Novel site-specific DNA modification in Streptomyces:: analysis of preferred intragenic modification sites present in a 5.7 kb amplified DNA sequence
Novel site-specific DNA modification in Streptomyces:: analysis of preferred intragenic modification sites present in a 5.7 kb amplified DNA sequence
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DOI：
10.1093/nar/26.14.3364
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发表时间：
1998-07-15
影响因子：
14.9
通讯作者：
Dyson, PJ
中科院分区：
文献类型：
--
作者：
Boybek, A;Ray, TD;Dyson, PJ
变铅青链霉菌和阿维链霉菌编码复制后DNA修饰的相似系统，这种修饰作用于两条链上紧密相对的鸟嘌呤上。由于它们在体外与Tris的氧化衍生物反应，导致链切割，因此可以检测到这些修饰。先前对pLJ101的首选修饰位点的分析表明，需要大量的侧翼序列，包括正向和反向重复结构，才能在体内指导在一个6bp的中心回文内的修饰。我们现在研究了一种染色体元件的首选修饰位点，即在某些S、利维丹突变体中发现的5.7kb扩增DNA序列(ADS(5.7))。与plJ101位点相比，每个ADS(5.7)位点都是基因内的，并且修改的频率降低了10倍。然而，真正的双链修饰需要相似程度的侧翼序列；缺失突变体表现出不同的修饰图谱，包括移位的双链或单链修饰。对不同修饰位点的比较表明，中心核心序列是保守的，但侧翼序列之间没有显著的相似性。在ADS(5.7)的克隆区域中检测到了增强的修饰，这表明局部DNA拓扑可能受到DNA超螺旋和侧翼序列性质的影响，从而影响修饰活性。
Streptomyces lividans and Streptomyces avermitilis encode similar systems of post-replicative DNA modification which act site-specifically on closely opposed guanines on either strand. The modifications can be detected since they react in vitro with an oxidative derivative of Tris, resulting in strand cleavage. Previous analysis of the preferred modification site of plasmid plJ101 indicated that extensive amounts of flanking sequence, including direct and inverted repeat structures, are required to direct modification in vivo within a central 6 bp palindrome, We have now examined the preferred modification sites of a chromosomal element, the 5.7 kb amplified DNA sequence (ADS(5.7)) found in certain S,lividans mutants. In contrast to the plJ101 site, each of the ADS(5.7) sites is intragenic and modified with a 10-fold reduced frequency. However, similar extents of flanking sequence are required for authentic double-strand modification; deletion mutants exhibited different modification profiles, including displaced double-stranded or single-stranded modification. Comparison of different modification sites reveals conservation of the central core sequence, but no significant similarities between flanking sequences. Enhanced modification was detected in a cloned region of the ADS(5.7), suggesting that local DNA topology, probably influenced by both DNA supercoiling and the nature of flanking sequences, can influence the modifying activity.