Accessory proteins for DNA polymerase alpha activity with single-strand DNA templates.

Lamothe, Pedro A.; Baril, Betty; Chi, Alice; Lee, Loretta; Baril, Earl F.

doi:10.1073/pnas.78.8.4723

Cited by 86 publications

(67 citation statements)

References 35 publications

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“…The remarkable correlation between nuclear patterns of cyclin (PCNA) antigen distribution and topographical patterns of DNA synthesis argue for a role for this protein in some aspect of DNA replication [20,2 11 [23,29,30], DNA topoisomerases [31,32] and DNA polymerase cy cofactors C1C2 [33,34]. Singlestrand-binding proteins of similar molecular masses have been described [35,36], but their relation to cyclin is unclear.…”

Section: Discussionmentioning

confidence: 99%

S‐phase patterns of cyclin (PCNA) antigen staining resemble topographical patterns of DNA synthesis

1985

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The sequence of cyclin (proliferating cell nuclear antigen, PCNA), antigen staining throughout the cell cycle of African green monkey kidney cells (BS‐C‐1) has been determined by indirect immunofluorescence using PCNA autoantibodies specific for this protein. Patterns of cyclin staining observed between the beginning of S‐phase and maximum DNA synthesis are similar to those reported in human AMA cells [(1985) Proc. Natl. Acad. Sci. USA 82, 3262‐3266], while those detected thereafter are significantly different; the most striking feature being the continuous staining of the nucleoli up to or very near the S/G2 border of the cell cycle. Using [3H]thymidine autoradiography and indirect immunofluorescence of the same cells we show a remarkable correlation between cyclin antigen distribution and topographical patterns of DNA synthesis. In addition, we present evidence showing that DNase I treatment of Triton‐extracted monolayers abolishes cyclin antigen staining but does not result in a substantial release of this protein. Taken together the above observations argue for a role of cyclin in some aspect of DNA replication.

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Section: Discussionmentioning

confidence: 99%

S‐phase patterns of cyclin (PCNA) antigen staining resemble topographical patterns of DNA synthesis

1985

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“…500 [7,281 230 (3) cf. 220 [6] 800 (4) cf. 640 [6] + + Since results were then negative, we conclude that all DNA polymerases were contained in the separation gel under standard conditions.…”

Section: Cenerulmentioning

confidence: 99%

“…220 [6] 800 (4) cf. 640 [6] + + Since results were then negative, we conclude that all DNA polymerases were contained in the separation gel under standard conditions. In most samples DNA polymerases migrated in positions corresponding to molecular masses between 200 kDa and 1300 kDa (Figs 1-6 and Table 1).…”

Section: Cenerulmentioning

confidence: 99%

Non‐disruptive detection of DNA polymerases in nondenaturing polyacrylamide gels

Holler

Fischer

Simek

1985

European Journal of Biochemistry

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A non-disruptive method is described with which DNA polymerases can be detected in homogeneous preparations and unfractionated cell extracts after electrophoresis in nondenaturing polyacrylamide gradient gels. The technique involves diffusion of DNA polymerase activity into an overlay assay agarose gel, the synthesis of radioactive DNA, removal of excess substrates and autoradiography. Cell extracts from a variety of organisms were studied using this method. The activity from Escherichia coli crude extracts migrated in a postition corresponding to a higher molecular mass than did purified preparations of DNA polymerase I. DNA polymerases of higher organisms generally migrated in positions corresponding to 400 -900 kDa, in some cases, close to 200 kDA.Recent analysis of purified samples has shown that replicating DNA polymerases (type CI in eucaryotes and type 111 in Escherichia coli) are in fact multiprotein complexes [l -1 I]. Because such complexes are delicate, they frequently do not survive purification, and their occurrence is often not detected. A non-disruptive method is needed that allows detection of such complexes in cell extracts and during purification. A previous approach along these lines was an electrophoretic assay in nondenaturing polyacrylamide gels containing activated calf thymus DNA [12].DNA polymerase activity has also been assayed after electrophoresis in denaturing SDS/polyacrylamide gels [13] and molecular masses of single polypeptides in crude preparations could be estimated. Although innovative, this method cannot be used for detection of complexes.We present here a technique which allows non-disruptive detection of DNA polymerase activity in polyacrylamide gels. Separation is achieved according to molecular masses of proteins by electrophoresis in polyacrylamide gradient gels under nondenaturing conditions according to established techniques [14-171. The method was applied to unfractionated extracts from bacteria, fungi, plant and mammalian cells. EXPERIMENTAL PROCEDURE MaterialsCells of Escherichia coli MRE 600 were purchased from Merck (Darmstadt). E. coli superproducing strain W3110 carrying the temperature-inducible prophage NM879 polACorrespondence to

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“…A multiprotein complex with molecular weight of 640 kDa has been isolated from the HeLa cells. This complex contains DNA polymerase 0~, DNA primase, 3'-5' exonuclease, Ap,~A binding protein and primer recognition accessory proteins C2 and C3 [5,6]. It is very likely that specific protein-protein interactions of components in this multiprotein complex determine the specificity and efficiency of DNA replication.…”

Section: Introductionmentioning

confidence: 99%

Photoaffinity labeling of DNA polymerase α DNA primase complex based on the catalytic competence of a dNTP reactive analog

1992

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FABdCTP was found to be a sabstrate of DNA polymerization catalyzed b), a DNA p01ymerafie ~-DNA primase complex on the 5"-GTGAG. TAAGTGGAGqTIGGCACGAT-3' template and 3'-CTCAAACCGT.5' primer. After compleie primer extension in the presence of FABdCTP under UV-irradiation of the reaction mixture, 70% of the template was cowileatly linked to the primer. Labeling of the 165 kDa subunit of the DNA polymcrase ~, 59 kDa and 49 kDa subunits of the DNA primase and an unknown protein with apparent molecular weight of 31 kDa was observed. By another way of protein labeling FABdCTP wa~ ¢ovalently bound to the subunit.~ of the enzyme under UV irradiation attd th~n this moiety wa~ introduced into the 3'-end of the 5'-[~-'P]primei by the catalytic activity of DNA i~olymerase or DNA primase. In this ~se covalent labeling of the 165 kDa, 49 kDa arid 31 kDa ~t~bunits war observed DNA pol),merase a: Pkotoaffinity modification

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Accessory proteins for DNA polymerase alpha activity with single-strand DNA templates.

Cited by 86 publications

References 35 publications

S‐phase patterns of cyclin (PCNA) antigen staining resemble topographical patterns of DNA synthesis

S‐phase patterns of cyclin (PCNA) antigen staining resemble topographical patterns of DNA synthesis

Non‐disruptive detection of DNA polymerases in nondenaturing polyacrylamide gels

Photoaffinity labeling of DNA polymerase α DNA primase complex based on the catalytic competence of a dNTP reactive analog

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