On the Processivity of Polymerases<sup>a</sup>

Hippel, Peter H. von; Fairfield, Frederic R.; Dolejsi, Mary Kay

doi:10.1111/j.1749-6632.1994.tb52803.x

Cited by 85 publications

(84 citation statements)

References 19 publications

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“…Processivity of Pol -Processivity is a measure of how many deoxynucleotides a DNA polymerase incorporates in a single DNA binding event (15,18). To ensure that we were observing deoxynucleotide incorporation resulting from a single DNA binding event, we monitored DNA synthesis in the presence of an excess of nonradiolabeled, sonicated herring sperm DNA as a trap (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For each deoxynucleotide incorporation n, processivity, P n , is the probability that the polymerase will incorporate the next deoxynucleotide rather than dissociating (15) and is given by the following equation (Equation 4). …”

Section: Methodsmentioning

confidence: 99%

“…Analysis of Processivity-The processivity, P n , after each deoxynucleotide incorporation was calculated by a method derived from one previously described (15). Briefly, gel band intensities of the deoxynucleotide incorporation products at the 240 s incubation time were quantitated using the PhosphorImager and ImageQuant software.…”

Section: Methodsmentioning

confidence: 99%

“…the DNA template (15). First, the percentage of active polymerase molecules incorporating at least n deoxynucleotides was calculated using Equation 3 (see "Materials and Methods").…”

Section: Fidelity Of Yeast Pol 36836mentioning

confidence: 99%

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Fidelity and Processivity of Saccharomyces cerevisiae DNA Polymerase η

Washington

Johnson

Prakash

et al. 1999

Journal of Biological Chemistry

169

159

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The yeast RAD30 gene functions in error-free replication of UV-damaged DNA, and RAD30 encodes a DNA polymerase, pol , that has the ability to efficiently and correctly replicate past a cis-syn-thymine-thymine dimer in template DNA. To better understand the role of pol in damage bypass, we examined its fidelity and processivity on nondamaged DNA templates. Steadystate kinetic analyses of deoxynucleotide incorporation indicate that pol has a low fidelity, misincorporating deoxynucleotides with a frequency of about 10 ؊2 to 10 ؊3 . Also pol has a low processivity, incorporating only a few nucleotides before dissociating. We suggest that pol 's low fidelity reflects a flexibility in its active site rendering it more tolerant of DNA damage, while its low processivity limits its activity to reduce errors.Mutations in the RAD30 gene of Saccharomyces cerevisiae confer moderate sensitivity to ultraviolet (UV) radiation, and genetic studies have indicated the involvement of this gene in error-free bypass of UV-damaged DNA (1-3). RAD30 encodes a DNA polymerase, pol , 1 that can efficiently and correctly bypass a cis-syn-thymine-thymine (T-T) dimer in template DNA by inserting two adenines opposite the two thymines of the dimer (4). The Rad30 DNA polymerase activity is essential for resistance to UV radiation and for its role in error-free bypass (3). Recently, the human homologue of pol has been identified, and mutational alterations in this protein are responsible for the variant form of the cancer-prone genetic disorder xeroderma pigmentosum (XP-V) (5, 6).Most DNA polymerases misincorporate deoxynucleotides with very low frequencies, in part because they prefer to insert deoxynucleotides that form correct Watson-Crick base pairs and which do not distort the Watson-Crick geometry (7). Thus, even though the capacity for base pairing recognition is retained in the cyclobutane pyrimidine dimer (8, 9), a T-T dimer is still a block to most DNA polymerases, including the eukaryotic replicative polymerase pol ␦ (4), presumably because of the intolerance of their active site to DNA distortion caused by the dimer (10, 11). In contrast, the ability of pol for efficient and correct bypass of a T-T dimer may derive from an unusual active site that is more tolerant of DNA distortions. In that case, pol would be expected to have a low fidelity (see also the "Discussion"). Here we determine the fidelity of pol by measuring the steady-state kinetics of correct and incorrect deoxynucleotide incorporation and also examine its processivity. We find that pol is a low fidelity polymerase, and it incorporates only a few nucleotides before dissociating from the primer-template DNA substrate. This low processivity may restrict pol synthesis to short patches, thereby minimizing the error frequency, and thus accounting for its role in error-free bypass of UV-damaged DNA. MATERIALS AND METHODSDNA Substrates-The following four (53-mer) oligodeoxynucleotides were used as templates, and they differ only in the underlined sequences: Template G, 5Ј-ATGC...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…the DNA template (15). First, the percentage of active polymerase molecules incorporating at least n deoxynucleotides was calculated using Equation 3 (see "Materials and Methods").…”

Section: Fidelity Of Yeast Pol 36836mentioning

confidence: 99%

See 2 more Smart Citations

Fidelity and Processivity of Saccharomyces cerevisiae DNA Polymerase η

Washington

Johnson

Prakash

et al. 1999

Journal of Biological Chemistry

169

159

View full text Add to dashboard Cite

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“…Processivity has been observed in several enzymes but is most commonly identified with the DNA polymerases (19)(20)(21)(22)(23)(24). Some typical methods designed to establish processivity in this enzyme family rely on trapping free polymerase by using agents such as heparin (25), poly(A)⅐oligo(dT) (26), poly(dI-dC) (27), and activated calf thymus DNA (28).…”

mentioning

confidence: 99%

Processive phosphorylation of alternative splicing factor/splicing factor 2

Aubol

Chakrabarti

Ngo

et al. 2003

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

102

133

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SR proteins, named for their multiple arginine͞serine (RS) dipeptide repeats, are critical components of the spliceosome, influencing both constitutive and alternative splicing of pre-mRNA. SR protein function is regulated through phosphorylation of their RS domains by multiple kinases, including a family of evolutionarily conserved SR protein-specific kinases (SRPKs). The SRPK family of kinases is unique in that they are capable of phosphorylating repetitive RS domains with remarkable specificity and efficiency. Here, we carried out kinetic experiments specially developed to investigate how SRPK1 phosphorylates the model human SR protein, ASF͞SF2. By using the start-trap strategy, we monitored the progress curve for ASF͞SF2 phosphorylation in the absence and presence of an inhibitor peptide directed at the active site of SRPK1. ASF͞SF2 modification is not altered when the inhibitor peptide (trap) is added with ATP (start). However, when the trap is added first and allowed to incubate for a specific delay time, the decrease in phosphate content of the enzyme-substrate complex follows a simple exponential decline corresponding to the release rate of SRPK1. These data demonstrate that SRPK1 phosphorylates a specific region within the RS domain of ASF͞SF2 by using a fully processive catalytic mechanism, in which the splicing factor remains ''locked'' onto SRPK1 during RS domain modification.B oth the assembly of the spliceosome and the specificity of splice-site selection in mammalian cells require SR proteins, which contain one or two RNA-recognition motifs (RRMs) in the N terminus and a signature domain enriched with arginine͞ serine dipeptide repeats (RS) in the C terminus (1, 2). The SR proteins are phosphorylated and activated by the SRPK and Clk͞Sty families of protein kinases (3)(4)(5). This posttranslational modification is required for translocation of the SR protein from the cytoplasm to the nucleus (6, 7) and recruitment of the SR proteins from nuclear speckles (also known as splicing-factor compartments) to nascent transcripts for cotranscriptional splicing (8-10). Phosphorylated SR proteins are believed to facilitate 5Ј splice-site recognition through interaction with the RS domain in U1-70 K (a component of the U1 small nuclear ribonucleoprotein) and 3Ј splice-site selection by means of the RS domains present in the U2AF heterodimer (11-13). Finally, reversal of the kinase action by protein phosphatases appears to be essential for spliceosome activation and the return of splicing factors to nuclear speckles (5). ASF͞SF2 is a prototypical SR protein with two RRM domains for RNA binding and a Cterminal RS domain for mediating protein-protein interactions during spliceosome assembly (see Fig. 1C). A total of 20 serine residues lie in the RS domain and are potential sites for phosphorylation. Mutational studies have verified that phosphorylation occurs exclusively in the RS domain (3,7,14).Although the importance of phosphorylation of SR proteins is well documented for their biological functions in splici...

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