Purification of a 9S DNA polymerase .alpha. species from calf thymus

Grosse, Frank; Krauss, Gerhard

doi:10.1021/bi00522a019

Cited by 74 publications

(43 citation statements)

References 26 publications

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“…Thus, stimulation greater than that we observed would be surprising. However, preparations of conventionally purified polymerase-primase complex [7] with a rathcr low efficiency for singlc-stranded DNA replication were stimulated by RNase H by up to a factor of 10 (F. Grosse, unpublished experiments).…”

Section: Discussionmentioning

confidence: 99%

“…Sucrose gradient centrifugation. Ultracentrifugation was carried out with an isokinetic 6-30% sucrose gradient in 30 mM potassium phosphate, pH 7.8, 10 mM Na2S205, 1 mM EDTA, 7 mM 2-mercaptoethanol, in an SW40 rotor (Beckman) for 40 h at 40000 rpm at 4°C as describcd in [7].…”

Section: Methodsmentioning

confidence: 99%

“…The glands were frozen in liquid nitrogen and kept at -80'C until usage. Preparation of the crude cytosolic extract and chromatography on DEAE cellulose, phosphocellulose, and heparin-Sepharose wcrc exactly as described for the purification of the DNA-polymerase-aprimasc coinplex by utilizing the assay for DNA polymerase ct as the only criterion for collccting active fractions [7].…”

Section: Methodsmentioning

confidence: 99%

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A distinct form of ribonuclease H from calf thymus stimulates its homologous DNA‐polymerase‐α–primase complex

Hagemeier

Grosse

1989

European Journal of Biochemistry

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A ribonuclease H which degrades RNA specifically in K N A-DNA hybrids and, moreover, stimulates its homologous DNA-polymerase-primase complex was purified from calf thymus. The enzyme consists of a single polypeptide of molecular mass 78 kDa. It requires divalent cations for activity, and prefers MgZ+ over Mn2+.Ribonuclease H is optimally active at neutral pH and in 75 mM potassium acetate and is strongly sensitive to N-ethylmaleimide.[3H]Poly(rA) . poly(dT), [3H]poly(rC) . poly(dI), and [3H]KNA . MI 3-DNA are degraded to 3 -9-mer oligoribonucleotides with similar kinetics, whereas double-or single-stranded DNA, and double-and single-stranded RNA remain unaffected. The enzyme stimulates in vitro DNA synthesis by the immunoaffinitypurified calf-thymus DNA-polymerase-a -primase complex threefold. When ribonuclease H is present in a threefold molar excess to the polymerase-primase complex, twice as much primer is formed as in the absence of ribonuclease H. Kibonuclease H also stimulates the elongation rate of DNA polymerase L Y by a factor of 2-3. independent of whether primase-primed DNA templates or templates primed with oligonucleotides are used. Our rcsults suggest that this form of ribonuclease H is a likely candidate for a genuine primer-removing enzyme in maininalian cells.DNA replication is a complex process which requires at least 20 -30 different proteins. Central activities for lagging strand synthesis involve a DNA primase for the formation o f short pieces of RNA, typically 5 -10 nucleotides long. which serve as primers for the replicative DNA polymerase (for a review see 11, 21). In eukaryotic organisnis RNA primer formation and DNA synthesis are performed by a multisubunit complex, named DNA polymerase-or -primase complex (for a review see [3]). The removal of RNA primers is a prerequisite for the subsequent sealing of Okazaki fragments by DNA ligation. Most likely, a ribonuclease specific for the RNA strand of a DNA-RNA hybrid, ribonuclease H (RNase H; EC 3.1.4.34), is responsible for primer removal. Such an enzyme was first detected by Skin and Hausen in calf thymus 141. Since then, the puritication and characterization of several different forms of RNase H from many organisms has been reported (for a review see [S]). However, until now it was not clear which of the known forms of RNase H were involved in the process of DNA replication.Our laboratory is concerned with the identification and isolation of proteins involved in DNA replication in calf thymus cells in order to reconstitute a replication complex in vitro. These studies began with the isolation of the DNA polymerase cc [6, 71 and were continued by the isolation and Corresponderrce to F. Grosse, Abteilung Chemie.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A distinct form of ribonuclease H from calf thymus stimulates its homologous DNA‐polymerase‐α–primase complex

Hagemeier

Grosse

1989

European Journal of Biochemistry

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“…In our recent work we have purified and characterized many replicative proteins from calf thymus glands, including the DNA-polymerase-a -primase complex [13,141, the DNA polymerases 6 and F [I 51, a polymerase-a-stimulating ribonuclease H [16], DNA topoisomerases [17], and two DNA helicases [I 81. Because of the species-specific interaction between SSB and polymerase a and probably other components of the replicative apparatus as well, we attempted to purify SSB from bovine tissue.…”

mentioning

confidence: 99%

Single‐stranded DNA binding protein from calf thymus

Atrazhev¹,

Zhang²,

Grosse³

1992

European Journal of Biochemistry

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A binding protein for single-stranded DNA (ssDNA) was purified from calf thymus to near homogeneity by chromatography on DEAE-cellulose, blue-Sepharose, ssDNA -cellulose and FPLC Mono Q. The most purified fraction consisted of four polypeptides with molecular masses of 70, 55, 30, and 11 kDa. The polypeptide with the molecular mass of 55 kDa is most likely a degraded form of the largest polypeptide. The complex migrated as a whole on both glycerol gradient ultracentrifugation (s = 5.1 S) and gel filtration (Stokes' radius z 5.1 nm). Combining these data indicates a native molecular mass of about 110 kDa, which is in accord with a 1 : 1 : 1 stoichiometry for the 70 + 55/30/ 11 -kDa complex. The ssDNA binding protein (SSB) covered approximately 20 -25 nucleotides on M13mp8 ssDNA, as revealed from both band shift experiments and DNase I digestion studies. The homologous DNA-polymerase-a -primase complex was stimulated by the ssDNA binding protein 1.2-fold on poly(dA) . (dT)14 and 10-13-fold on singly primed M13mp8 DNA. Stimulation was mainly due to facilitated DNA synthesis through stable secondary structures, as demonstrated by the vanishing of many, but not all, pausing sites. Processivity of polymerase-primase was not affected on poly(dA) . (dT)14; with poly(dT) . (rA)lo an approximately twofold increase in product lengths was observed when SSB was present. The increase was attributed to a facilitated rebinding of polymerase GI to an already finished DNA fragment rather than to an enhancement of the intrinsic processivity of the polymerase. Similarly, products 300 -600 nucleotides long were formed on singly primed M13 DNA in the presence of SSB, in contrast to 20-120 nucleotides when SSB was absent. DNA-primase-initiated DNA replication on M13 DNA was inhibited by SSB in a concentrationdependent manner. However, with less sites available to begin with RNA priming, more homogeneous products were formed.In the course of DNA replication, the double-stranded parental DNA has to be opened in order to provide a singlestranded template for the replicative DNA polymerases [l]. Zn vivo these single-stranded DNA (ssDNA) intermediates will be covered by single-strand-specific DNA binding proteins (SSB), mainly in order to prevent rehybridization to the energetically favored duplex structure and to prevent attacks from single-strand-specific nucleases. SSB proteins have been isolated from many different organisms, such as bacteria, bacteriophages, fungi, and viruses (for recent reviews see [2, 31).

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“…Concentration of stock solution of MLV reverse transcriptas¢ (Gibco-BRL, USA) was 100 unit~/~l. DNA polymerase u and Bollam terminal transferase from calf thymus were purified according to modified methods of Grosse et al [6] and budt;m [7]. Specific activity of the enzymes were 5000 units/me and 20,00:) units/me, respectively.…”

Section: Methodsmentioning

confidence: 99%

3′‐Mercapto‐2′,3′‐dideoxynucleotides are high effective terminators of DNA synthesis catalyzed by HIV reverse transcriptase

1992

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Four Y-mcrcapto-2",3'-dideoxynucleoside 5'-triphosphates (A, G, C and T) were tested as DNA chain terminator substmtes for calf thymus cz-DNA polymerase, E. coil DNA polymerase I Klenow fragment, terminal deoxyn ucleotidyl transferase and reverse transeripta~es oEAMV, HIV and MLV viruses. It was sl~own that the analogues selectively and irreversibly terminated DNA chain elon~ttion by AMV and HIV reverse transeriptases and the terminal transferase. Other DNA polymerases tested did not use the nucleotide analogues as chain terminator substrate.

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Purification of a 9S DNA polymerase .alpha. species from calf thymus

Cited by 74 publications

References 26 publications

A distinct form of ribonuclease H from calf thymus stimulates its homologous DNA‐polymerase‐α–primase complex

A distinct form of ribonuclease H from calf thymus stimulates its homologous DNA‐polymerase‐α–primase complex

Single‐stranded DNA binding protein from calf thymus

3′‐Mercapto‐2′,3′‐dideoxynucleotides are high effective terminators of DNA synthesis catalyzed by HIV reverse transcriptase

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