Breier Adam M, Grossman Alan D
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Mol Microbiol. 2007 May;64(3):703-18. doi: 10.1111/j.1365-2958.2007.05690.x.
We investigated the genome-wide DNA binding of the chromosome partitioning and sporulation protein and ParB family member Spo0J in Bacillus subtilis using chromatin immunoprecipitation and DNA microarrays. We identified 10 parS loci to which Spo0J binds, two of which were unexpectedly distant (> 1 Mb) from the origin of replication. We used all 10 sites to refine the consensus sequence for parS. We found that Spo0J spreads along the DNA around each site. Binding was near maximal levels up to 1.6 kb away from parS, and significantly above background as far away as 18 kb. Deletion of soj (parA) had little or no effect on spreading. In contrast, the spo0J93 allele appeared to cause a significant decrease in spreading in vivo, without significantly affecting the DNA binding affinity in vitro. spo0J93 causes a phenotype similar to that of a spo0J null mutant and alters the region thought to be involved in interaction between Spo0J dimers. Our findings indicate that spreading is important for in vivo function of Spo0J. Gene expression in areas near parS sites was similar in wild type and a spo0J null mutant, indicating that binding and spreading of Spo0J on DNA does not normally silence transcription of nearby genes.
我们利用染色质免疫沉淀和DNA微阵列技术，研究了枯草芽孢杆菌中染色体分配和孢子形成蛋白以及ParB家族成员Spo0J在全基因组范围内的DNA结合情况。我们鉴定出10个Spo0J结合的parS位点，其中两个位点与复制起点的距离意外较远（> 1 Mb）。我们利用所有10个位点来完善parS的共有序列。我们发现Spo0J会沿着每个位点周围的DNA扩散。在距离parS达1.6 kb处，结合水平接近最高，在18 kb远处仍显著高于背景水平。缺失soj（parA）对扩散几乎没有影响。相比之下，spo0J93等位基因似乎在体内导致扩散显著减少，而在体外对DNA结合亲和力没有显著影响。spo0J93导致的表型与spo0J缺失突变体相似，并改变了被认为参与Spo0J二聚体相互作用的区域。我们的研究结果表明，扩散对Spo0J的体内功能很重要。野生型和spo0J缺失突变体中parS位点附近区域的基因表达相似，这表明Spo0J在DNA上的结合和扩散通常不会使附近基因的转录沉默。
