The main strategy used by pro- and eukaryotic cells for replication of damaged DNA is translesion synthesis (TLS). Here, we investigate the TLS process catalyzed by DNA polymerases beta and lambda on DNA substrates using mono- or dinucleotide gaps opposite damage located in the template strand. An analog of a natural apurinic/apyrimidinic site, the 3-hydroxy-2-hydroxymetylthetrahydrofuran residue (THF), was used as damage. DNA was synthesized in the presence of either Mg2+ or Mn2+. DNA polymerases beta and lambda were able to catalyze DNA synthesis across THF only in the presence of Mn2+. Moreover, strand displacement synthesis was not observed. The primer was elongated by only one nucleotide. Another unusual aspect of the synthesis is that dTTP could not serve as a substrate in all cases. dATP was a preferential substrate for synthesis catalyzed by DNA polymerase beta. As for DNA polymerase lambda, dGMP was the only incorporated nucleotide out of four investigated. Modified on heterocyclic base photoreactive analogs of dCTP and dUTP showed substrate specificity for DNA polymerase beta. In contrast, the dCTP analog modified on the exocyclic amino group was a substrate for DNA polymerase lambda. We also observed that human replication protein A inhibited polymerase incorporation by both DNA polymerases beta and lambda on DNA templates containing damage.