DNA-Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.
DNA-Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK
Nucleic Acids Res. 2014 May;42(9):5633-43. doi: 10.1093/nar/gku188. Epub 2014 Mar 15.
In all domains of life, the resection of double-stranded DNA breaks to form long 3'-ssDNA overhangs in preparation for recombinational repair is catalyzed by the coordinated activities of DNA helicases and nucleases. In bacterial cells, this resection reaction is modulated by the recombination hotspot sequence Chi. The Chi sequence is recognized in cis by translocating helicase-nuclease complexes such as the Bacillus subtilis AddAB complex. Binding of Chi to AddAB results in the attenuation of nuclease activity on the 3'-terminated strand, thereby promoting recombination. In this work, we used stopped-flow methods to monitor the coupling of adenosine triphosphate (ATP) hydrolysis and DNA translocation and how this is affected by Chi recognition. We show that in the absence of Chi sequences, AddAB translocates processively on DNA at ∼2000 bp s(-1) and hydrolyses approximately 1 ATP molecule per base pair travelled. The recognition of recombination hotspots results in a sustained decrease in the translocation rate which is accompanied by a decrease in the ATP hydrolysis rate, such that the coupling between these activities and the net efficiency of DNA translocation is largely unchanged by Chi.
在所有生命领域中，双链 DNA 断裂的切除以形成用于重组修复的长 3'-ssDNA 突出端，这是由 DNA 解旋酶和核酸酶的协调活动催化的。在细菌细胞中，这种切除反应受重组热点序列 Chi 的调节。Chi 序列通过迁移解旋酶-核酸酶复合物（如枯草芽孢杆菌 AddAB 复合物）在顺式中被识别。Chi 与 AddAB 的结合导致 3'-端终止链上核酸酶活性的衰减，从而促进重组。在这项工作中，我们使用停流方法来监测三磷酸腺苷 (ATP) 水解和 DNA 易位的偶联，以及 Chi 识别如何影响这种偶联。我们表明，在不存在 Chi 序列的情况下，AddAB 以约 2000 bp s(-1) 的速度在 DNA 上连续易位，并且每移动一个碱基对水解约 1 个 ATP 分子。重组热点的识别导致易位速率持续下降，同时水解速率下降，使得这些活性之间的偶联和 DNA 易位的净效率基本不受 Chi 的影响。
