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 Tyr(141), Ala(142), Cys(143), Gln(144) and Arg(145) in Polµ, and by Trp(239), Val(240), Cys(241), Ala(242) and Gln(243) 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.
Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), 28049 Madrid, Spain.
Maria Jose
MJ
Martin
Maria Victoria
MV
Garcia-Ortiz
Ana
A
Gomez-Bedoya
Veronica
V
Esteban
Susana
S
Guerra
Luis
L
Blanco
D000595:Amino Acid Sequence; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D059766:DNA End-Joining Repair; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D004247:DNA; D018844:Cyclin-Dependent Kinases; C415367:DNA polymerase beta2; C407743:DNA polymerase mu; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase