Gel retardation electrophoresis

Poly(ADP-ribose) polymerase is a chromatin bound enzyme which catalyzes the covalent attachment of ADP-ribose units from the coenzyme NAD to various nuclear proteins (1-3). Poly(ADP-ribosyl)ation is a posttranslational modification which appears to be involved in DNA excision repair, cellular proliferation and differentiation (1-3) and in modulation of chromatin structure (4, 5). This DNA dependent enz3mie is inactive unless stimulated by DNA strand breaks. Although the DNA stmctures which activate the enzyme have been identified (6,7) the basis for the DNA requirement as weU as the stimulation of the enzyme activity are not yet understood. We have studied the interaction between poly(ADP-ribose) polymerase and different DNAs using electron microscopy and gel retardation electrophoresis. 

For agarose gel retardation electrophoresis, aliquots corresponding to 0.5 pg of DNA in the complex, were mixed with 5 pi of sample buffer containing 25% glycerol and bromophenol blue, deposited onto a 1% agarose gel and ran in Tris-phosphate-EDTA buffer at 30 V for 14-16 h. 

Fig. 1. Formation of poly(ADP-ribose) polymerase DNA complexes studied by gel retardation electrophoresis. pBR322 form I DNA (containing about 10% form II DNA) was mixed to purified cdf thymus poly(ADP-ribose) polymerase at different ratios r (mole/mole).

Both the electron microscopy and gel retardation electrophoresis results indicate that in the absence of single or double strand breaks, purified poly(ADP-ribose) polymerase preferentially binds to supeihelical DNA. By electron microscopy we have found a high frequency of enzyme molecules at intersections of DNA duplexes, independently of the type of DNA used. 

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See also in sourсe #XX -- [ , , ]

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