Hormeño Silvia, Ramos Cristina, Mendia-Garcia Javier, Aicart-Ramos Clara, Ayora Silvia, Moreno-Herrero Fernando
Department of Macromolecular Structures, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, 28049, Madrid, Spain.
Department of Microbial Biotechnology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, 28049, Madrid, Spain.
Nucleic Acids Res. 2025 Jun 6;53(11). doi: 10.1093/nar/gkaf459.
RecD2 is a superfamily 1B helicase involved in DNA replication and repair, modulating replication restart, fork progression, and RecA recombinase activity. In this work, we have characterized the functions of Bacillus subtilis RecD2 using biochemical and single-molecule approaches. ATPγS binding and low MgCl2 concentrations enhance DNA association, with a preference for forked structures and unstructured DNA longer than 30 nucleotides. RecD2 binds to end-less single-stranded DNA stretched at 8-20 pN and translocates through ATP hydrolysis over long distances (>20 kb) with 5'-3' polarity at high rates. RecD2 shows limited unwinding activity on fork structures, strongly dependent on protein concentration and duplex length, reflecting low processivity. However, processivity improves significantly when force is applied to the translocating strand or unwound DNA ends, enabling the unwinding of thousands of base pairs at rates up to 160 bp/s. Single-molecule assays reveal frequent strand switching on fork substrates, resulting in a non-productive cycle of unwinding and rewinding, likely mediated by the N-terminal domain. This behavior explains the low helicase activity observed in bulk assays. We propose that regulation of strand-switching activity may be relevant for RecD2's in vivo function.
RecD2是一种参与DNA复制和修复的超家族1B解旋酶，可调节复制重启、叉状进程和RecA重组酶活性。在这项工作中，我们使用生化和单分子方法对枯草芽孢杆菌RecD2的功能进行了表征。ATPγS结合和低浓度MgCl2可增强DNA结合，对叉状结构和长度超过30个核苷酸的无结构DNA具有偏好性。RecD2结合在8-20 pN张力下拉伸的无端单链DNA，并通过ATP水解以5'-3'极性高速长距离（>20 kb）转运。RecD2对叉状结构显示出有限的解旋活性，强烈依赖于蛋白质浓度和双链长度，反映出低持续性。然而，当对转运链或解旋的DNA末端施加力时，持续性会显著提高，能够以高达160 bp/s的速度解旋数千个碱基对。单分子分析揭示了在叉状底物上频繁的链切换，导致解旋和重新缠绕的非生产性循环，这可能由N端结构域介导。这种行为解释了在大量分析中观察到的低保旋酶活性。我们认为，链切换活性的调节可能与RecD2的体内功能相关。
