EPR & RESONANCE RAMAN OF CHLAMYDOMONAS CHLOROPLAST HEMOGLOBIN (HB) & MUTANTS

DNA转座涉及一个可转座DNA的整合 将转座子插入新的靶染色体。 述方法是 突变和基因组重排的共同来源，是必不可少的 抗生素耐药性的机制以及 逆转录病毒 细菌病毒Mu利用四聚体蛋白 MuA转座酶既切割DNA又连接DNA的两端， 转座子转移到宿主染色体上。 二价金属阳离子 镁或锰是DNA切割和连接所必需的， 但对金属结合的细节知之甚少。 研究 将利用多频（4-18，140 GHz）cw和脉冲EPR， 解决了Mu转座酶中金属结合的问题。 初始 X波段连续波研究将确定其数目和离解常数 在蛋白质-DNA组装和活性的各个阶段结合的Mn（II） (i.e.单体阶段，四聚体形成-DNA结合阶段，裂解 和加入阶段）。 这些实验之后， 多频脉冲EPR研究（ESEEM和ENDOR），以确定 特定金属结合位点的结构方面。 网站 在特定氨基酸残基（Asp 269，Asp 336， Glu 392）严重损害DNA切割和链转移 MuA的活性，为金属结合提供间接证据3 绝佳的价钱 ESEEM/ENDOR/2 H，13 C，15 N，17 O标记的高频EPR Asp和Glu将提供这些结合的直接证据。 通过测量的超精细相互作用的残留物。 再次，这些 可以在组装/活动的不同阶段进行测量， 确定活性部位结构的变化。 其他二价金属 （Co（II）、Cu（II）、VO（II））可用作所述探针的互补探针。 金属结合位点。

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).