Effects of a high-affinity antibody fragment on DNA polymerase reactions near a (6-4) photoproduct site

Pyrimidine (6-4) pyrimidone photodimers are major photoproducts that have m utagenic and carcinogenic consequences, One major reason for these biologic al effects of (6-4) photoproducts may be base mispairing/DNA replication er rors due to hydrogen bonding to bases apposite these damaged sites. We synt hesized a modified 41-mer DNA containing a (6-4) photoproduct using a prefo rmed building block, then employed it as a template far primer extension re actions catalyzed by Klenow fragment and DNA polymerases alpha, beta and de lta (pol alpha, pol beta and pol delta), None of these DNA polymerases were able to bypass the (6-4) photoproduct and elongation terminated at or near the 3'-pyrimidone of the photoproduct, depending on the dNTP concentration . When a single-chain Fv (scFv) with high affinity for the (6-4) photoprodu ct was included in the polymerization reaction, DNA synthesis was inhibited at base positions four, six, eight or eight nucleotides prior to the 3'-py rimidone by Klenow fragment, pol alpha, pol beta or pol delta, respectively . These results suggest that the scFv can bind to the template DNA containi ng a (6-4) photoproduct and inhibit extension reactions by polymerases.