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

Human DNA polymerases mu (Polµ) and lambda (Polλ) 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 Polß-like polymerase core, and formed by Tyr141, Ala142, Cys143, Gln144 and Arg145 in Polµ, and by Trp239, Val240, Cys241, Ala242 and Gln243 in Polλ. 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 Polµ. 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 Polµ and polymerase lambda to perform non-homologous end joining reactions in vitro

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

Human DNA polymerases mu (Polμ) and lambda (Polλ) 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 Polß-like polymerase core, and formed by Tyr141, Ala142, Cys 143, Gln144 and Arg145 in Polμ, and by Trp239, Val240, Cys241, Ala242 and Gln243 in Polλ. 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 Polμ. 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 Polμ and polymerase lambda to perform non-homologous end joining reactions in vitro. © 2013 The Author(s) 2013.Ministerio de Ciencia y Tecnologıa (BFU2009-10085) and CONSOLIDER (CSD2007-00015); Fundacion Ramon Areces; Comunidad Autonoma de MadridPeer Reviewe