Abstract
A human DNA damage binding protein implicated in the DNA excision repair disorder xeroderma pigmentosum E was purified to near homogeneity from HeLa cells. The protein is abundant (approximately 10(5) copies/cell) and has a native molecular weight of 154,000-163,000 as estimated by gel filtration and glycerol gradient sedimentation. DNA damage binding activity copurified with polypeptides of 124 and 41 kDa. Based on the native molecular weight, cosedimentation of both polypeptides with DNA damage binding activity on glycerol gradients, and a molar ratio of approximately 1:1 for the two polypeptides, it appears that p124 and p41 are subunits of a heterodimeric protein. Binding to damaged DNA was resistant to K+ concentrations approaching 1 M, but showed anion-specific sensitivity to Cl- concentrations above 0.5 M, suggesting that the majority of the binding energy is contributed by nonionic interactions. In contrast to previous reports, the DNA damage binding protein was shown to recognize cyclobutane pyrimidine dimers in addition to a nonphotoreactivable lesion(s), most likely the pyrimidine-pyrimidone (6-4) photoproduct.
Highlights
It appears thatp124 and p41are subunitsof a heterodi- X P is a rare genetic disease characterizedby a clinical and meric protein
Substantially less is known about the mechanisms of DNA damage recognition or excision of bulky adducts, but recent studies have identified several activities that appear tobe involved in theprocess
Several human proteins that bind to DNA that hasbeen damaged with the antitumor drug cisplatin have been identifiedo,f wonheich is the chromosomal protein HMG-ll (Hughes et al, 1992; Pi1 matological abnormalities.ChuandChang (1988) originally observed that cells from some XP-E patients were missing a DNA damagebinding (DDB) activity, butsubsequently we (Keeney et al, 1992) and Kataoka and Fujiwara(1991) found that cells from most XP-E patients have normal levels of the activity
Summary
Cated fractions were assayed by band mobility shift. Damage-specific bands are indicated by the arrows. Protein-Fraction VI contained two major polypeptides of 124 and 41 kDa as estimated by SDS-PAGE (Fig. E ) Both polypeptides cosedimented with the peak of DDB activity during glycerol gradient ultracentrifugation (Fig. 3), suggesting that they form a complex in solution. (The appearance of the two polypeptides as doublets wasvariable from preparationto preparation and may represent partial degradation or differential post-translational modifications.) The peak of DDB ac-. An alternative interpretation of the sedimentation data would be that the DDB protein consists of p124 only and that p41 is a homotetrameric contaminant (M, 160,000) This interpretation seemsunlikely,since DNA binding activity in the glycerol gradient profile correlates only with the presence of p124, suggesting that p41 does not by itself have DNA binding activity under standard assay conditions.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
