RecQ helicases are believed to function in repairing replication forks stalled by DNA damage and may also play a role in the intra-S-phase checkpoint, which delays the replication of damaged
DNA, thus permitting repair to occur. Since little is known regarding the effects of DNA damage on
RecQ helicases, and because the replication and recombination defects in
Werner syndrome cells may reflect abnormal processing of damaged
DNA associated with the replication fork, we examined the effects of specific bulky, covalent adducts at N(6) of
deoxyadenosine (dA) or N(2) of
deoxyguanosine (dG) on Werner (WRN) syndrome helicase activity. The adducts are derived from the optically active 7,8-diol 9,10-epoxide (DE) metabolites of the
carcinogen benzo[a]pyrene (BaP). The results demonstrate that WRN helicase activity is inhibited in a strand-specific manner by BaP DE-dG adducts only when on the translocating strand. These adducts either occupy the minor groove without significant perturbation of
DNA structure (trans adducts) or cause base displacement at the adduct site (cis adducts). In contrast, helicase activity is only mildly affected by intercalating BaP DE-dA adducts that locally perturb
DNA double helical structure. This differs from our previous observation that intercalating dA adducts derived from
benzo[c]phenanthrene (BcPh) DEs inhibit WRN activity in a strand- and stereospecific manner. Partial unwinding of the
DNA helix at BaP DE-dA adduct sites may make such adducted DNAs more susceptible to the action of helicase than
DNA containing the corresponding BcPh DE-dA adducts, which cause little or no destabilization of duplex
DNA. The
single-stranded DNA binding protein RPA, an auxiliary factor for WRN helicase, enabled the
DNA unwinding
enzyme to overcome inhibition by either the trans-R or cis-R BaP DE-dG adduct, suggesting that WRN and RPA may function together to unwind duplex
DNA harboring specific covalent adducts that otherwise block WRN helicase acting alone.