In vitro conversion of a calf thymus 8S DNA polymerase to a 7.3S species

Holmes, Andrew M.; Hesslewood, Ian P.; Johnston, Irving R.

doi:10.1038/255420a0

Cited by 28 publications

(29 citation statements)

References 5 publications

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“…Since non-denaturing electrophoresis can result in removal of considerable amounts of protein impurity [19], the band at M , 135 000 may represent enzyme protein (compare M , from calculated molar mass). Finally, preincubation o f enzyme D with 2.8 M urea for urea for 30 min did not alter either its sedimentation coefficient or its elution position on DEAEcellulose [13].…”

Section: Physical Propertiesmentioning

confidence: 83%

“…1). Fractions were pooled to avoid inclusion of terminal transferase (step IV) and then chromatographed on DEAE-cellulose as previously [6,13]. Enzyme A1 eluted from the DEAE-cellulose column (50 x 1.6 cm) between 0.055 M and 0.075 M, A2 between 0.075 M and 0.095 M, D between 0.1 M and 0.125 M and C between 0.125 M and 0.160 M potassium phosphate, pH 7.8.…”

Section: Enzyme D Preparationmentioning

confidence: 99%

“…A further point of interest concerns the heterogeneity of DNA polymerase-a reported in several instances, for example in calf thymus [4-71 rat liver and spleen [5], Chinese hamster cells [8], mouse myeloma [9-111 and baby hamster kidney (BHK) cells [12]. When preparations of DNA polymerase-a from calf thymus, freed of DNA polymerase-p and terminal transferase, are fractionated on DEAE-cellulose, several peaks of polymerase-a activity can be detected using activated DNA in assays carried out at pH 7.8 [5,13]. From tissue frozen shortly after removal from the animal these are enzymes A1, A2 and C. A further enzyme B appears occasionally and is probably derived by proteolysis from enzyme C [6] (see also [14]).…”

mentioning

confidence: 99%

“…This activity, designated enzyme D [6,13], has been further purified and some of its properties compared with similarly purified samples of enzymes A1 and C. The relationships of the various DNA polymerases of the a-polymerase fraction is discussed.…”

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confidence: 99%

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Studies on the Purification and Properties of a 6.8‐S DNA Polymerase Activity Found in Calf‐Thymus DNA Polymerase‐α Fraction

Hesslewood

Holmes

Wakeling

et al. 1978

European Journal of Biochemistry

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The heterogeneity of calf thymus DNA polymerase-a has been further investigated. In particular, an enzyme (enzyme D) which exhibits higher activity on poly(dA) . (dT)lo (A : T = 20 : 1) compared with that on activated DNA, has been further purified and its properties compared with two other activities of the DNA polymerase-a fraction (enzymes A1 and C) which do not show a preference for poly (dA) . (dT)lo over activated DNA.As with A1 and C, enzyme D was shown to have many of the characteristic properties of DNA polymerase-a in that it is an acidic protein as judged by its binding to DEAE-cellulose, has a molecular weight of about 140000, does not use a poly(A) . (dT)Io template-initiator complex and is inhibited by N-ethylmaleimide. It exhibits anomalous gel filtration behaviour on Sepharose 6B and it binds relatively weakly to DNA-cellulose compared with DNA polymerase-p. The extreme sensitivity of enzyme D to inhibition by N-ethylmaleimide distinguishes it from A1 and C, as does its elution position from a DEAE-cellulose column. On the other hand enzymes C and D are readily inactivated by heating at 45 "C unlike enzyme Al.The possible interrelationships of the multiple activities of calf thymus DNA polymerase-a are discussed.Several different types of DNA polymerase activity have been described in mammalian cells [1,2]. Of these DNA polymerase-a is of particular interest since its activity is highest in situations where DNA synthesis is occurring [3]. A further point of interest concerns the heterogeneity of DNA polymerase-a reported in several instances, for example in calf thymus [4-71 rat liver and spleen [5], Chinese hamster cells [8], mouse myeloma [9-111 and baby hamster kidney (BHK) cells [12]. When preparations of DNA polymerase-a from calf thymus, freed of DNA polymerase-p and terminal transferase, are fractionated on DEAE-cellulose, several peaks of polymerase-a activity can be detected using activated DNA in assays carried out at pH 7.8 [5,13]. From tissue frozen shortly after removal from the animal these are enzymes A1, A2 and C. A further enzyme B appears occasionally and is probably derived by proteolysis from enzyme C [6] (see also [14]). However, assay of the same DEAEcellulose profile with the synthetic template-initiator complex poly(dA) . (dT)lo at pH 7.0 reveals a further

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Section: Physical Propertiesmentioning

confidence: 83%

Section: Enzyme D Preparationmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Studies on the Purification and Properties of a 6.8‐S DNA Polymerase Activity Found in Calf‐Thymus DNA Polymerase‐α Fraction

Hesslewood

Holmes

Wakeling

et al. 1978

European Journal of Biochemistry

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“…Whether this treatment eliminates a true sub-unit or a pre-existing fragment created by proteolytic cleavage of the A enzymes either during preparation or in vitro is still not clear [.5] . The additional components is evidently fairly basic in character [6].…”

Section: Introductionmentioning

confidence: 91%