A specific N-terminal extension of the 8 kDa domain is required for DNA end-bridging by human Pol mu and Pol lambda

摘要

Human DNA polymerases mu (Pol mu) and lambda (Pol lambda) are X family members involved in the repair of double-strand breaks in DNA during non-homologous end joining. Crucial abilities of these enzymes include bridging of the two 3' single-stranded overhangs and trans-polymerization using one 3' end as primer and the other as template, to minimize sequence loss. In this context, we have studied the importance of a previously uncharacterised sequence ('brooch'), located at the N-terminal boundary of the Pol ss-like polymerase core, and formed by Tyr(141), Ala(142), Cys(143), Gln(144) and Arg(145) in Pol mu, and by Trp(239), Val(240), Cys(241), Ala(242) and Gln(243) in Pol lambda. The brooch is potentially implicated in the maintenance of a closed conformation throughout the catalytic cycle, and our studies indicate that it could be a target of Cdk phosphorylation in Pol mu. The brooch is irrelevant for 1 nt gap filling, but of specific importance during end joining: single mutations in the conserved residues reduced the formation of two ended synapses and strongly diminished the ability of Pol mu and polymerase lambda to perform non-homologous end joining reactions in vitro.