Biomedical subjects

Richard D Wood

Publications and source records attributed to Richard D Wood.

4 recordsLinked to original sources

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↗