Access Type

Open Access Dissertation

Date of Award

January 2014

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Louis J. Romano

Abstract

The two major forms of DNA adducts from the carcinogen N-acetoxyacetyl-2-aminofluorene, N-(deoxygunanonsin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), are both known to impede replication, though in different ways. AAF is a strong block to replication leading to frameshift mutations, while the AF adduct is more easily bypassed, causing base substitutions. Surface plasmon resonance (SPR) was used to study the binding of exonuclease deficient E. coli polymerase I, Klenow fragment (KF), to DNA modified with AF or AAF at two locations: as a templating base or in the last formed base pair. KF binding to the modified DNA bases was also monitored to DNA that contained a terminal mismatch. When the templating guanine is modified with either AF or AAF, KF binds tightly and the addition of the next correct nucleotide does not increase binding, nor does an incorrect nucleotide decrease binding. However when an AAF or AF modified guanine in the terminal base pair, bound KF is unable to form a stable structure. Interestingly, when the dG-AF was part of a terminal mismatch, KF bound better to mismatched DNA than correctly base paired DNA, both in the presence and absence of nucleotide. Incorrect dNTP showed the least KF destabilization binding to the dG-AF mismatched DNA constructs. It is possible that the bulky, mismatched primer template terminus rearranges the active site allowing the modified dG to rotate and serve as a template for the incoming dCTP. SPR is a useful label free technique to determine both equilibrium binding constants (KD), and dissociation rates (koff) from the same set of data. The dissociation rates reveal that for most constructs, KF dissociates at two rates, a fast and a slow rate, relating to KF dissociating from different conformations.

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