Functional roles of DNA polymerases beta and gamma.

Abstract

The physiological functions of DNA polymerases (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) (3 and y were investigated by using neuronal nuclei and synaptosomes isolated from rat brain. UV irradiation of neuronal nuclei from 60-day-old rats resulted in a 7-to 10-fold stimulation of DNA repair synthesis attributable to DNA polymerase # which, at this developmental stage, is virtually the only DNA polymerase present in the nuclei. No repair synthesis could be elicited by treating the nuclei with N-methyl-N-nitrosourea, but this was probably due to the inability of brain tissue to excise alkylated bases from DNA. The role of DNA polymerase y was studied in synaptosomes by using a system mimicking in vivo mitochondrial DNA synthesis. By showing that, under these conditions, DNA The direct assignment of functions to the three mammalian DNA polymerases (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) (a, (, and y) in DNA replication and repair is hampered by the lack of conditional mutants defective in DNA synthesis. To date, one is forced to admit that the evidence presented is at best circumstantial. Despite these reservations, some progress has been made concerning the function of DNA polymerase a. A wealth of information indicates that this enzyme has a major role in DNA replication (reviewed in refs. 1 and 2). Recently, this idea has been further strengthened by the finding that DNA polymerase (v is the major polymerase associated with replicating simian virus 40 chromosomes (3-5). In addition, all aspects of in vitro replicative synthesis by these chromosomes (3) or HeLa cell lysates (6) were found to be resistant to 2',3'-dideoxythymidine 5'-triphosphate (d2TTP), a specific inhibitor of DNA polymerases ( and By but not of DNA polymerase a (3, 6). Conversely, aphidicolin, a selective inhibitor of DNA polymerase at, prevents mitotic division of sea urchin embryos, which requires replicative DNA synthesis, but it has no effect on meiotic division in starfish oocytes, which is not dependent on DNA replication (7). Taken together these observations strongly indicate that DNA polymerase ae is the nuclear replicating enzyme.In contrast, the physiological functions of DNA polymerases (14) and synaptosomea (15) were prepared from the forebrain cortex of 60-day-old and 14-day-old rats (SIV-50 strain), respectively. The preparations were frozen immediately at -70'C until use. Nuclei were counted by using a flow cytophotometer (Phywe, Gottingen, Germany), and protein concentrations were determined by the method of Lowry et al. (16). Repair DNA synthesis in neuronal nuclei and synaptosomes was assayed as described in Table 1.In Vitro System Supporting mtDNA Synthesis in Synaptosomes. Synaptosomes were suspended in 0.32 M sucrose/1 mM KH2PO4, pH 7.5/0.1 mM EDTA and were made permeAbbreviations: BrdUTP, 5-bromo-2'-deoxyuridine 5'-triphosphate; d2TTP, 2',3'-dideoxythymidine 5'-triphosphate; MNU,KN-methyl-N-nitrosourea. t Present address:

DNA polymerase beta null mouse embryonic fibroblasts harbor a homozygous null mutation in DNA polymerase iota

“…Of the four eukaryotic DNA polymerases identified by 1977, pol β soon earned the moniker of 'the' DNA repair polymerase [8][9][10][11]. These early studies defined a role for pol β in repair using isolated nuclei or nuclear extracts, monitoring the incorporation of radioactive nucleosides following ultraviolet (UV) light-induced DNA damage [8][9][10][11]. A more definitive role for pol β in excision repair in vitro was demonstrated in 1983, in which complete removal of pyrimidine dimers in DNA was conducted by purified enzyme preparations of pol β, T4 denV (UV endonuclease) and HeLa AP endonuclease II [12].…”

“…Since its initial discovery in 1971 [1], the enzyme that was to eventually be designated pol β [2,3] was unique in its enzymatic properties [4][5][6] as compared to the other newly characterized mammalian DNA polymerases alpha (pol α), gamma (pol γ) and delta (polδ) [1,7]. Of the four eukaryotic DNA polymerases identified by 1977, pol β soon earned the moniker of 'the' DNA repair polymerase [8][9][10][11]. These early studies defined a role for pol β in repair using isolated nuclei or nuclear extracts, monitoring the incorporation of radioactive nucleosides following ultraviolet (UV) light-induced DNA damage [8][9][10][11].…”

DNA repair synthesis in plant protoplasts is aphidicolin‐resistant

“…This synthesis is inhibited by aphidicolin, similarly to that shown in cultured plant cells [17] and may therefore be attributed to the activity of the replicative a-like DNA polymerase. However, a 1-2% residual incorporation of the radioactive DNA precursor, observed even in the presence of saturating doses of the drug, hinders the attempts to show a UV-induced [3H]thymidine incorporation into nuclear DNA since the rate of DNA repair synthesis in higher organisms is expected to be 1>3 orders of magnitude lower than that found in nuclear DNA replication [5]. A similar problem with animal cells was approached by exploiting the unique property of brain neurons which, in adult rats, do not carry out nuclear DNA replication, thus allowing a study of the UV-induced DNA repair synthesis [3,5 ].…”

“…Animal ceils contain 3 distinct DNA polymerases, which, on the basis of studies with isolated •rgane••es and with specific inhibitors [1][2][3][4][5][6][7][8][9] have been assigned the following main, but possibly not exclusive, functions: ct-polymerase is involved in nuclear DNA replication;/3-polymerase is involved in nuclear DNA repair synthesis; and 7-polymerase is involved in mitochondrial DNA replication.…”

Mouse models of DNA polymerases

“…Single base oxidative and methyl damage is repaired by the base excision repair (BER) pathway. The main polymerases operating during BER are Pol ß, Pol g, and Pol k [Hubscher et al, 1979;Braithwaite et al, 2010]. Adducts that distort the DNA helix are removed by the nucleotide excision repair (NER) pathway.…”