Identification of DNA polymerase .delta. in CV-1 cells: studies implicating both DNA polymerase .delta. and DNA polymerase .alpha. in DNA replication

Hammond, Russell A.; Byrnes, John; Miller, Michael R.

doi:10.1021/bi00395a035

Cited by 72 publications

(38 citation statements)

References 44 publications

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“…The data presented here rather indicate that both of the DNA polymerases are involved in DNA replication in living cells. Dresler and Frattini (20) and Hammond et al (25) also reached a similar conclusion from their studies on the sensitivity of chromosomal replication to BuPdGTP. Moredirect evidence on the involvement of DNA polymerase 8 must await the isolation of DNA polymerase 8 mutants or the introduction of anti-DNA polymerase 8 antibodies into living cells.…”

Section: Resultssupporting

confidence: 60%

Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

Zuber¹,

Tan²,

Ryoji³

1989

Mol. Cell. Biol.

107

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Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase 8 but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase 8 is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase a. Anti-DNA polymerase a alone inhibited plasmid replication by 63%. Thus, DNA polymerase a is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase a antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase 8. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase a, that the structure of DNA polymerase a holoenzyme for chromosome replication is significantly different from that for plasmid replication.Proliferating cell nuclear antigen (PCNA) was initially recognized as a nuclear autoantigen which reacts with autoimmune sera from a certain population of patients with systemic lupus erythematosus (47; for recent reviews, see references 60 and 61). This antigen is an acidic nuclear protein with a molecular weight of about 36,000 (13,42,51,58). It is readily extractable from whole-cell or nuclear preparations and is expressed mainly in mitotically active cells of various tissues and cell lines (58). Independently, Celis and his colleagues identified, by two-dimensional gel electrophoresis, a protein called cyclin which is abundant in proliferating cells (for reviews, see references 13 and 16). It was later shown that cyclin and PCNA are identical (42). The complete amino acid sequence of this protein was determined by cDNA cloning in two independent laboratories (1,43). Immunofluorescence studies with human autoantibodies against PCNA have demonstrated that PCNA expression increases during late Gl and early S phase, immediately preceding the onset of DNA synthesis (14,36,48,55,58). During S phase, the distribution of PCNA changes dramatically within the nucleus (5,7,14,36,41,55,58,61), following closely the sites of chromosomes which are pulselabeled with [3H]thymidine or bromodeoxyuridine (8, 41).These observations strongly suggest an association of PCNA with the DNA replication apparatus. It has also been proposed that PCNA might be involved in DNA repair synthesis, because UV damage of DNA caused an increase in the nuclear PCNA staining of non-S-phas...

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Section: Resultssupporting

confidence: 60%

Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

Zuber¹,

Tan²,

Ryoji³

1989

Mol. Cell. Biol.

107

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“…As shown in lane 5, the repair polymerase can incorporate ddCTP into the nick, which can be efficiently utilized by polymerases (3, 'y, and, to a lesser extent, 8 (15), but produces an unligatable nick that appears at the position of the HincII marker. The ddCTP incorporation, as well as the repair reaction can be inhibited with a rabbit anti-rat DNA polymerase (3 antiserum (lanes 4 and 6), btit not with the preimmune serum (data not shown), while the repair reaction remained unaffected by the addition of 25 ,uM aphidicolin, a specific polymerase a (and polymerase 8) inhibitor (16) (10), show that in nuclear extracts from HeLa cells the initial step in the correction of G-T mispairs arising through the deamination of 5-methylcytosine is the removal of the mispaired thymine by a DNA glycosylase to generate an AP site opposite the guanine. Like all other glycosylases characterized to date, our enzyme has no requirement for Mg2" or ATP.…”

Section: Resultsmentioning

confidence: 92%

Mismatch-specific thymine DNA glycosylase and DNA polymerase beta mediate the correction of G.T mispairs in nuclear extracts from human cells.

Wiebauer

Jiricny

1990

Proc. Natl. Acad. Sci. U.S.A.

245

149

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To avoid the mutagenic effect of spontaneous hydrolytic deamination of 5-methylcytosine, G-T inspirs, arising in DNA as a result of this process, should always be corrected to G-C pairs. We describe here the identification of a DNA glycosylase activity present in nuclear extracts from HeLa cells, which removes the mispaired thymine to generate an apyrimidinic (AP) site opposite the guanine. We further show, using a specific antibody and inhibitors, that the single nucleotide gap, created upon processing of the AP site, is filled in by DNA polymerase (3. This rmding substantiates the proposed role of this enzyme in short-patch DNA repair.Efficient correction of base-base mismatches, arising as errors of DNA polymerase or through recombination, requires that the cellular machinery be able to direct the repair to the strand carrying the original information. For this purpose, secondary signals such as adenine methylation or strand nicks are used (for a comprehensive review, see ref. 1). The GOT mispair is the only one that can arise also by an alternative pathway in "resting" (i.e., nonreplicating, nonrecombining) DNA-through the spontaneous hydrolytic deamination of 5-methylcytosine. In the correction of this latter type of G-T mismatch, no strand discrimination is required, as it should always be corrected to a G-C. Indeed, repair pathways thought to be dedicated to the correction of the deamination-associated GOT mispairs have been identified in Escherichia coli (2-4) as well as in mammalian cells (5, 6).In all organisms studied to date, correction of deamination damage is mediated by base-specific glycosylases, which remove the deaminated base from the sugar-phosphate backbone by cleaving the glycosylic bond. Uracdil DNA glycosylase and hypoxanthine DNA glycosylase remove the products of cytosine and adenine deamination, uracil (U) and hypoxanthine (H), respectively (for review, see refs. 7 and 8). Both enzymes are highly substrate specific and can remove their respective substrates from matched (A-U and C-H) as well as mismatched (G-U and T.H) duplexes. As neither U nor H is a natural DNA base, their excision can be mediated by the base-specific enzymes from both double-and singlestranded DNA.In our previous studies, we reported that G-T mispairs, incorporated in the simian virus 40 genome and transfected into monkey CV-1 (5, 6) or human (9) cells, were corrected with high efficiency and mostly to G-C pairs. Our in vitro experiments (10) demonstrated that in nuclear extracts from human (HeLa) cells, the mispaired thymidine was excised from DNA to generate a single nucleotide gap. Preliminary evidence also suggested that the initial step of this repair process was mediated by a DNA glycosylase. This was an unexpected finding. Thymine is a natural DNA base and any enzyme responsible for its removal from G-T mispairs would have to be, unlike the uracil and hypoxanthine glycosylases, fully inactive on single-stranded and matched doublestranded substrates. It therefore seemed likely that a thymine DNA glyco...

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“…59 The DNA polymerase alpha assay was performed according to Hammond et al, 60 with the following modifications. 20 l cell extract was incubated with 4 mg protein A-sepharose (Pharmacia, Freiburg, Germany), and 25 l of purified MAB SJK 237-71 (0.1 mg/ml) in phosphate-buffered saline (PBS) for 2 h at 4°C.…”

Section: Dna Polymerase ␣ Assaymentioning

confidence: 99%

Cytogenetic subgroups in acute myeloid leukemia differ in proliferative activity and response to GM-CSF

et al. 2001

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The current study was undertaken to search for differences in the biology of cytogenetic subgroups in patients with de novo acute myeloid leukemia (AML). In addition, factors influencing the metabolism of cytosine arabinoside (araC) as the key agent of antileukemic activity were assessed. Bone marrow aspirates from 91 patients with newly diagnosed AML in whom karyotypes were successfully obtained were analyzed: (1) for spontaneous proliferative activity by 3 H-thymidine ( 3 H-TdR) incorporation; (2) proliferative response to GM-CSF by in vitro incubation of blasts for 48 h with or without GM-CSF (100 U/ml) followed by an additional 4-h exposure to 3 H-TdR (0.5 Ci/ml); and (

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Identification of DNA polymerase .delta. in CV-1 cells: studies implicating both DNA polymerase .delta. and DNA polymerase .alpha. in DNA replication

Cited by 72 publications

References 44 publications

Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

Mismatch-specific thymine DNA glycosylase and DNA polymerase beta mediate the correction of G.T mispairs in nuclear extracts from human cells.

Cytogenetic subgroups in acute myeloid leukemia differ in proliferative activity and response to GM-CSF

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