Human DNA Polymerase Mu post-catalytic complex

4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID

Sustained active site rigidity during synthesis by human DNA polymerase mu.

GSAAAPLSPAWMPAYACQRPTPLTHHNTGLSEALEILAEAAGFEGSEGRLLTFCRAASVLKALPSPVTTLSQLQGLPHFG
EHSSRVVQELLEHGVCEEVERVRRSERYQTMKLFTQIFGVGVKTADRWYREGLRTLDDLREQPQKLTQQQKAGLQHHQDL
STPVLRSDVDALQQVVEEAVGQALPGATVTLTGGFRRGKLQGHDVDFLITHPKEGQEAGLLPRVMCRLQDQGLILYHQHQ
HSCCESPTRLAQQSHMDAFERSFCIFRLPQPGSWKAVRVDLVVAPVSQFPFALLGWTGSKLFQRELRRFSRKEKGLWLNS
HGLFDPEQKTFFQAASEEDIFRHLGLEYLPPEQRNA

Modified version of the pGEX4T3 vector backbone, where a TEV protease cleavage site has been inserted downstream of the thrombin cleavage site, and the sequence of the multicloning site has been replaced by that of the pMALX vector (Moon et al, 2010). Three TGA stop codons have been inserted downstream of the multicloning site, each utilizing a different reading frame.

Crystals were grown by mixing 1uL protein/DNA mixture with 1uL mother liquor (85-90mM HEPES pH 7.5, 17-18% PEG4000), using the sitting drop vapor diffusion technique. Crystals were transferred in two steps to a cryoprotectant solution containing 0.1M HEPES pH 7.5, 10mM MgCl2, 50mM NaCl, 20% PEG4000, 5% glycerol, 15% ethylene glycol, 10mM dTTP for 16 hours. The crystals were then backsoaked in cryoprotectant containing 2mM pyrophosphate. , VAPOR DIFFUSION, SITTING DROP, temperature 295K

polymerase, DNA break repair, TRANSFERASE-DNA complex

PLSPAWMPAYACQRPTPLTHHNTGLSEALEILAEAAGFEGSEGRLLTFCRAASVLKALPSPVTTLSQLQGLPHFGEHSSR
VVQELLEHGVCEEVERVRRSERYQTMKLFTQIFGVGVKTADRWYREGLRTLDDLREQPQKLTQQQKAGLQHHQDLSTPVL
RSDVDALQQVVEEAVGQALPGATVTLTGGFRRGKLQGHDVDFLITHPKEGQEAGLLPRVMCRLQDQGLILYHQHQHSCCE
SPTRLAQQSHMDAFERSFCIFRLPQPPGAAVGGSTRPCPSWKAVRVDLVVAPVSQFPFALLGWTGSKLFQRELRRFSRKE
KGLWLNSHGLFDPEQKTFFQAASEEDIFRHLGLEYLPPEQRNA