Improved conditions for activity gel analysis of DNA polymerase catalytic polypeptides

Karawya, Essam; Swack, Judith A.; Wilson, Samuel H.

doi:10.1016/0003-2697(83)90689-9

Cited by 62 publications

(30 citation statements)

References 18 publications

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“…In Situ Gel Analysis of DNA Polymerase Activity-The assay was carried out essentially as described previously (35). The protein samples were electrophoresed in 7-20% gradient SDS-PAGE gels containing 1.5 mg/ml activated calf thymus DNA (Pharmacia Biotech Inc.) as template-primer, followed by in situ renaturation of proteins and incubation of the gel in a DNA polymerase assay mixture.…”

Section: Nucleotides and Proteins-unlabeledmentioning

confidence: 99%

Characterization of an African Swine Fever Virus 20-kDa DNA Polymerase Involved in DNA Repair

Oliveros

Yáñez²,

Salas

et al. 1997

Journal of Biological Chemistry

118

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African swine fever virus (ASFV) encodes a novel DNA polymerase, constituted of only 174 amino acids, belonging to the polymerase (pol) X family of DNA polymerases. Biochemical analyses of the purified enzyme indicate that ASFV pol X is a monomeric DNA-directed DNA polymerase, highly distributive, lacking a proofreading 3-5-exonuclease, and with a poor discrimination against dideoxynucleotides. A multiple alignment of family X DNA polymerases, together with the extrapolation to the crystal structure of mammalian DNA polymerase ␤ (pol ␤), showed the conservation in ASFV pol X of the most critical residues involved in DNA binding, nucleotide binding, and catalysis of the polymerization reaction. Therefore, the 20-kDa ASFV pol X most likely represents the minimal functional version of an evolutionarily conserved pol ␤-type DNA polymerase core, constituted by only the "palm" and "thumb" subdomains. It is worth noting that such an "unfingered" DNA polymerase is able to handle templated DNA polymerization with a considerable high fidelity at the base discrimination level. Base excision repair is considered to be a cellular defense mechanism repairing modified bases in DNA. Interestingly, the fact that ASFV pol X is able to conduct filling of a single nucleotide gap points to a putative role in base excision repair during the ASFV life cycle.Despite the variety of existing DNA polymerases, there are a few basic principles that are common to all these enzymes, irrespective of their role either in DNA replication or in DNA repair. The basic chemistry of each individual reaction always involves a pair of divalent metal ions that are coordinated by carboxylate residues. Such a two-metal ion mechanism, originally proposed by Beese and Steitz (1) and probably extrapolative to all nucleotidyltransferases (2-4), appears to be either evolutionarily conserved or acquired by convergent evolution of nonhomologous proteins. In addition to the general deoxynucleotidyl transfer mechanism, it appears that some structural convergency could apply also for the interaction with DNA, a common substrate; an overall view of the crystal structures available for DNA-dependent polymerases always shows a hand-shaped structure, with "thumb," "palm," and "fingers" subdomains, defining at least one cleft for holding DNA (5-12). Moreover, both DNA replicases and DNA repair enzymes are often multienzymatic proteins, having built-in nucleolytic activities that exist as individual structural modules, separated from the polymerization domain. Thus, the paradigmatic Escherichia coli DNA polymerase I (pol I) 1 has a proofreading 3Ј-5Ј-exonuclease, and a 5Ј-3Ј-exonuclease to remove the RNA from the Okazaki fragments (13). Most DNA replicases are also endowed with a proofreading 3Ј-5Ј-exonuclease domain, having an evolutionarily conserved pol I-type active site (14). Reverse transcriptases have an RNase H activity, required for second strand DNA synthesis (reviewed in Telesnitsky and Goff (15)). Even pol ␤, the smallest of the known DNA polymerases (39 kDa)...

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Section: Nucleotides and Proteins-unlabeledmentioning

confidence: 99%

Characterization of an African Swine Fever Virus 20-kDa DNA Polymerase Involved in DNA Repair

Oliveros

Yáñez²,

Salas

et al. 1997

Journal of Biological Chemistry

118

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“…SDS-DNA activity gel analysis was carried out as described (10,11). Ten milliliters of induced cells was pelleted and resuspended in 0.3 ml of 50 mM Tris HCl (pH 6.8).…”

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confidence: 99%

A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides.

Tabor

Richardson

1995

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

318

178

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Bacteriophage T7 DNA polymerase efficiently incorporates a chain-terminating dideoxynucleotide into DNA, in contrast to the DNA polymerases from Escherichia coli and Thermus aquaticus. The molecular basis for this difference has been determined by constructing active site hybrids of these polymerases. A single hydroxyl group on the polypeptide chain is critical for selectivity. Replacing tyrosine-526 of T7 DNA polymerase with phenylalanine increases discrimination against the four dideoxynucleotides by >2000-fold, while replacing the phenylalanine at the homologous position in E. coli DNA polymerase I (position 762) or T. aquaticus DNA polymerase (position 667) with tyrosine decreases discrimination against the four dideoxynucleotides 250-to 8000-fold. These mutations allow the engineering of new DNA polymerases with enhanced properties for use in DNA sequence analysis.All known DNA polymerases can be placed in one of four families based on sequence homologies (1, 2). Although Escherichia coli DNA polymerase I, Thermus aquaticus DNA polymerase (Taq DNA polymerase), and bacteriophage T7 DNA polymerase are members of the DNA polymerase I (Pol I) family, a number of their properties differ significantly (3).First, T7 DNA polymerase is responsible for the replication of a genome and, as such, interacts with other replication proteins, whereas E. coli DNA polymerase I and Taq DNA polymerase are mainly responsible for repair and recombination. Second, the three polymerases have different exonuclease activities: T7 has only a 3' --5' activity, Taq has only a 5' --3' activity, and E. coli DNA polymerase I has both 3' --5' and 5' --3' activities. The exonuclease activities reside in separate domains at the amino termini and can be inactivated selectively by genetic modification. Third, the thermostability of Taq DNA polymerase distinguishes it from the other two. A fourth difference is their relative abilities to distinguish between a deoxy-and a dideoxyribose in the nucleoside triphosphate. E. coli DNA polymerase I (4-6) and Taq DNA polymerase (7) incorporate deoxyiucleotides at a rate that is several hundred to several thousand times that of dideoxynucleotides, while T7 DNA polymerase incorporates dideoxynucleotides much more efficiently, preferring deoxynucleotides by only a factor of three (5, 6). To determine the molecular basis for this difference we constructed active site hybrids of the DNA polymerases and examined their ability to use ddNTPs relative to dNTPs. Inasmuch as chain termination by dideoxynucleotides is the basis of all commonly used enzymatic methods of DNA sequencing (5, 8), an understanding of this difference has utility for improving the DNA polymerases used for this purpose.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6339METHODS Construction ofHybrid Genes. Hybrid genes were constructed by using synthetic oligonucleotides and...

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“…P-polymerase mRNA level was highest 4 h after exposure of cells to MMS and remained elevated for several hours after No P-polymerase mRNA increase was found; an example of this with UV irradiation is shown in Table 2. Finally, we examined the effect of MNNG and MMS treatment on the level of ,-polymerase protein in the crude cell extract by using activity gel analysis (12,14). During the 12-h period after the addition of MNNG (Fig.…”

mentioning

confidence: 99%

“…For example, MNNG, MMS, and H202 produced single-base damage repaired by excision and inb. sponding to the middle of the l1o gel is shown; enzymatic activity of renatured enzyme is used as the indicator of the number of protein molecules (12). Cells cultured in medium alone (control) or exposed to 30 ,uM MNNG for 4 h and then cultured in medium without alkylating agent (7 and 12 h) were collected, sonicated, and extracted with buffer containing 0.5 M NaCl.…”

mentioning

confidence: 99%

Induction of beta-polymerase mRNA by DNA-damaging agents in Chinese hamster ovary cells.

Fornace¹,

Zmudzka²,

Hollander³

et al. 1989

Mol. Cell. Biol.

151

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Studies of mammalian cells with DNA polymerase inhibitors have suggested that 3-polymerase has a role in DNA repair (1,3,13,18,24), and the in vitro activity of purified ,-polymerase points to an in vivo role in resynthesis of short gaps after damaged residues have been excised (4,9,15,19,21,22). Lesions produced by several DNA-damaging agents are repaired by single-base excision or short-gap excision followed by resynthesis to fill the gap (8). It is unknown whether regulation of P-polymerase gene expression is correlated with DNA repair, although it is clear that levels of 3-polymerase mRNA do not correlate with stages of the cell cycle or with replication of genomic DNA (25a). A rodent cDNA for ,-polymerase was isolated recently (26), and we have used this DNA as a probe to study regulation of P-polymerase mRNA in Chinese hamster ovary (CHO) cells after treatment with a variety of DNA-damaging agents.We found that the level of ,B-polymerase mRNA in exponentially growing CHO cells is elevated three-to fivefold after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), or N-acetoxy-2-acetylaminofluorene (AAAF) (Fig. 1). The results of quantitative RNA dot blot analyses (6, 7) after treatment with high levels of these and other agents are summarized in Table 1. For reference, dot blots also were probed with

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Improved conditions for activity gel analysis of DNA polymerase catalytic polypeptides

Cited by 62 publications

References 18 publications

Characterization of an African Swine Fever Virus 20-kDa DNA Polymerase Involved in DNA Repair

Characterization of an African Swine Fever Virus 20-kDa DNA Polymerase Involved in DNA Repair

A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides.

Induction of beta-polymerase mRNA by DNA-damaging agents in Chinese hamster ovary cells.

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