Eukaryotic Y-family polymerases bypass a 3-methyl-2'-deoxyadenosine analog in vitro and methyl methanesulfonate-induced DNA damage in vivo.

N3-methyl-adenine (3MeA) is the major cytotoxic lesion formed in DNA by S(N)2 methylating agents. The lesion presumably blocks progression of cellular replicases because the N3-methyl group hinders interactions between the polymerase and the minor groove of DNA. However, this hypothesis has yet to be rigorously proven, as 3MeA is intrinsically unstable and is converted to an abasic site, which itself is a blocking lesion. To circumvent these problems, we have chemically synthesized a 3-deaza analog of 3MeA (3dMeA) as a stable phosphoramidite and have incorporated the analog into synthetic oligonucleotides that have been used in vitro as templates for DNA replication. As expected, the 3dMeA lesion blocked both human DNA polymerases alpha and delta. In contrast, human polymerases eta, iota and kappa, as well as Saccharomyces cerevisiae poleta were able to bypass the lesion, albeit with varying efficiencies and accuracy. To confirm the physiological relevance of our findings, we show that in S. cerevisiae lacking Mag1-dependent 3MeA repair, poleta (Rad30) contributes to the survival of cells exposed to methyl methanesulfonate (MMS) and in the absence of Mag1, Rad30 and Rev3, human polymerases eta, iota and kappa are capable of restoring MMS-resistance to the normally MMS-sensitive strain.

Intramolecular carbolithiation reactions of chiral alpha-amino-organolithium species.

Enantiomerically enriched alpha-amino-organolithium species, in which the lithium atom is attached to a stereogenic carbon centre, have been found to be chemically stable at room temperature in a solvent of very low polarity and undergo intramolecular carbolithiation onto an unactivated alkene. The configurational stability of the chiral organolithium species, bearing a variety of N-alkenyl substituents, was probed by studying the enantiomeric purity of the cyclization products. With N-but-3-enyl-2-lithiopyrrolidine, cyclization to the five-membered ring is more rapid than racemization and a high yield of the pyrrolizidine alkaloid (+)-pseudoheliotridane was obtained with no loss of optical purity. In contrast, with N-pent-4-enyl-2-lithiopyrrolidine, cyclization to the six-membered ring was found to occur with significant loss of optical purity. The cyclization to the six-membered ring was determined to occur with a half-life, t(1/2) approximately 90 min at 23 degrees C. The epimerization of this organolithium species in hexane/Et2O 4:1 was calculated to have a half-life, t(1/2) approximately 30 min at 23 degrees C. Enhanced levels of enantioselectivity for the formation of the indolizidine ring system were obtained using an alkene bearing a terminal phenylthio substituent. With N-[(3-phenylthio)-prop-2-enyl]-2-lithiopyrrolidine, cyclization to the four-membered ring occurs with poor enantioselectivity at low temperature in THF but is highly enantioselective at room temperature in a solvent of very low polarity.