A DNA Polymerase with Specificity for Five Base Pairs

2001 ◽  
Vol 123 (8) ◽  
pp. 1776-1777 ◽  
Author(s):  
Alexander K. Showalter ◽  
Ming-Daw Tsai
Keyword(s):  
Dna Polymerase ◽  
Base Pairs

scholarly journals The fidelity of replication of the three-base-pair set adenine/thymine, hypoxanthine/cytosine and 6-thiopurine/5-methyl-2-pyrimidinone with T7 DNA polymerase

2004 ◽  
Vol 381 (3) ◽  
pp. 709-717 ◽  
Author(s):  
Harry P. RAPPAPORT
Keyword(s):  
Dna Polymerase ◽  
Rate Constant ◽  
Base Pair ◽  
Apparent Rate Constant ◽  
Polymerase Activity ◽  
Exonuclease Activity ◽  
Base Pairs ◽  
Error Frequency ◽  
Steady State Kinetics ◽  
Adenine Thymine

With the goal of constructing a genetic alphabet consisting of a set of three base pairs, the fidelity of replication of the three base pairs TH (5-methyl-2-pyrimidinone)/HS (6-thiopurine; thiohypoxanthine), C/H (hypoxanthine) and T/A was evaluated using T7 DNA polymerase, a polymerase with a strong 3′→5′ exonuclease activity. An evaluation of the suitability of a new base pair for replication should include both the contribution of the fidelity of a polymerase activity and the contribution of proofreading by a 3′→5′ exonuclease activity. Using a steady-state kinetics method that included the contribution of the 3′→5′ exonuclease activity, the fidelity of replication was determined. The method determined the ratio of the apparent rate constant for the addition of a deoxynucleotide to the primer across from a template base by the polymerase activity and the rate constant for removal of the added deoxynucleotide from the primer by the 3′→5′ exonuclease activity. This ratio was designated the eni (efficiency of net incorporation). The eni of the base pair C/H was equal to or greater than the eni of T/A. The eni of the base pair TH/HS was 0.1 times that of A/T for TH in the template and 0.01 times that of A/T for HS in the template. The ratio of the eni of a mismatched deoxynucleotide to the eni of a matched deoxynucleotide was a measure of the error frequency. The error frequencies were as follows: thymine or TH opposite a template hypoxanthine, 2×10−6; HS opposite a template cytosine, <3×10−4. The remaining 24 mismatched combinations of bases gave no detectable net incorporation. Two mismatches, hypoxanthine opposite a template thymine or a template TH, showed trace incorporation in the presence of a standard dNTP complementary to the next template base. T7 DNA polymerase extended the primer beyond each of the matched base pairs of the set. The level of fidelity of replication of the three base pairs with T7 DNA polymerase suggests that they are adequate for a three-base-pair alphabet for DNA replication.

scholarly journals Escherichia coli DNA Polymerase III Can Replicate Efficiently past a T-T cis-syn Cyclobutane Dimer if DNA Polymerase V and the 3′ to 5′ Exonuclease Proofreading Function Encoded by dnaQ Are Inactivated

2002 ◽  
Vol 184 (10) ◽  
pp. 2674-2681 ◽  
Author(s):  
Angela Borden ◽  
Paul I. O'Grady ◽  
Dominique Vandewiele ◽  
Antonio R. Fernández de Henestrosa ◽  
Christopher W. Lawrence ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Wild Type ◽  
Lesion Bypass ◽  
Base Pairs ◽  
Pol V ◽  
Pol I ◽  
Cyclobutane Dimer ◽  
Dna Polymerase V ◽  
Pol Iii

ABSTRACT Although very little replication past a T-T cis-syn cyclobutane dimer normally takes place in Escherichia coli in the absence of DNA polymerase V (Pol V), we previously observed as much as half of the wild-type bypass frequency in Pol V-deficient (ΔumuDC) strains if the 3′ to 5′ exonuclease proofreading activity of the Pol III ε subunit was also disabled by mutD5. This observation might be explained in at least two ways. In the absence of Pol V, wild-type Pol III might bind preferentially to the blocked primer terminus but be incapable of bypass, whereas the proofreading-deficient enzyme might dissociate more readily, providing access to bypass polymerases. Alternatively, even though wild-type Pol III is generally regarded as being incapable of lesion bypass, proofreading-impaired Pol III might itself perform this function. We have investigated this issue by examining dimer bypass frequencies in ΔumuDC mutD5 strains that were also deficient for Pol I, Pol II, and Pol IV, both singly and in all combinations. Dimer bypass frequencies were not decreased in any of these strains and indeed in some were increased to levels approaching those found in strains containing Pol V. Efficient dimer bypass was, however, entirely dependent on the proofreading deficiency imparted by mutD5, indicating the surprising conclusion that bypass was probably performed by the mutD5 Pol III enzyme itself. This mutant polymerase does not replicate past the much more distorted T-T (6-4) photoadduct, however, suggesting that it may only replicate past lesions, like the T-T dimer, that form base pairs normally.

Enzymatic synthesis of ligand-bearing DNAs for metal-mediated base pairing utilising a template-independent polymerase

2016 ◽  
Vol 52 (19) ◽  
pp. 3762-3765 ◽  
Author(s):  
Teruki Kobayashi ◽  
Yusuke Takezawa ◽  
Akira Sakamoto ◽  
Mitsuhiko Shionoya
Keyword(s):  
Dna Polymerase ◽  
Enzymatic Synthesis ◽  
Base Pairing ◽  
Base Pairs ◽  
Dna Oligomers ◽  
Artificial Dna

Ligand-bearing artificial DNA oligomers that form metal-mediated base pairs were enzymatically synthesised by utilising a template-independent DNA polymerase.

scholarly journals DNA polymerase β: effects of gapped DNA substrates on dNTP specificity, fidelity, processivity and conformational changes1

1998 ◽  
Vol 331 (1) ◽  
pp. 79-87 ◽  
Author(s):  
Jinwoo AHN ◽  
Vadim S. KRAYNOV ◽  
Xuejun ZHONG ◽  
Brian G. WERNEBURG ◽  
Ming-Daw TSAI
Keyword(s):  
Conformational Change ◽  
Dna Polymerase ◽  
Catalytic Efficiency ◽  
Duplex Dna ◽  
Gap Filling ◽  
Dna Polymerase Β ◽  
Base Pairs ◽  
Single Nucleotide ◽  
Dna Substrates ◽  
Gapped Dna

Pre-steady-state kinetic analysis was used to compare the catalytic properties of DNA polymerase β (Pol β) for single-base gap-filling and regular duplex DNA synthesis. The rate of polymerization (kpol) and the apparent equilibrium dissociation constant of dNTP (Kd) were determined with single-nucleotide gapped DNA substrates for all four possible correct base pairs and twelve possible incorrect base pairs, and the results were compared with those obtained previously with non-gapped primer/template duplex DNA substrates. For correct dNTP incorporation, the use of single-nucleotide gapped DNA led to significant decreases in the Kd of dNTP. Although kpol was little affected, the catalytic efficiency kpol/Kd increased significantly owing to the decreases in Kd. In contrast, for incorrect dNTP incorporation, the use of single-nucleotide gapped DNA substrates did not affect the Kd of dNTP appreciably but caused the kpol (and thus kpol/Kd) for incorrect dNTP incorporation to increase. As a consequence the fidelity of Pol β was not significantly affected by the use of single-nucleotide gapped DNA substrates. In addition we show that under processive polymerization conditions the processivity of Pol β increases in the gap-filling synthesis owing to a decreased rate of DNA dissociation. Finally, with a single-nucleotide gapped DNA substrate the rate-limiting conformational change step before chemistry was also observed. However, the preceding fast conformational change observed with duplex DNA substrates was not clearly detected. A possible cause is that in the complex with the gapped DNA, the 8 kDa N-terminal domain of Pol β already exists in a closed conformation. This interpretation was supported by tryptic digestion experiments.

scholarly journals Hemoglobin Birmingham and hemoglobin Galicia: two unstable beta chain variants characterized by small deletions and insertions

Blood ◽  
1990 ◽  
Vol 75 (9) ◽  
pp. 1883-1887 ◽  
Author(s):  
JB Wilson ◽  
BB Webber ◽  
H Hu ◽  
A Kutlar ◽  
F Kutlar ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Dna Polymerase ◽  
Beta Chain ◽  
Base Pairs ◽  
Spanish Patient ◽  
Local Distortion ◽  
Leucine Residue ◽  
Black Man ◽  
Unstable Hemoglobins ◽  
Dna Helix

Abstract Two unstable hemoglobins (Hbs) causing rather severe hemolytic anemia have been characterized. The beta chain of Hb Birmingham, found in an adult black man, is characterized by the loss of -Leu-Ala-His-Lys- at positions 141, 142, 143, and 144 and their replacement by one Gln residue. These changes are the result of a deletion of nine nucleotides, namely two base pairs (bp) of codon 141, all of codons 142 and 143, and one bp of codon 144; the remaining CAG triplet (C from codon 141 and AG from codon 144) codes for the inserted glutamine. In the beta chain of Hb Galicia from a Spanish patient, His and Val at positions 97 and 98 are replaced by one Leu residue. This is due to an ACG deletion in codons 97 and 98, which causes the removal of one His and one Val residue, while the remaining CTG triplet (C from codon 97 and TG from codon 98) codes for the inserted leucine residue. Two mechanisms, namely slipped mispairing in the presence of short repeats, and misreading by DNA polymerase due to a local distortion of the DNA helix, are considered in explaining the origin of the small deletions.

scholarly journals Mammalian DNA Polymerase Kappa Activity and Specificity

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2805 ◽  
Author(s):  
Hannah R. Stern ◽  
Jana Sefcikova ◽  
Victoria E. Chaparro ◽  
Penny J. Beuning
Keyword(s):  
Dna Polymerase ◽  
Genome Instability ◽  
Fragile Sites ◽  
Chemotherapy Response ◽  
Nucleotide Polymorphisms ◽  
Lesion Bypass ◽  
Base Pairs ◽  
Domains Of Life ◽  
Copy Dna ◽  
Y Family

DNA polymerase (pol) kappa is a Y-family translesion DNA polymerase conserved throughout all domains of life. Pol kappa is special6 ized for the ability to copy DNA containing minor groove DNA adducts, especially N2-dG adducts, as well as to extend primer termini containing DNA damage or mismatched base pairs. Pol kappa generally cannot copy DNA containing major groove modifications or UV-induced photoproducts. Pol kappa can also copy structured or non-B-form DNA, such as microsatellite DNA, common fragile sites, and DNA containing G quadruplexes. Thus, pol kappa has roles both in maintaining and compromising genomic integrity. The expression of pol kappa is altered in several different cancer types, which can lead to genome instability. In addition, many cancer-associated single-nucleotide polymorphisms have been reported in the POLK gene, some of which are associated with poor survival and altered chemotherapy response. Because of this, identifying inhibitors of pol kappa is an active area of research. This review will address these activities of pol kappa, with a focus on lesion bypass and cellular mutagenesis.

scholarly journals Preferential Incorporation of G Opposite Template T by the Low-Fidelity Human DNA Polymerase ι

2000 ◽  
Vol 20 (19) ◽  
pp. 7099-7108 ◽  
Author(s):  
Yanbin Zhang ◽  
Fenghua Yuan ◽  
Xiaohua Wu ◽  
Zhigang Wang
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Polymerase Activity ◽  
Abasic Site ◽  
Base Pairing ◽  
Base Pairs ◽  
Dna Lesion ◽  
Biological Source ◽  
Human Dna ◽  
Base Selection

ABSTRACT DNA polymerase activity is essential for replication, recombination, repair, and mutagenesis. All DNA polymerases studied so far from any biological source synthesize DNA by the Watson-Crick base-pairing rule, incorporating A, G, C, and T opposite the templates T, C, G, and A, respectively. Non-Watson-Crick base pairs would lead to mutations. In this report, we describe the ninth human DNA polymerase, Polι, encoded by the RAD30B gene. We show that human Polι violates the Watson-Crick base-pairing rule opposite template T. During base selection, human Polι preferred T-G base pairing, leading to G incorporation opposite template T. The resulting T-G base pair was less efficiently extended by human Polι compared to the Watson-Crick base pairs. Consequently, DNA synthesis frequently aborted opposite template T, a property we designated the T stop. This T stop restricted human Polι to a very short stretch of DNA synthesis. Furthermore, kinetic analyses show that human Polι copies template C with extraordinarily low fidelity, misincorporating T, A, and C with unprecedented frequencies of 1/9, 1/10, and 1/11, respectively. Human Polι incorporated one nucleotide opposite a template abasic site more efficiently than opposite a template T, suggesting a role for human Polι in DNA lesion bypass. The unique features of preferential G incorporation opposite template T and T stop suggest that DNA Polι may additionally play a specialized function in human biology.

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