CHARACTERIZATION OF THE XRCC1-DNA LIGASE-III COMPLEX IN-VITRO AND ITSABSENCE FROM MUTANT HAMSTER-CELLS

The human DNA repair protein XRCC1 was overexpressed as a histidine-ta gged polypeptide (denoted XRCC1-His) in Escherichia coli and purified in milligram quantities by affinity chromatography. XRCC1-His compleme nted the mutant Chinese hamster ovary cell line EM9 when constitutivel y expressed from a plasmid or when introduced by electroporation. XRCC 1-His directly interacted with human DNA ligase III in vitro to form a complex that was resistant to 2 M NaCl. XRCC1-His interacted equally well with DNA ligase III from Bloom syndrome, HeLa and MRC5 cells, ind icating that Bloom syndrome DNA ligase III is normal in this respect. Detection of DNA ligase III on far Western blots by radiolabelled XRCC 1-His indicated that the level of the DNA ligase polypeptide was reduc ed similar to 4-fold in the mutant EM9 and also in EM-C11, a second me mber of the XRCC1 complementation group. Decreased levels of polypepti de thus account for most of the similar to 6-fold reduced DNA ligase I II activity observed previously in EM9. Immunodetection of XRCC1 on We stern blots revealed that the level of this polypeptide was also decre ased in EM9 and EM-C11 (>10-fold), indicating that the XRCC1-DNA ligas e III complex is much reduced in the two CHO mutants.