Novel symmetric and asymmetric DNA scission determinants for Streptococcus pneumoniae topoisomerase IV and gyrase are clustered at the DNA breakage site
Novel symmetric and asymmetric DNA scission determinants for Streptococcus pneumoniae topoisomerase IV and gyrase are clustered at the DNA breakage site
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DOI：
10.1074/jbc.m500156200
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发表时间：
2005-04-08
影响因子：
4.8
通讯作者：
Fisher, LM
中科院分区：
文献类型：
--
作者：
Leo, E;Gould, KA;Fisher, LM
拓扑异构酶(TOPO)IV和旋转酶是细菌IIA型DNA拓扑异构酶，对DNA复制和染色体分离至关重要，它们通过涉及共价酶-DNA“切割复合体”的瞬时双链DNA断裂发挥作用。尽管它们在机制上很重要，但对于任何细菌II型酶来说，DNA断裂决定因素都不是很清楚。我们研究了肺炎链球菌Topo IV对DNA的切割，以及双氧氟沙星和其他抗肺炎链球菌氟喹诺酮类药物稳定的旋转酶。Topo IV和旋转酶诱导不同但重叠的双链DNA断裂位点，这些双链DNA断裂位点对于七种不同的喹诺酮类药物来说基本上是相同的，并通过正负超螺旋(在强度上)得到增强。对双氧氟沙星在肺炎球菌DNA上启动的180个Topo IV和126个旋转酶位点进行了序列分析，发现了各自的共同序列：G(G/C)(A/T)A*GNNCT(T/A)N(C/A)和GN(4)G(G/C)(A/C)G*GNNCTTN(C/A)(下划线是首选碱基；不喜欢的碱基是小写的；N表示没有偏爱；星号表示DNA在-1和+1位置之间发生断裂)。这两种酶都表现出强烈的对DNA断裂位点周围对称聚集的碱基的偏好，即+1G/+4C、-4G/+8C，特别是新的-2A/+6T，但在交错的4个碱基突出处没有+2/+3的偏好。不对称元素包括-3G和几个不受欢迎的碱基。这些切割偏好是第一次针对革兰氏阳性IIA拓扑异构酶，与已报道的大肠杆菌topo IV(共识(A/G)*T/A)和旋转酶的切割偏好明显不同，后者基于较少的位点。然而，这两种肺炎球菌酶都裂解了一个大肠杆菌旋转酶位点，表明旋转酶决定因素有重叠。我们提出了一个拓扑酶IV/旋转酶切割复合体的模型，该模型考虑了独特的-2A/+6T和其他偏好。
Topoisomerase ( topo) IV and gyrase are bacterial type IIA DNA topoisomerases essential for DNA replication and chromosome segregation that act via a transient double- stranded DNA break involving a covalent enzyme-DNA " cleavage complex." Despite their mechanistic importance, the DNA breakage determinants are not understood for any bacterial type II enzyme. We investigated DNA cleavage by Streptococcus pneumoniae topo IV and gyrase stabilized by gemifloxacin and other antipneumococcal fluoroquinolones. Topo IV and gyrase induce distinct but overlapping repertoires of double- strand DNA breakage sites that were essentially identical for seven different quinolones and were augmented ( in intensity) by positive or negative supercoiling. Sequence analysis of 180 topo IV and 126 gyrase sites promoted by gemifloxacin on pneumococcal DNA revealed the respective consensus sequences: G( G/ C)( A/ T) A* GNNCT( T/ A) N( C/ A) and GN(4)G( G/ C)( A/ C) G* GNNCTTN( C/ A) ( preferred bases are underlined; disfavored bases are in small capitals; N indicates no preference; and asterisk indicatesDNAscission between - 1 and + 1 positions). Both enzymes show strong preferences for bases clustered symmetrically around the DNA scission site, i. e. + 1G/ + 4C, - 4G/ + 8C, and particularly the novel - 2A/ + 6T, but with no preference at + 2/ + 3 within the staggered 4- bp overhang. Asymmetric elements include - 3G and several unfavored bases. These cleavage preferences, the first for Gram- positive type IIA topoisomerases, differ markedly from those reported for Escherichia coli topo IV ( consensus ( A/ G)* T/ A) and gyrase, which are based on fewer sites. However, both pneumococcal enzymes cleaved an E. coli gyrase site suggesting overlap in gyrase determinants. We propose a model for the cleavage complex of topo IV/ gyrase that accommodates the unique - 2A/ + 6T and other preferences.