A PROCESSIVE VERSUS A DISTRIBUTIVE MECHANISM OF ACTION CORRELATES WITH DIFFERENCES IN ABILITY OF NORMAL AND XERODERMA-PIGMENTOSUM GROUP-A ENDONUCLEASES TO INCISE DAMAGED NUCLEOSOMAL DNA

A DNA endonuclease, isolated from the nuclei of normal human and xerod erma pigmentosum complementation group A (XPA) cells, which recognizes predominately pyrimidine dimers, was examined for the mechanism by wh ich it locates sites of damage on UVC-irradiated DNA, In reaction mixt ures with low ionic strengths (i.e. lacking KCI), the normal and XPA e ndonuclease locate sites of UV damage on both naked and reconstituted nucleosomal DNA by different mechanisms, On both of these substrates, the normal endonuclease acts by a processive mechanism, meaning that i t binds non-specifically to DNA and scans the DNA for sites of damage, whereas the XPA endonuclease acts by a distributive one, meaning that it randomly locates sites of damage on DNA, However, while both the n ormal and XPA endonucleases can incise UVC irradiated naked DNA, they differ in ability to incise damaged nucleosomal DNA. The normal endonu clease showed increased activity on UVC treated nucleosomal DNA compar ed with naked DNA, whereas the XPA endonuclease showed decreased activ ity on the damaged nucleosomal substrate. Since a processive mechanism of action is sensitive to the ionic strength of the micro-environment , the KCl concentration of the reaction was increased, At 70 mM KCl, t he normal endonuclease snitched to a distributive mechanism of action and its ability to incise damaged nucleosomal DNA also decreased, Thes e studies show that there is a correlation between the ability of thes e endonucleases to act by a processive mechanism and their ability to incise damaged nucleosomal DNA; the normal endonuclease, which acts pr ocessively, can incise damaged nucleosomal DNA, whereas the XPA endonu clease, which acts distributively, is defective in ability to incise t his substrate.