Enzymatic Repair of DNA Damage in Plants

近年来，平流层臭氧的显著消耗导致到达地球表面的紫外线(UV)辐射增加。随着紫外线辐射的增加，人们对紫外线辐射对生物体细胞过程的具体影响产生了关注和兴趣。其中一种效应是DNA损伤，在细菌和哺乳动物系统中得到了广泛的研究；然而，关于紫外线损伤对植物DNA的影响及其通过细胞机制进行体内修复的信息有限。这项建议描述了在植物细胞中发现的环饱和的嘧啶切割活性的初步研究，该活性针对紫外线照射或氧化的DNA。通过离子交换、亲和层析和凝胶过滤层析等常规技术从甘蓝(花椰菜)中纯化这种酶活性。对紫外线辐射或氧化的放射性标记多脱氧核糖核苷酸的底物专一性将用高压液相色谱(HPLC)来表征。利用纯化的酶作为探针，DNA碱基序列对这些损伤的形成和随后的体内修复的影响将被量化。由于叶绿体基因组也容易受到破坏，因此将调查叶绿体中是否存在环饱和的嘧啶-DNA修复酶。这应该确定是否有一种方法可以有效地从叶绿体DNA中去除这种受损的损伤，以保持这种遗传物质的完整性。这些研究的结果应该有助于更好地理解构成植物细胞对DNA损伤的反应的生化过程。其目的是最终利用从这个项目中获得的知识来分离具有更强的修复辐射和氧化造成的损害的能力并因此具有更强的生存能力的植物菌株。

In recent years, a marked depletion of stratospheric ozone has caused an increase in ultraviolet (UV) radiation reaching the surface of the Earth. With this increase, concern and interest has been raised as to what specific effects UV radiation has on cellular processes in organisms. One such effect, the damage of DNA, has been extensively investigated in bacterial and mammalian systems; however, limited information exists on the effects of UV damage to plant DNA and its in vivo repair by cellular mechanisms. This proposal describes the initial study of a ring- saturated pyrimidine incising activity found in plant cells against UV irradiated or oxidized DNA. This enzymatic activity will be purified from Brassica oleracea (cauliflower) by conventional techniques including ion-exchange, affinity, and gel filtration chromatography. The substrate specificity against UV- irradiated or oxidized, radiolabeled polydeoxyribonucleotides will be characterized by high pressure liquid chromatography (HPLC). Using the purified enzyme as a probe, the effects of DNA base sequence on the formation of these lesions and their subsequent in vivo repair will be quantified. Since the chloroplast genome is also susceptible to damage, the presence of the ring-saturated pyrimidine-DNA repair enzyme in chloroplasts will be investigated. This should determine if there is a means to effectively remove this damaged lesion from chloroplast DNA to maintain the integrity of this genetic material. The results of these studies should lead to a better understanding of the biochemical processes that constitute the plant cellular response to DNA damage. The aim is to ultimately use the knowledge obtained from this project to isolate plant strains that have an increased ability to repair damage caused by radiation and oxidation and therefore have an increased survival capacity.