DNA polymerase lambda (pol λ) is a novel family X DNA polymerase that has been suggested to play a role in meiotic recombination and DNA repair. The recent demonstration of an intrinsic 5′-deoxyribose-5-phosphate lyase activity in pol λ supports a function of this enzyme in base excision repair. However, the biochemical properties of the polymerization activity of this enzyme are still largely unknown. We have cloned and purified human pol λ to homogeneity in a soluble and active form, and we present here a biochemical description of its polymerization features. In support of a role in DNA repair, pol λ inserts nucleotides in a DNA template-dependent manner and is processive in small gaps containing a 5′-phosphate group. These properties, together with its nucleotide insertion fidelity parameters and lack of proofreading activity, indicate that pol λ is a novel β-like DNA polymerase. However, the high affinity of pol λ for dNTPs (37-fold over pol β) is consistent with its possible involvement in DNA transactions occurring under low cellular levels of dNTPs. This suggests that, despite their similarities, pol β and pol λ have nonredundantin vivo functions.

Short interfering RNAs (siRNAs) targeting viral or cellular genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, the emergence of mutations in the gene being targeted could lead to the rapid escape from the siRNA. Here, we simulate viral escape by systematically introducing single-nucleotide substitutions in all 19 HIV-1 residues targeted by an effective siRNA. We found that all mutant viruses that were tested replicated better in the presence of the siRNA than in the presence of the wild-type virus. The antiviral activity of the siRNA was completely abolished by single substitutions in 10 (positions 4 to 11, 14, and 15) out of 16 positions tested (substitution at 3 of the 19 positions explored rendered nonviable viruses). With the exception of the substitution observed at position 12, substitutions at either the 5′ end or the 3′ end (positions 1 to 3, 16, and 18) were better tolerated by the RNA interference machinery and only in part affected siRNA inhibition. Our results show that optimal HIV-1 gene silencing by siRNA requires a complete homology within most of the target sequence and that substitutions at only a few positions at the 5′ and 3′ ends are partially tolerated.