Yancey-Wrona J E, Camerini-Otero R D
National Institutes of Health, NIDDK, Bethesda, Maryland 20892-1810, USA.
Curr Biol. 1995 Oct 1;5(10):1149-58. doi: 10.1016/s0960-9822(95)00231-4.
The basic molecular mechanisms that govern the search for DNA homology and subsequent homologous pairing during genetic recombination are not understood. RecA is the central homologous recombination protein of Escherichia coli; because several RecA homologues have been identified in eukaryotic cells, it is likely that the mechanisms employed by RecA are conserved throughout evolution. Analysis of the kinetics of the homologous search and pairing reactions catalyzed by RecA should therefore provide insights of general relevance into the mechanisms by which macromolecules locate, and interact with, specific DNA targets.
RecA forms three-stranded synaptic complexes with a single-stranded oligonucleotide and a homologous region in duplex DNA. The kinetics of this initial pairing reaction were characterized using duplex DNA molecules of various concentrations and complexities containing a single target site, as well as various concentrations of homologous single-stranded oligonucleotides. The formation of the synaptic complex follows apparent second-order reaction kinetics with a rate proportional to the concentrations of both the homologous single-stranded oligonucleotide and the target sites within the duplex DNA. The reaction rate is independent of the complexity of duplex DNA in the reaction. We propose a kinetic scheme in which the RecA-single-stranded DNA filament interacts with duplex DNA and locates its target in a relatively fast reaction. We also suggest that complex conformational changes occur during the subsequent rate-limiting step.
We conclude that, during the formation of synaptic complexes by RecA, the search for homology is not rate-limiting, and that the iteration frequency of the search is around 10(2)-10(3) s-1. This value agrees well with what has been calculated as the minimum number for such a frequency in genome-wide searches, and limits the possible structures involved in the search for homology to those involving very soft (low energy) interactions. Furthermore, from the order of the reaction at the DNA concentrations found in eukaryotic nuclei, and the rate constant of the overall reaction, we predict that the search for homology is also not the rate-limiting step in the genome-wide searches implicated in meiosis and in gene targeting.
在基因重组过程中，控制寻找DNA同源性以及随后同源配对的基本分子机制尚不清楚。RecA是大肠杆菌中的核心同源重组蛋白；由于在真核细胞中已鉴定出几种RecA同源物，因此RecA所采用的机制很可能在整个进化过程中是保守的。因此，对RecA催化的同源搜索和配对反应动力学的分析应该能够为大分子定位特定DNA靶点并与之相互作用的机制提供具有普遍相关性的见解。
RecA与单链寡核苷酸和双链DNA中的同源区域形成三链突触复合物。使用含有单个靶位点的各种浓度和复杂度的双链DNA分子以及各种浓度的同源单链寡核苷酸来表征这种初始配对反应的动力学。突触复合物的形成遵循明显的二级反应动力学，其速率与同源单链寡核苷酸和双链DNA内靶位点的浓度成正比。反应速率与反应中双链DNA的复杂度无关。我们提出了一种动力学方案，其中RecA-单链DNA细丝与双链DNA相互作用，并在相对快速的反应中定位其靶点。我们还表明，在随后的限速步骤中会发生复杂的构象变化。
我们得出结论，在RecA形成突触复合物的过程中，寻找同源性不是限速步骤，并且搜索的迭代频率约为10² - 10³ s⁻¹。该值与在全基因组搜索中计算出的这种频率的最小值非常吻合，并将参与寻找同源性的可能结构限制为那些涉及非常弱（低能量）相互作用的结构。此外，从真核细胞核中发现的DNA浓度下的反应级数以及总反应的速率常数来看，我们预测在减数分裂和基因靶向中涉及的全基因组搜索中，寻找同源性也不是限速步骤。
