DNA adducts formed by aromatic amines such as N-acetyl-2-aminofluorene (AAF) and N-2-aminofluorene (AF) are known to cause mutations by interfering with the process of DNA replication. To understand this phenomenon better, a gel retardation assay was used to measure the equilibrium dissociation constants for the binding of an exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment) to DNA primertemplates modified with an AAF or AF adduct. The results indicate that the nature of the adduct as well as the presence and nature of an added dNTP have a significant influence on the strength of the binding of the polymerase to the DNA. More specifically, it was found that the binding is 5-10-fold stronger when an AAF adduct, but not an AF adduct, is positioned in the enzyme active site. In addition, the polymerase was found to bind the unmodified primer-template less strongly in the presence of a noncomplementary dNTP than in the presence of the correct nucleotide. The same trend holds true for the primer-template having an AF adduct, although the magnitude of this difference was lower. In the case of the AAF adduct, the interaction of the polymerase with the primer-template was stronger and almost independent of the nucleotide present.It is well established that the presence of DNA adducts in the template strand can impede or block DNA synthesis at a replication fork. Although most bulky adducts inhibit DNA synthesis strongly, some can be bypassed readily in vitro. The well studied carcinogen N-acetyl-2-aminofluorene (AAF) 1 can form both types of adducts in DNA; N-(deoxyguanosin-8-yl)-2-acetylaminofluorene adducts (dG-C8-AAF) are known to be strong blocks to DNA synthesis, whereas N-(deoxyguanosin-8-yl)-2-aminofluorene adducts (dG-C8-AF) can be bypassed by all polymerases tested (1). The mutagenic consequences of each adduct are also quite distinct. The dG-C8-AAF adduct results in mostly frameshift mutations in bacteria, whereas the dG-C8-AF adduct produces predominantly base substitution mutations (2-4). These different properties are likely to be the result of differences in the structures that these adducts assume in DNA in the active site of the DNA polymerase. Structural and enzymatic studies on duplex DNA molecules have demonstrated that the AF adduct produces less distortion in the DNA helix than the AAF adduct (2, 5). Multidimensional NMR experiments show that the guanine bearing the C8-AAF adduct rotates from anti to syn conformation in the doublestranded DNA helix with the fluorene ring inserted into the helix (base displacement model (6)). This contrasts with the AF adduct, which can adopt interchangeable conformations: in one the fluorene remains outside the helix (outside binding model), whereas the other has the fluorene ring stacked within the helix (5). The ratio of these conformations seems to be dependent on the sequence within which the adduct lies (7). Although it has been assumed that the structural differences between the AAF and AF adducts are responsible for the observed in the...

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...

The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3 -hydroxyl of the growing DNA chain. Here we report a singlemolecule approach to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution. As each nucleotide is incorporated, the single-molecule Fö rster resonance energy transfer intensity drops in discrete steps to values consistent with single-nucleotide incorporations. Purines and pyrimidines are incorporated with comparable rates. A mismatched primer/template junction exhibits dynamics consistent with the primer moving into the exonuclease domain, which was used to determine the fraction of primer-termini bound to the exonuclease and polymerase sites. Most interestingly, we observe a structural change after the incorporation of a correctly paired nucleotide, consistent with transient movement of the polymerase past the preinsertion site or a conformational change in the polymerase. This may represent a previously unobserved step in the mechanism of DNA synthesis that could be part of the proofreading process.Klenow Fragment ͉ polymerase and exonuclease site ͉ single molecule fluorescence ͉ single nucleotide resolution ͉ structural dynamics T he catalytic mechanism of Escherichia coli DNA polymerase I has been rigorously studied for more than 40 years (1, 2). The E. coli DNA polymerase I (Klenow fragment [KF]), an active truncated form of polymerase I, is composed of two domains: a polymerase domain that incorporates nucleotides, and a 3Ј-5Ј exonuclease domain that excises misincorporated nucleotides. The polymerase domain consists of three subdomains: the fingers, the palm, and the thumb. The fingers subdomain is primarily involved in interactions with the singlestranded region of the DNA template and the incoming nucleotide; the palm forms the active site of the polymerase upon interaction with the incoming dNTP; and the thumb is responsible for binding double-stranded DNA. The exonuclease domain, located Ϸ30 Å from the polymerase domain, binds to the 3Ј-terminus of the primer when a mismatched base is incorporated (3).Like other high-fidelity polymerases, KF achieves its extraordinary accuracy through a series of steps that discriminate between a correct and incorrect dNTP. A minimal reaction pathway for KF has been proposed with much of the data obtained from chemical quench experiments (4, 5) (Fig. 1A). The rate-limiting step (k 3 ) that precedes the phosphoryl-transfer step (k 4 ) had been tentatively attributed to a conformational change of the fingers domain (6). A comparison of the crystal structures of the binary polymerase-DNA complexes with those of the ternary polymerase-DNA-dNTP complexes reveals a substantial movement upon nucleotide binding, supporting the model that fingers closing was the rate-limiting step (7). However, recent results have shown this step is much too fast to be rate limiting, suggesting additional noncovalent steps th...

The mechanism by which DNA polymerases achieve their extraordinary accuracy has been intensely studied because of the linkage between this process and mutagenesis and carcinogenesis. Here, we have used single-molecule fluorescence microscopy to study the process of nucleotide selection and exonuclease action. Our results show that the binding of Escherichia coli DNA polymerase I (Klenow fragment) to a primer-template is stabilized by the presence of the next correct dNTP, even in the presence of a large excess of the other dNTPs and rNTPs. These results are consistent with a model where nucleotide selection occurs in the open complex prior to the formation of a closed ternary complex. Our assay can also distinguish between primer binding to the polymerase or exonuclease domain and, contrary to ensemble-averaged studies, we find that stable exonuclease binding only occurs with a mismatched primer terminus.

Y-family DNA polymerases play a crucial role in translesion DNA synthesis. Here, we have characterized the binding kinetics and conformational dynamics of the Y-family polymerase Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) using single-molecule fluorescence. We find that in the absence of dNTPs, the binary complex shuttles between two different conformations within ∼1 s. These data are consistent with prior crystal structures in which the nucleotide binding site is either occupied by the terminal base pair (preinsertion conformation) or empty following Dpo4 translocation by 1 base pair (insertion conformation). Most interestingly, on dNTP binding, only the insertion conformation is observed and the correct dNTP stabilizes this complex compared with the binary complex, whereas incorrect dNTPs destabilize it. However, if the n+1 template base is complementary to the incoming dNTP, a structure consistent with a misaligned template conformation is observed, in which the template base at the n position loops out. This structure provides evidence for a Dpo4 mutagenesis pathway involving a transient misalignment mechanism.

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The presence of benzo[a]pyrene diol epoxide (B[a]PDE) adducts in DNA is known to interfere with DNA replication. Kinetic studies of nucleotide insertion by exonuclease-deficient E. coli DNA polymerase I (Klenow fragment) across from either the (+)-trans- or the (+)-cis-B[a]P-N(2)-dG adduct in the 5'-CGT-3' sequence context indicated that the rate of nucleotide incorporation followed the order: dAMP > dGMP > dTMP > dCMP, which did not correlate with the mutational spectrum observed for these adducts in this sequence in E. coli (mostly G-->A transitions). Interestingly, a kinetic analysis of extension past the adduct showed that, unlike other sequences studied, the primer-template was extended best when dT was positioned at the 3'-terminus of the primer across from either a (+)-trans- or a (+)-cis-B[a]P-N(2)-dG adduct. In contrast, when the (+)-trans-B[a]P-N(2)-dG adduct was positioned in the 5'-TGC-3' sequence context, which gives predominantly G-->T mutations in E. coli, extension was detectable only when dA was positioned across from the adduct. These data provide the first in vitro evidence that may explain why G-->A transitions, rather than the G-->T transversions found in other sequences, are preferred in the 5'-CGT-3' sequence in vivo.