Feng S, Parrish D D, Lambert M W
Department of Pathology and Laboratory Medicine, UMDNJ-New Jersey Medical School, Newark 07103, USA.
Carcinogenesis. 1997 Feb;18(2):279-86. doi: 10.1093/carcin/18.2.279.
A DNA endonuclease, isolated from the nuclei of normal human and xeroderma pigmentosum complementation group A (XPA) cells, which recognizes predominately pyrimidine dimers, was examined for the mechanism by which it locates sites of damage on UVC-irradiated DNA. In reaction mixtures with low ionic strengths (i.e. lacking KCl), the normal and XPA endonuclease 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 it 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 normal and XPA endonucleases can incise UVC irradiated naked DNA, they differ in ability to incise damaged nucleosomal DNA. The normal endonuclease showed increased activity on UVC treated nucleosomal DNA compared with naked DNA, whereas the XPA endonuclease showed decreased activity 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 KCI, the normal endonuclease switched to a distributive mechanism of action and its ability to incise damaged nucleosomal DNA also decreased. These studies show that there is a correlation between the ability of these endonucleases to act by a processive mechanism and their ability to incise damaged nucleosomal DNA; the normal endonuclease, which acts processively, can incise damaged nucleosomal DNA, whereas the XPA endonuclease, which acts distributively, is defective in ability to incise this substrate.
从正常人及着色性干皮病A互补组（XPA）细胞的细胞核中分离出一种主要识别嘧啶二聚体的DNA内切酶，并对其在紫外线C（UVC）照射的DNA上定位损伤位点的机制进行了研究。在低离子强度（即不含KCl）的反应混合物中，正常内切酶和XPA内切酶通过不同机制在裸露的和重组核小体DNA上定位紫外线损伤位点。在这两种底物上，正常内切酶通过连续作用机制起作用，即它非特异性地结合到DNA上并扫描DNA寻找损伤位点，而XPA内切酶通过分布作用机制起作用，即它在DNA上随机定位损伤位点。然而，虽然正常内切酶和XPA内切酶都能切割UVC照射的裸露DNA，但它们切割受损核小体DNA的能力有所不同。与裸露DNA相比，正常内切酶对UVC处理的核小体DNA的活性增加，而XPA内切酶对受损核小体底物的活性降低。由于连续作用机制对微环境的离子强度敏感，因此增加了反应中的KCl浓度。在70 mM KCl时，正常内切酶转变为分布作用机制，其切割受损核小体DNA的能力也降低。这些研究表明，这些内切酶通过连续作用机制起作用的能力与其切割受损核小体DNA的能力之间存在相关性；正常内切酶以连续方式起作用，能够切割受损核小体DNA，而XPA内切酶以分布方式起作用，在切割这种底物的能力上存在缺陷。
