EPR INVESTIGATIONS OF METAL BINDING IN MU TRANSPOSASE

DNA转座涉及可转座的DNA的整合 元素(转座子)进入新的靶染色体。这个过程是一个 突变和基因组重排的共同来源，并且是必不可少的 对抗生素耐药机制和对抗生素的作用 逆转录病毒。细菌病毒Mu利用四聚体蛋白 MUA转座酶既可以切割DNA，也可以连接DNA的两端 转座子进入宿主染色体。二价金属阳离子 DNA切割和连接都需要镁或锰， 但人们对金属结合的细节知之甚少。这项研究 将利用多频(4-18,140 GHz)连续波和脉冲EPR来 解决与Mu转座酶中金属结合有关的问题。首字母 X波段连续波研究将确定分子的数目和离解常数 在蛋白质-DNA组装和活性的不同阶段结合的Mn(II) (即单体阶段、四聚体形成-DNA结合阶段、切割阶段 和加入阶段)。这些实验之后将会有 多频脉冲EPR研究(ESEEM和ENDOR)以确定 特定金属结合部位的结构方面(S)。立地 特定氨基酸残基(Asp269、Asp336、 Glu392)严重影响DNA切割和链转移 MUA的活动，为金属结合提供了间接证据 地点。ESEEM/Endor/高频EPR，带2H、13C、15N、17O标记 天冬氨酸和谷氨酸将提供这些结合的直接证据 通过测量的超精细相互作用产生残留物。再说一次，这些 可以在组装/活动的不同阶段进行测量 确定活动站点结构中的更改。其他二价金属 (Co(II)，Cu(II)，VO(II))可用作 金属结合部位(S)。

DNA transposition involves the integration of a transposable DNA element (transposon) into a new target chromosome. The process is a common source of mutations and genome rearrangements, and is essential to the mechanism of antibiotic resistance and to the function of retroviruses. The bacterial virus Mu utilizes the tetrameric protein MuA transposase both to cleave DNA and to join the two ends of the transposon to the host chromosome. The divalent metal cations magnesium or manganese are required for both DNA cleavage and joining, but details of the metal binding are poorly understood. The study will utilize multifrequency (4-18, 140 GHz) cw and pulsed EPR to address issues regarding metal binding in Mu transposase. Initial X-band cw studies will determine the number and dissociation constants of Mn(II) bound at various stages of protein-DNA assembly and activity (i.e. monomer stage, tetramer formation-DNA binding stage, cleavage and joining stages). These experiments will be followed by multifrequency pulsed EPR studies (ESEEM and ENDOR) to determine structural aspects of the specific metal binding site(s). Site specific mutagenesis at specific amino acid resideues (Asp269, Asp336, Glu392) severely compromises both DNA cleavage and strand transfer activities of MuA, giving indirect evidence3 for a metal bvinding site. ESEEM/ENDOR/High frequency EPR with 2H, 13C, 15N, 17O-labeled Asp and Glu will provide direct evidence of the binding of these residues via measured hyperfine interactions. Again, these measurements can be made at different stages of assembly/activity to determine changes in active site structure. Other divalent metals (Co(II), Cu(II), VO(II)) may be used as complementary probes of the metal binding site(s).