Molecular heterogeneity of DNA polymerase α from P815 mouse mastocytoma cells

Bieri-Bonniot, Françoise; Schuerch, Alfred R.

doi:10.1016/0014-5793(78)81092-8

FEBS Letters

1978

DOI: 10.1016/0014-5793(78)81092-8

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Molecular heterogeneity of DNA polymerase α from P815 mouse mastocytoma cells

Françoise Bieri-Bonniot¹,

Alfred R. Schuerch²

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1979

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Cited by 7 publications

(2 citation statements)

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“…As for one of the essential factors in DNA replication, DNA polymerase, DNA polymerase a is suggested to be involved in DNA replication among the three DNA polymerases, a, ß, and 7, found in mammalian cells (Weissbach, 1977). In various eukaryotic cells, the existence of multiple forms of DNA polymerase a, which differ in their chromatographic behavior, has been demonstrated (Yoshida et al, 1974;Hachmann & Lezius, 1975; Craig & Keir, 1975;Matsukage et al, 1976;PedraliNoy & Weissbach, 1977;Nishioka et al, 1977;Bieri-Bonniot & Schuerch, 1978;Hesselewood et al, 1978;Brakel & Blumenthal, 1978;Chen et al, 1979;Lamothe et al, 1981). We have also reported that HeLa cells or their isolated nuclei contain two active forms of DNA polymerase a (Ono et al, 1978(Ono et al, , 1979Tanuma et al, 1980).…”

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Purification and characterization of two forms of DNA polymerase .alpha. from HeLa cell nuclei

Enomoto¹,

Tanuma²,

Yamada³

1983

Biochemistry

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Isolated nuclei contained two active forms of DNA polymerase alpha (form I and form II). Form II was extracted from nuclei by KCl at concentrations lower than 0.18 M. Above 0.18 M selective extraction of form I was observed. The purified two active forms differed in chromatographic and electrophoretic behaviors, in their salt requirement for optimal activity, and in preference of template-primers, although both forms exhibited properties characteristic of DNA polymerase alpha such as sensitivity of N-ethylmaleimide, 1-beta-D-arabino-furanosylcytosine triphosphate, and aphidicolin. Marked difference between the two forms was preference of template-primer that form I was more active with poly(dT).(rA)10 than poly(dA).(dT)12 whereas form II exhibited higher activity with poly(dA).(dT)12 than poly(dT).(rA)10. Possible roles of two forms of DNA polymerase alpha in the processes of DNA replication will be discussed.

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confidence: 99%

Purification and characterization of two forms of DNA polymerase .alpha. from HeLa cell nuclei

Enomoto¹,

Tanuma²,

Yamada³

1983

Biochemistry

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“…The occurrence of multiple forms of DNA polymerase (pol) a (DNA nucleotidyltransferase, EC 2.7.7.7) activity in calf thymus and rat liver (1)(2)(3)(4)(5)(6), mouse myeloma (7-9), mouse mastocytoma (10), and rat ascites hepatoma cells (11) has been reported in recent years. Very little work has been done on characterization of the various forms of DNA pol a from cultured human cells until recently (12)(13)(14).…”

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Differential inhibition of multiple forms of DNA polymerase alpha from IMR-32 human neuroblastoma cells.

Bhattacharya¹,

Simet²,

Basu³

1981

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

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Three forms of DNA polymerase (pol) a from human neuroblastoma IMR-32 were separated by DEAE column chromatography. All sedimented at -7 S in 5-20% continuous sucrose density gradients. All were heat labile, with pol a2 the most (90% inactivated) and pol a3 the least (50% inactivated) sensitive to heating for 5 min at 50TC. pol a, and a2 efficiently utilized activated calf thymus DNA as template. The most active form, pol a2, used both poly(dA)-(dT)j2_j8 and poly(dT)-(dA)12_18 as template at equal rates. Differential inhibition of DNA polymerase at activities was examined in the presence of ricin, hemin, and a nonhistone chromatin protein. All three polymerases were inhibited by both ricin (nonreduced) and hemin,. with pol a2 the most (80-90%) and pol a3 the least (60%) sensitive in each case. In contrast, only pol a2 and a3 activities were inhibited (80-85%) by rat liver nonhistone chromatin protein.The occurrence of multiple forms of DNA polymerase (pol) a (DNA nucleotidyltransferase, EC 2.7.7.7) activity in calf thymus and rat liver (1-6), mouse myeloma (7-9), mouse mastocytoma (10), and rat ascites hepatoma cells (11) has been reported in recent years. Very little work has been done on characterization of the various forms of DNA pol a from cultured human cells until recently (12)(13)(14). Moreover, it is not known whether the different forms of DNA pol a have different reaction rates during replication of eukaryotic genomes. Each of these DNA pol a forms can generate low molecular weight species (6). Whether the occurrence of multiple forms is due to some modification of a common catalytic subunit or to the presence of different primary structures has yet to be established with purified enzymes isolated from normal and tumor cells. Tryptic peptide maps of individual subunits of two forms of highly purified mouse myeloma DNA pol a (9) indicate no structural homology between the two forms. One ofthese forms contains both 3'--5' and 5'--3' exonuclease activities (9). The different template specificities of different forms of DNA pol a in mouse myeloma solid tumors (9, 15) suggest that these forms may play different biological roles in the recognition of base sequences, thereby regulating cell division and differentiation.We have reported the inhibition of RNA-primed (20). A mixture of template and primer (20:1) in 50 mM NaCl/ 5 mM MnCl2 was heated for 10 min at 550C, maintained at room temperature for 1 hr, and stored at -18'C. Subscripts denote chain lengths of polynucleotide primers.Isolation of Multiple Forms of DNA pol a. Human neuroblastoma clone IMR-32 cells were maintained in our laboratory as described (17,21). Confluent monolayers (5-7 x 106 cells per 250-ml Falcon plastic flask) were harvested with 7.0 mM potassium phosphate, pH 7.2/0.14 M NaCV0. 1% EDTA. To isolate and purify DNA pol -x from neuroblastoma, IMR-32 frozen cells were thawed and homogenized in 50 mM Tris'HCl, pH 7.5/1 mM MgClJ6 mM KCV1 mM 2-mercaptoethanol. The homogenate was sonicated for four 15-sec intervals at 40C. The son...

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The role of eucaryotic cell surface glycoconjugates in DNA replication

1983

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Molecular heterogeneity of DNA polymerase α from P815 mouse mastocytoma cells

Cited by 7 publications

References 17 publications

Purification and characterization of two forms of DNA polymerase .alpha. from HeLa cell nuclei

Purification and characterization of two forms of DNA polymerase .alpha. from HeLa cell nuclei

Differential inhibition of multiple forms of DNA polymerase alpha from IMR-32 human neuroblastoma cells.

The role of eucaryotic cell surface glycoconjugates in DNA replication

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