DNA damage generated by oxidant byproducts of cellular metabolism has been proposed as a key factor in cancer and aging. Oxygen free radicals cause predominantly base damage in DNA, and the most frequent mutagenic base lesion is 7,8-dihydro-8-oxoguanine (8-oxoG). This altered base can pair with A as well as C residues, leading to a greatly increased frequency of spontaneous G.C-->T.A transversion mutations in repair-deficient bacterial and yeast cells. Eukaryotic cells use a specific DNA glycosylase, the product of the OGG1 gene, to excise 8-oxoG from DNA. To assess the role of the mammalian enzyme in repair of DNA damage and prevention of carcinogenesis, we have generated homozygous ogg1(-/-) null mice. These animals are viable but accumulate abnormal levels of 8-oxoG in their genomes. Despite this increase in potentially miscoding DNA lesions, OGG1-deficient mice exhibit only a moderately, but significantly, elevated spontaneous mutation rate in nonproliferative tissues, do not develop malignancies, and show no marked pathological changes. Extracts of ogg1 null mouse tissues cannot excise the damaged base, but there is significant slow removal in vivo from proliferating cells. These findings suggest that in the absence of the DNA glycosylase, and in apparent contrast to bacterial and yeast cells, an alternative repair pathway functions to minimize the effects of an increased load of 8-oxoG in the genome and maintain a low endogenous mutation frequency.

Gene-targeted knockout mice have been generated lacking the major uracil-DNA glycosylase, UNG. In contrast to ung- mutants of bacteria and yeast, such mice do not exhibit a greatly increased spontaneous mutation frequency. However, there is only slow removal of uracil from misincorporated dUMP in isolated ung-/- nuclei and an elevated steady-state level of uracil in DNA in dividing ung-/- cells. A backup uracil-excising activity in tissue extracts from ung null mice, with properties indistinguishable from the mammalian SMUG1 DNA glycosylase, may account for the repair of premutagenic U:G mispairs resulting from cytosine deamination in vivo. The nuclear UNG protein has apparently evolved a specialized role in mammalian cells counteracting U:A base pairs formed by use of dUTP during DNA synthesis.

TREX1, originally designated DNase III, was isolated as a major nuclear DNA-specific 335 exonuclease that is widely distributed in both proliferating and nonproliferating mammalian tissues. The cognate cDNA shows homology to the editing subunit of the Escherichia coli replicative DNA polymerase III holoenzyme and encodes an exonuclease which was able to serve a DNA-editing function in vitro, promoting rejoining of a 3 mismatched residue in a reconstituted DNA base excision repair system. Here we report the generation of genetargeted Trex1؊/؊ mice. The null mice are viable and do not show the increase in spontaneous mutation frequency or cancer incidence that would be predicted if Trex1 served an obligatory role of editing mismatched 3 termini generated during DNA repair or DNA replication in vivo. Unexpectedly, Trex1 ؊/؊ mice exhibit a dramatically reduced survival and develop inflammatory myocarditis leading to progressive, often dilated, cardiomyopathy and circulatory failure.Two distinct nuclear exonucleases account for the major part of the total exonucleolytic activity on DNA observed in mammalian cell extracts (24, 25). They were identified as a 3Ј35Ј exonuclease acting preferentially on single-stranded DNA and a 5Ј33Ј exonuclease specific for double-stranded DNA that could remove a single-stranded 5Ј overhang as an oligonucleotide. These nuclear enzymes were designated DNase III and DNase IV, as they are distinct from the pancreatic and macrophage lysosomal DNA endonucleases DNase I and DNase II. DNase IV was later renamed flap endonuclease 1 (FEN1) (23), and its main function is processing displaced 5Ј single strands that arise during lagging-strand DNA replication, as well as during DNA repair, recombination, and triplet repeat expansion. The elimination of Fen1 activity leads to early embryonic lethality in mice, consistent with an essential role of the enzyme in DNA replication (20). In contrast, DNase III is expressed at similar levels in nonproliferating and proliferating tissues; it was isolated as the major nuclear 3Ј35Ј DNA exonuclease from the adult rabbit liver (14) and also from the calf thymus and human myoblasts, where it was designated TREX1 (37, 38).The human TREX1/DNase III cDNA (14, 29) shares amino acid sequence homology with the Escherichia coli DnaQ/MutD editing subunit of the replicative DNA polymerase III holoenzyme, which can increase the fidelity of an exonuclease-deficient mammalian DNA polymerase in vitro (36). The TREX1/ DNase III cDNA encodes a nonprocessive 3Ј35Ј DNA-specific exonuclease, with a preference for single-stranded DNA or mispaired 3Ј termini; like the native protein isolated from mammalian cells, it forms homodimers (14, 29, 30). Since two of the major mammalian nuclear DNA polymerases, Pol␣ and Pol␤, do not have an intrinsic 3Ј exonuclease function, it was proposed that TREX1/DNase III may serve to edit mismatched deoxyribonucleotides during lagging-strand DNA synthesis or gap filling in DNA base excision repair, which are conducted by Pol␣ and Pol␤, respectively...

Human polydeoxyribonucleotide kinase is an enzyme that has the capacity to phosphorylate DNA at 5-hydroxyl termini and dephosphorylate 3-phosphate termini and, therefore, can be considered a putative DNA repair enzyme. The enzyme was purified from HeLa cells. Amino acid sequence was obtained for several tryptic fragments by mass spectrometry. The sequences were matched through the dbEST data base with an incomplete human cDNA clone, which was used as a probe to retrieve the 5-end of the cDNA sequence from a separate cDNA library. The complete cDNA, which codes for a 521-amino acid protein (57.1 kDa), was expressed in Escherichia coli, and the recombinant protein was shown to possess the kinase and phosphatase activities. Comparison with other sequenced proteins identified a P-loop motif, indicative of an ATP-binding domain, and a second motif associated with several different phosphatases. There is reasonable sequence similarity to putative open reading frames in the genomes of Caenorhabditis elegans and Schizosaccharomyces pombe, but similarity to bacteriophage T4 polynucleotide kinase is limited to the kinase and phosphatase domains noted above. Northern hybridization revealed a major transcript of approximately 2.3 kilobases and a minor transcript of approximately 7 kilobases. Pancreas, heart, and kidney appear to have higher levels of mRNA than brain, lung, or liver. Confocal microscopy of human A549 cells indicated that the kinase resides predominantly in the nucleus. The gene encoding the enzyme was mapped to chromosome band 19q13.4.Transient DNA strand breaks and short gaps are frequently observed in cellular DNA. Many arise during regular cellular activity such as DNA replication, recombination, or differentiation. Others occur as a consequence of exposure to endogenous or exogenous DNA damaging agents. Repair of these strand interruptions is usually mediated by DNA ligases and polymerases. Both of these classes of enzymes require 3Ј-hydroxyl DNA termini, and the DNA ligases also require 5Ј-phosphate termini. However, the termini generated by nucleases, such as DNase II, and many produced by ionizing radiation bear 3Ј-phosphate and 5Ј-hydroxyl groups (1-4), and therefore must be processed before they can be acted upon by DNA ligases or polymerases.One enzyme that possesses the capacity to both phosphorylate 5Ј-hydroxyl termini and dephosphorylate 3Ј-phosphate termini is polynucleotide kinase (PNK).1 The PNK from T4 phage has found widespread application in molecular biology, especially for radiolabeling DNA and oligonucleotides (5). It can act on DNA and RNA and even phosphorylate nucleoside 3Ј-monophosphates. However, the main cellular function of the T4 enzyme is not to repair DNA, but rather to counter the action of a phage endoribonuclease that cleaves tRNA (6). Eukaryotic PNKs fall into two categories depending on whether their preferred substrate is DNA or RNA (7). While both can phosphorylate 5Ј-termini, only the former have an associated 3Ј-phosphatase activity (8 -12).Mammalian DNA kinases have ...

Eukaryotic DNA ligases are ATP-dependent DNA strand-joining enzymes that participate in DNA replication, repair, and recombination. Whereas mammalian cells contain several different DNA ligases, encoded by at least three distinct genes, only one DNA ligase has been detected previously in either budding yeast or fission yeast. Here, we describe a newly identified nonessential Saccharomyces cerevisiae gene that encodes a DNA ligase distinct from the CDC9 gene product. This DNA ligase shares significant amino acid sequence homology with human DNA ligase IV; accordingly, we designate the yeast gene LIG4. Recombinant LIG4 protein forms a covalent enzyme-AMP complex and can join a DNA single-strand break in a DNA/RNA hybrid duplex, the preferred substrate in vitro. Disruption of the LIG4 gene causes only marginally increased cellular sensitivity to several DNA damaging agents, and does not further sensitize cdc9 or rad52 mutant cells. In contrast, lig4 mutant cells have a 1000-fold reduced capacity for correct recircularization of linearized plasmids by illegitimate end-joining after transformation. Moreover, homozygous lig4 mutant diploids sporulate less efficiently than isogenic wild-type cells, and show retarded progression through meiotic prophase I. Spore viability is normal, but lig4 mutants appear to produce a higher proportion of tetrads with only three viable spores. The mutant phenotypes are consistent with functions of LIG4 in an illegitimate DNA end-joining pathway and ensuring efficient meiosis.