence↗

PubMed2001

A human DNA helicase homologous to the DNA cross-link sensitivity protein Mus308.

Repair of DNA interstrand cross-links is a challenging problem for cells. Many human gene products influence sensitivity to DNA cross-linking agents, but the mechanisms of cross-link repair are unknown. In Drosophila melanogaster, the mus308 mutation leads to marked sensitivity to DNA cross-linking agents. The C-terminal portion of the Mus308 polypeptide encodes a DNA polymerase, whereas a putative DNA helicase is encoded by the N-terminal portion. As a step toward isolating proteins involved in DNA cross-link repair, we searched for mammalian genes similar to the DNA helicase portion of Mus308. Human and mouse homologs were isolated from cDNA expression libraries and designated HEL308. Human HEL308 is on chromosome 4q21 and encodes a polypeptide of 1101 amino acids. The protein was expressed in insect cells and purified. HEL308 is a single-stranded DNA-dependent ATPase and DNA helicase. Mutation of a highly conserved lysine to methionine in helicase domain I eliminated both activities. The protein readily displaces 20- to 40-mer duplex oligonucleotides. Displacement of longer substrates was less efficient but was stimulated by the single-stranded DNA-binding protein RPA. Activity was supported by ATP or dATP but not other nucleotide triphosphates. The enzyme translocates on DNA with 3' to 5' polarity and behaves as a multimer upon gel filtration.

Adenosine Triphosphatases↗