A u t h o r ' s p e r s o n a l c o p y
-deoxyguanosin-8-yl)-2-aminofluorene(AF) (Figure 1(a)), along with the minor 3-(2
These bulky DNA lesions, if notrepaired, can produce mutations during replicationthat initiate the process of carcinogenesis. In bacteria AF-adducts mostly induce G
T transver-sions, whereas AAF-adducts cause mostly deletionmutations.
In mammalian cells, however, bothadducts promote sequence-dependent base substi-tution mutations.
The bulky AF and AAF adducts have long beenused as model systems for investigation of thestructure/functionion relationship in the arylaminemutagen family.
However, the molecular under-standing of how they exert mutation
isgenerally poor and is complicated by adduct-induced conformational heterogeneities. The AAF-adduct exists predominantly in the base-displaced
Because it lacksWatson
Crick base-pairs at the lesion site, theAAF-adduct produces a major structural distortionin DNA. In contrast, the
-deacetylated AF-adductpossesses flexiblility around the glycosidyl bond(
), enabling it to adopt multiple conformationalmotifs depending upon the location of the amino-fulorene moiety, including a major-groove binding
(B) conformation and a minor-groove binding
(W) conformation in additionto the S conformation (Figure 1(b)).
In general,fully base-paired DNA duplexes that have incorpo-rated AF are present in an S/B equilibrium in whichthe tendencyforthe molecules tofavorthe Sor theBconformation is dependent upon the flanking-sequence context.
Consistent with this observa-tion, the mutational specificity and frequency of theAF-adduct in mammalian cells vary dependingupon thesequence context inwhich itisembedded.
The W-conformer has only been observed in AF-modified duplexes with dA and dG-mismatches atthe lesion site;
these mispairings apparentlyunderlieG
Ctransversions, respectively.Normally, DNA replication in a replicative poly-merase proceeds with high fidelity and processivityon a natural DNA template.
The process, how-ever, is interupted significantly when a damaged base is present in the template. Continued replica-tion of the adduct-containing template is termed astranslesion synthesis (TLS), which is a major sourceof point mutations.
The TLS events are modu-latedbyvariousfactorsincludingthelesion-inducedconformational changes of the template, its sur-rounding base sequence context, and by the natureof DNA polymerases.
The slowing of replicationin a replicative polymerase is now understood topredominantly produce switch to a lesion bypasspolymerase
which is frequently error prone.However, many specific details of this paradigmremains unknown, i.e. how does the polymeraseswitch at a site of DNA damage really occur?
Mutagenic outcomes in replicative polymerasesmay alsocontribute tothe overall mutagenic burden
Although the steady-state kinetics of nucleotideinsertion opposite the lesion have been studiedextensively for many DNA adducts,
data deal-ing with the long-range effects of a lesion on TLS arelimited.
Lindsley & Fuchs
have shown thatthe rate of primer extension by T7 DNA polymerase both at (n) and adjacent to (n+1) the lesion site witha DNA template containing an AF-lesion is reducedsignificantly (
) relative to unmodified controlDNA. A greater rate reduction (
) was ob-served in the presence of an AAF-adduct. Miller &Grollman
subsequently extended the scope of theinvestigation, probing the long-range effects of AF and other DNA adducts on the exo
DNA polymerase I.Polymerase activity was affected as far as four bases downstream (n
) of the dG[AF]:dA mismatch lesion. The effect was much lesspronounced (10
) for extension of dG [AF]:dC. Similar results were obtained with Pol II & III.
The slowed replication rate even after incorporationof bases opposite the lesion provides sufficient timefor template alignment, a general model to predictdeletion mutations by various bulky DNA adductsincluding AF and AAF-adducts.
In the present study we have used the well-defined AF-induced B/S/W-conformational hetero-geneity model
as a basis for investigating theadduct structures at various positions relative to theprimer terminus during simulated AF-induced TLS.Temperature-dependent
F NMR spectra wereobtained for two distinct TLS models: extension of dG [FAF]:dC-match and dG [FAF]:dA mismatch.The dynamic NMR results coupled with the model-ing work with the thermophilic DNA polymerase I
were examined in thecontext of previous kinetic parameters
in orderto gain insight into the long-range effects of theconformationally flexible AF-adduct on DNA poly-merase function.
The synthesis and purification of an FAF-mod-ified 12-mer oligodeoxynucleotide and time-of-flight-mass spectrometry characterization were car-ried out using the procedures described.
Themodified template strand was annealed withappropriate primers in order to produce variousTLS models of the primer extension reaction (n
1,n, n+1, n+3, and n+6, n=primer terminus) (Figure1(c)).Figures 2 and 3show the dynamic
F NMRresults for the dC-match and dA-mismatch exten-sion models, respectively. We have recently demon-strated the utility of
F NMR/CD procedure forprobing the AF-induced B-S-W-conformationalheterogeneities.
F NMR method takesadvantage of the sensitivity of the fluorine nucleusto the tertiary structure of DNA, thereby requiringfluorine-tagged FAF as a model probe (Figure1(a)).
Induced circular dichroism in the 290
360 nm FAF-absorbing range (ICD
Long-range Effect of Aminofluorene-Heterogeneity