Accumulation of (5'S)-8,5'-cyclo-2'-deoxyadenosine in organs of Cockayne syndrome complementation group B gene knockout mice

Cockayne syndrome (CS) is a human genetic disorder characterized by sensitivity to UV radiation, neurodegeneration, premature aging among other phenotypes. CS complementation group B (CS-B) gene (csb) encodes the CSB protein (CSB) that is involved in base excision repair of a number of oxidatively induced lesions in genomic DNA in vivo. We hypothesized that CSB may also play a role in cellular repair of the DNA helix-distorting tandem lesion (5'S)-8,5'-cyclo-2'-deoxyadenosine (S-cdA). Among many DNA lesions, S-cdA is unique in that it represents a concomitant damage to both the sugar and base moieties of the same nucleoside. Because of the presence of the C8-C5' covalent bond, S-cdA is repaired by nucleotide excision repair unlike most of other oxidatively induced lesions in DNA, which are subject to base excision repair. To test our hypothesis, we isolated genomic DNA from brain, kidney and liver of wild type and csb knockout (csb(-/-)) mice. Animals were not exposed to any exogenous oxidative stress before the experiment. DNA samples were analysed by liquid chromatography/mass spectrometry with isotope-dilution. Statistically greater background levels of S-cdA were observed in all three organs of csb(-/-) mice than in those of wild type mice. These results suggest the in vivo accumulation of S-cdA in genomic DNA due to lack of its repair in csb(-/-) mice. Thus, this study provides, for the first time, the evidence that CSB plays a role in the repair of the DNA helix-distorting tandem lesion S-cdA. Accumulation of unrepaired S-cdA in vivo may contribute to the pathology associated with CS.     

Functional importance of the conserved N-terminal domain of the mitochondrial replicative DNA helicase

The mitochondrial replicative DNA helicase is an essential cellular protein that shows high similarity with the bifunctional primase-helicase of bacteriophage T7, the gene 4 protein (T7 gp4). The N-terminal primase domain of T7 gp4 comprises seven conserved sequence motifs, I, II, III, IV, V, VI, and an RNA polymerase basic domain. The putative primase domain of metazoan mitochondrial DNA helicases has diverged from T7 gp4 and in particular, the primase domain of vertebrates lacks motif I, which comprises a zinc binding domain. Interestingly, motif I is conserved in insect mtDNA helicases. Here, we evaluate the effects of overexpression in Drosophila cell culture of variants carrying mutations in conserved amino acids in the N-terminal region, including the zinc binding domain. Overexpression of alanine substitution mutants of conserved amino acids in motifs I, IV, V and VI and the RNA polymerase basic domain results in increased mtDNA copy number as is observed with overexpression of the wild type enzyme. In contrast, overexpression of three N-terminal mutants W282L, R301Q and P302L that are analogous to human autosomal dominant progressive external ophthalmoplegia mutations results in mitochondrial DNA depletion, and in the case of R301Q, a dominant negative cellular phenotype. Thus whereas our data suggest lack of a DNA primase activity in Drosophila mitochondrial DNA helicase, they show that specific N-terminal amino acid residues that map close to the central linker region likely play a physiological role in the C-terminal helicase function of the protein.     

Conserved motifs II to VI of DNA helicase II from Escherichia coli are all required for biological activity.

There are seven conserved motifs (IA, IB, and II to VI) in DNA helicase II of Escherichia coli that have high homology among a large family of proteins involved in DNA metabolism. To address the functional importance of motifs II to VI, we employed site-directed mutagenesis to replace the charged amino acid residues in each motif with alanines. Cells carrying these mutant alleles exhibited higher UV and methyl methanesulfonate sensitivity, increased rates of spontaneous mutagenesis, and elevated levels of homologous recombination, indicating defects in both the excision repair and mismatch repair pathways. In addition, we also changed the highly conserved tyrosine(600) in motif VI to phenylalanine (uvrD309, Y600F). This mutant displayed a moderate increase in UV sensitivity but a decrease in spontaneous mutation rate, suggesting that DNA helicase II may have different functions in the two DNA repair pathways. Furthermore, a mutation in domain IV (uvrD307, R284A) significantly reduced the viability of some E. coli K-12 strains at 30 degrees C but not at 37 degrees C. The implications of these observations are discussed.     

UvrD2 is essential in Mycobacterium tuberculosis, but its helicase activity is not required

UvrD is an SF1 family helicase involved in DNA repair that is widely conserved in bacteria. Mycobacterium tuberculosis has two annotated UvrD homologues; here we investigate the role of UvrD2. The uvrD2 gene at its native locus could be knocked out only in the presence of a second copy of the gene, demonstrating that uvrD2 is essential. Analysis of the putative protein domain structure of UvrD2 shows a distinctive domain architecture, with an extended C terminus containing an HRDC domain normally found in SF2 family helicases and a linking domain carrying a tetracysteine motif. Truncated constructs lacking the C-terminal domains of UvrD2 were able to compensate for the loss of the chromosomal copy, showing that these C-terminal domains are not essential. Although UvrD2 is a functional helicase, a mutant form of the protein lacking helicase activity was able to permit deletion of uvrD2 at its native locus. However, a mutant protein unable to hydrolyze ATP or translocate along DNA was not able to compensate for lack of the wild-type protein. Therefore, we concluded that the essential role played by UvrD2 is unlikely to involve its DNA unwinding activity and is more likely to involve DNA translocation and, possibly, protein displacement.     

Misincorporation of dAMP opposite 2-hydroxyadenine, an oxidative form of adenine.

Nucleotide incorporation opposite an oxidative form of adenine, 2-hydroxyadenine (2-OH-Ade) was investigated. When a primed template with 2-OH-Ade was treated with an exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I (KFexo-), recombinant rat DNA polymerase beta (pol beta) or calf thymus DNA polymerase alpha (pol alpha), incorporation of dTMP and dAMP was observed. In addition, KFexo- inserted dGMP as well. A steady-state kinetic study indicated that the insertion of dAMP and dTMP opposite the DNA lesion occurred with similar frequency with KFexo- and pol beta. Insertion of dTMP opposite 2-OH-Ade was favored to that of dAMP by pol alpha. Chain extension from the A.2-OH-Ade pair is less favored than that from the T.2-OH-Ade pair by all three DNA polymerase. Analysis of full-length products of in vitro DNA synthesis showed that dTMP and dAMP were incorporated by DNA polymerases and that exonuclease-proficient and -deficient Klenow fragments also inserted dGMP opposite 2-OH-Ade. These results suggest that formation of 2-OH-Ade from A in DNA will induce A-->T and A-->C transversions in cells.     

A mutation in the C-terminal putative Zn2+ finger motif of UL52 severely affects the biochemical activities of the HSV-1 helicase-primase subcomplex

Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex that is composed of the products of the UL5, UL52, and UL8 genes. A subcomplex consisting of the UL5 and UL52 proteins retains all the enzymatic activities exhibited by the holoenzyme in vitro. The UL52 protein contains a putative zinc finger at its C terminus which is highly conserved among both prokaryotic and eukaryotic primases. We constructed a mutation in which two highly conserved cysteine residues in the zinc finger motif were replaced with alanine residues. A UL52 expression plasmid containing the mutation in the zinc finger region is unable to support the growth of a UL52 mutant virus in a transient complementation assay. Wild type and mutant UL5.UL52 subcomplexes were purified from insect cells infected with recombinant baculoviruses. Surprisingly, the mutant protein was severely affected in all biochemical activities tested; no helicase or primase activities could be detected, and the mutant protein retains only about 9% of wild type levels of single-stranded DNA-dependent ATPase activity. Gel mobility shift assays showed that DNA binding is severely affected as well; the mutant subcomplex only retains approximately 8% of wild type levels of binding to a forked substrate. On the other hand, the mutant protein retains its ability to interact with UL5 as indicated by copurification and with UL8 as indicated by a supershifted band in the gel mobility shift assay. In addition, the ability of individual subunits to bind single-stranded DNA was examined by photo cross-linking. In the wild type UL5.UL52 subcomplex, both subunits are able to bind an 18-mer of oligo(dT). The mutant subcomplex was severely compromised in the ability of both UL5 and UL52 to bind the oligonucleotide; total cross-linking was only 2% of wild type levels. These results are consistent with the proposal that the putative zinc binding motif of UL52 is required not only for binding of the UL52 subunit to DNA and for primase activity but also for optimal binding of UL5 to DNA and for the subsequent ATPase and helicase activities.     

A conserved helicase motif of the AddA subunit of the Bacillus subtilis ATP-dependent nuclease (AddAB) is essential for DNA repair and recombination

Various mutations were introduced in a conserved helicase domain (motif VI) of the AddA subunit of the Bacillus subtilis ATP-dependent nuclease (AddAB) by site-directed mutagenesis. These mutations affected the helicase activity and the ATP-dependent exonuclease activity on double-stranded DNA (dsDNA) as the substrate to various degrees, but had hardly any effect on the exonuclease activity on single-stranded DNA (ssDNA), suggesting that exonuclease activity on dsDNA of the enzyme requires unwinding of the DNA. This idea was supported by the finding that, initially, the rate and extent of unwinding of the DNA were higher than those of its degradation to acid-soluble products by the exonucleolytic activity. The effects of the mutations on DNA repair and recombination correlated strongly with their effects on helicase activity. Taken together, these results suggest that motif VI is essential for the helicase activity, and that this activity is required for DNA repair and recombination.