Interaction of Escherichia coli DNA Polymerase I (Klenow Fragment) with Primer-Templates ContainingN-Acetyl-2-aminofluorene or N-2-Aminofluorene Adducts in the Active Site

Dzantiev, Leonid; Romano, Louis J.

doi:10.1074/jbc.274.6.3279

Cited by 39 publications

(124 citation statements)

References 37 publications

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“…2 and 3). It has been noted previously that increasing the concentration of KF results in the spontaneous shift in the retardation pattern of the enzyme-TP complex species from a regular to supershift position (15,25,(37)(38)(39). It has been suggested that the supershifted species of enzyme-TP complex corresponds to the binding of two or more enzymes to the same template-primer.…”

Section: Template-primer Binding By the Mutant Species Of H-bonding Tmentioning

confidence: 96%

Presence of 18-Å Long Hydrogen Bond Track in the Active Site of Escherichia coli DNA Polymerase I (Klenow Fragment)

Singh

Modak

2003

Journal of Biological Chemistry

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The analysis of the active site region in the crystal structures of template-primer-bound KlenTaq (Klenow fragment equivalent of Thermus aquaticus polymerase I) shows the presence of an ϳ18-Å long H-bonding track contributed by the Klenow fragment equivalent of Asn 845 , Gln 849 , Arg 668 , His 881 , and Gln 677 . Its location is nearly diagonal to the helical axis of the templateprimer. Four base pairs in the double stranded region proximal to 3 OH end of the primer terminus appear to interact with individual amino acid components of the track through either the bases or sugar moieties. To understand the functional significance of this H-bonding network in the catalytic function of Klenow fragment (KF), we generated N845A, N845Q, Q849A, Q849N, R668A, H881A, H881V, Q677A, and Q677N mutant species by site-directed mutagenesis. All of the mutant enzymes showed low catalytic activity. The kinetic analysis of mutant enzymes indicated that K m.dNTP was not significantly altered, but K D.DNA was significantly increased. Thus the mutant enzymes of the H-bonding track residues had decreased affinity for template-primer, although the extent of decrease was variable. Most interestingly, even the reduced binding of TP by the mutant enzymes occurs in the nonproductive mode. These results demonstrate that an H-bonding track is necessary for the binding of template-primer in the catalytically competent orientation in the pol I family of enzymes. The examination of the interactive environment of individual residues of this track further clarifies the mode of cooperation in various functional domains of pol I.Sequence alignment of various nucleic acid polymerases has revealed the presence of several conserved motifs (motifs A-E) in diverse DNA polymerase families (1-3). Two of these motifs (motifs A and C) are absolutely conserved in all DNA polymerase families, whereas the conservation of other motifs is restricted to individual families. Two conserved aspartates, one belonging, respectively, to motifs A and C each, serve as ligands for divalent cations (4 -6) and tags for the active site location of DNA polymerases (1, 2, 7).The Klenow fragment (KF) 1 of Escherichia coli DNA polymerase I has been extensively used as a model system for understanding the structural basis for the polymerase reaction mechanism. The crystal structures have shown the 68-kDa KF folds into two distinct domains, a ϳ200-amino acid long 3Ј-5Ј exonuclease domain at the N-terminal, and a ϳ400-residue polymerase domain at the C-terminal (8 -10). The polymerase domain, which anatomically resembles a half-open right-hand, consists of a cleft formed by three subdomains designated as fingers, palm, and thumb (8, 10). These subdomains have been implicated in different functions of polymerases (7). The active site constituted by motifs A and C resides at the palm subdomain, the dNTP binding site at the fingers subdomain, and the template-primer binding site at the thumb subdomain (7). The crystal structures of several DNA polymerases complexed with template-prim...

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Section: Template-primer Binding By the Mutant Species Of H-bonding Tmentioning

confidence: 96%

Presence of 18-Å Long Hydrogen Bond Track in the Active Site of Escherichia coli DNA Polymerase I (Klenow Fragment)

Singh

Modak

2003

Journal of Biological Chemistry

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“…The dG-AF adduct predominately produces randomly distributed base-substitution mutations (5,6), whereas the dG-AAF adduct results in frameshift mutations that frequently target specific repetitive sequences (4,(7)(8)(9). In vitro studies using templates modified with either a dG-AF or dG-AAF adduct have shown that the 2-aminofluorene (AF) adduct is bypassed much more readily than the corresponding AAF adduct by a variety of DNA polymerases (10,11). It has been suggested that the differences in mutagenic effects may be related to the diverse replication properties of these adducts.…”

mentioning

confidence: 99%

“…Nucleotide insertion is strongly blocked across from a dG-AAF adduct during primer extension reactions catalyzed by the Klenow fragment of E. coli DNA polymerase I, T7 DNA polymerase, and T4 DNA polymerase (10,11,27), and misinsertion of dAMP is favored over insertion of the correct nucleotide (dCMP) (28,29). The dG-AF lesion dramatically slows DNA synthesis, but this lesion is eventually bypassed (10,11). Although the correct nucleotide (dCTP) is preferentially incorporated across from a dG-AF adduct, dATP also is incorporated, to a lesser extent, at this site (29).…”

mentioning

confidence: 99%

“…Compared with the insertion of dCMP opposite dG, the incorporation across from either a dG-AAF or dG-AF adduct by a proofreading-deficient T7 DNA polymerase is Ϸ10 6 -or 10 4 -fold slower, respectively (30). Interestingly, the Klenow fragment polymerase binds to DNA containing a dG-AAF adduct with 5-to 10-fold higher affinity than it does to unmodified DNA or DNA containing a dG-AF adduct (10). The addition of nucleotide substrates that can base-pair with the templating base normally enhances DNA binding affinity by inducing the closure of the polymerase active site around the substrates (10,31).…”

mentioning

confidence: 99%

“…Interestingly, the Klenow fragment polymerase binds to DNA containing a dG-AAF adduct with 5-to 10-fold higher affinity than it does to unmodified DNA or DNA containing a dG-AF adduct (10). The addition of nucleotide substrates that can base-pair with the templating base normally enhances DNA binding affinity by inducing the closure of the polymerase active site around the substrates (10,31). However, the affinity of the Klenow fragment for DNA containing a dG-AAF adduct is unaffected by the addition of dCTP or any other nucleotide, suggesting that the lesion does not support base-pairing interactions within the polymerase active site (10,32).…”

mentioning

confidence: 99%

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Crystal structures of 2-acetylaminofluorene and 2-aminofluorene in complex with T7 DNA polymerase reveal mechanisms of mutagenesis

Dutta

Liu

Johnson

et al. 2004

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

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106

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The carcinogen 2-acetylaminofluorene forms two major DNA adducts: N-(2-deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and its deacetylated derivative, N-(2-deoxyguanosin-8-yl)-2-aminofluorene (dG-AF). Although the dG-AAF and dG-AF adducts are distinguished only by the presence or absence of an acetyl group, they have profoundly different effects on DNA replication. dG-AAF poses a strong block to DNA synthesis and primarily induces frameshift mutations in bacteria, resulting in the loss of one or two nucleotides during replication past the lesion. dG-AF is less toxic and more easily bypassed by DNA polymerases, albeit with an increased frequency of misincorporation opposite the lesion, primarily resulting in G 3 T transversions. We present three crystal structures of bacteriophage T7 DNA polymerase replication complexes, one with dG-AAF in the templating position and two others with dG-AF in the templating position. Our crystallographic data suggest why a dG-AAF adduct blocks replication more strongly than does a dG-AF adduct and provide a possible explanation for frameshift mutagenesis during replication bypass of a dG-AAF adduct. The dG-AAF nucleoside adopts a syn conformation that facilitates the intercalation of its fluorene ring into a hydrophobic pocket on the surface of the fingers subdomain and locks the fingers in an open, inactive conformation. In contrast, the dG-AF base at the templating position is not well defined by the electron density, consistent with weak binding to the polymerase and a possible interchange of this adduct between the syn and anti conformations. N umerous carcinogenic aromatic amines, including a variety of environmental and dietary carcinogens and heterocyclic aromatic amines present in tobacco smoke condensate, are known to react with DNA to form adducts at the C8 position of guanine (1). 2-Acetylaminofluorene (AAF) is the best-studied example of this class of carcinogen (2). Originally developed as a pesticide, toxicity tests showed that this compound and related derivatives are potent liver carcinogens (3). Thus, the compound was never introduced as a pesticide. Instead, AAF has become a model compound for the study of the mutagenic and carcinogenic effects of aromatic amines (4).Metabolic activation of AAF in vivo generates intermediates that form two related adducts bound to the C8 position of guanine DNA: the N-(2Ј-deoxyguanosin-8-yl)-AAF (dG-AAF) adduct and the corresponding deacetylated N-(2Ј-deoxyguanosin-8-yl)-2-aminofluorene (dG-AF) derivative (Fig. 1) (3). The mutagenic consequences of these adducts are quite distinct in Escherichia coli. The dG-AF adduct predominately produces randomly distributed base-substitution mutations (5, 6), whereas the dG-AAF adduct results in frameshift mutations that frequently target specific repetitive sequences (4, 7-9). In vitro studies using templates modified with either a dG-AF or dG-AAF adduct have shown that the 2-aminofluorene (AF) adduct is bypassed much more readily than the corresponding AAF adduct by a variety of DNA pol...

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Single‐molecule methods, activation‐induced cytidine deaminase, and quantum mechanical approach to explore and prevent carcinogenesis

Ullah,

Mabood,

Ullah

et al. 2024

VIEW

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Recent advancements in single‐molecule methods have not only made it possible to obtain precise measurements for complex biological processes but also to produce simple mathematical models for intricate biochemical mechanisms, which would otherwise be speculative. These developments have strengthened our ability to respond through mathematical modeling to concepts of protein‒protein and protein‒DNA interactions on a nanometer level and address‐related questions. In this article, we examine an intriguing biological phenomenon in which a protein and an enzyme co‐jointly encounter carcinogenic adducts during transcription. We are focusing mainly on the dysregulation of the protein involved and the possible consequences that may arise. By providing a quantum mechanical model, we have demonstrated that the presence of carcinogenic adducts in a transcriptional bubble deregulates the protein that could cause lethal mutations. Next, we present a case study to explore carcinogenesis by suggesting an alternative experimental design. Our quantum mechanical model emphasizes the use of a quantized energies approach for specific mechanisms within the living cells. Radiation‐induced carcinogenicity can be prevented if radiation interacting with tissue is not given the energies that satisfy the quantization conditions.

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Interaction of Escherichia coli DNA Polymerase I (Klenow Fragment) with Primer-Templates ContainingN-Acetyl-2-aminofluorene or N-2-Aminofluorene Adducts in the Active Site

Cited by 39 publications

References 37 publications

Presence of 18-Å Long Hydrogen Bond Track in the Active Site of Escherichia coli DNA Polymerase I (Klenow Fragment)

Presence of 18-Å Long Hydrogen Bond Track in the Active Site of Escherichia coli DNA Polymerase I (Klenow Fragment)

Crystal structures of 2-acetylaminofluorene and 2-aminofluorene in complex with T7 DNA polymerase reveal mechanisms of mutagenesis

Single‐molecule methods, activation‐induced cytidine deaminase, and quantum mechanical approach to explore and prevent carcinogenesis

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