Polycyclic aromatic hydrocarbon (PAH)-
DNA adducts pervert the execution or fidelity of enzymatic
DNA transactions and cause mutations and
cancer. Here, we examine the effects of intercalating PAH-
DNA adducts on the religation reaction of
vaccinia DNA topoisomerase, a prototypal type IB topoisomerase (TopIB), and the 3' end-resection reaction of Escherichia coli
exonuclease III (ExoIII),
a DNA repair
enzyme.
Vaccinia TopIB forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p / N(-1) in duplex
DNA. The rate of the forward cleavage reaction is suppressed to varying degrees by
benzo[a]pyrene (BP) or
benzo[c]phenanthrene (BPh) adducts at
purine bases within the 3'-G(+5)G(+4)G(+3)A(+2)A(+1)T(-1)A(-2) sequence of the nonscissile strand. We report that BP adducts at the +1 and -2 N6-deoxyadenosine (dA) positions flanking the scissile phosphodiester slow the rate of
DNA religation to a greater degree than they do the cleavage rate. By increasing the cleavage equilibrium constant > or = 10-fold, the BPdA adducts, which are intercalated via the major groove, act as TopIB
poisons. With respect to ExoIII, we find that (i) single BPdA adducts act as durable roadblocks to ExoIII digestion, which is halted at sites 1 and 2
nucleotides prior to the modified base; (ii) single BPhdA adducts, which also intercalate via the major groove, elicit a transient pause prior to the lesion, which is eventually resected; and (iii) BPh adducts at N2-deoxyguanosine, which intercalate via the minor groove, are durable impediments to ExoIII digestion. These results highlight the sensitivity of repair outcomes to the structure of the PAH ring system and whether intercalation occurs via the major or minor groove.