Structural Features Of RecA/Rad51 Recombinase Family And Mechanistic Insights Into Rad51 Inhibitors Through Molecular Dynamics Simulation

Homologous Recombination(HR)is an important and evolutionary conserved DNA repair pathway that plays a key role in double-strand break repair,stalled replication fork repair and meiosis,and other processes.RecA/Rad51 family DNA recombinases are the core proteins in the HR process,and they are responsible for facilitating the exchange of DNA strands during the repair process: Recombinases bind to single stranded DNA(ss DNA)at the site of DNA double strand breaks or DNA damage,forming a conserved recombinase-ss DNA helical structure called the presynaptic filament in the presence of ATP;The presynaptic filament then binds to double stranded DNA(ds DNA)to form a synaptic filament;When matched to a homologous ds DNA,the complementary strand of the donor ds DNA pairs with the original ss DNA to form a postsynaptic filament,which finally realizes the chain exchange process.Although existing studies have confirmed the functional and structural conservation of this family of proteins during HR,there is little scientific knowledge of the dynamic mechanisms of molecular structure during their biological functions.Meanwhile,human Rad51 is an important target for the treatment of a variety of cancers,but the mechanism of action of existing Rad51 inhibitors has not been elucidated,and there is also a lack of efficient and large-scale screening for inhibitor development.Using molecular dynamics simulation technology,we can deeply analyse the dynamic structure,substrate binding,and mechanism of the RecA/Rad51 family DNA recombinases at the molecular level,and then deeply understand their biological functions.Based on these features,molecular dynamics simulation of Rad51 inhibitor systems and virtual screening of compounds can help to elucidate the inhibition mechanism of Rad51 inhibitors and obtain potential therapeutic drugs targeting Rad51.The specific research content and results of this paper are as follows:1.Firstly,five representative RecA/Rad51 recombinase family members covering all major organisms(prokaryotes,eukaryotes,archaea and viruses)were selected to explore by a variety of techniques,including whole-atom molecular dynamics simulation,perturbation response scanning,and protein structure network analysis.To explore the molecular structure and dynamic behavior behind the conserved biological significance but low sequence consistency of each recombinant enzyme presynaptic filament.The results showed that the conserved molecular dynamics properties of RecA/Rad51 recombinase family members are manifested in:(1)In terms of co-motion,the core region of monomer I of each recombinase is negatively correlated with the motion of the core region of monomer II,and positively correlated with the motion of the N-terminal or C-terminal of monomer II;the P-loop of the ATP-binding region is positively correlated with that of the DNA-binding region in the L2 of monomer I,and negatively correlated with that of monomer II.The conserved dynamic inter-correlation between monomers reflects that the recombinant enzyme aggregates on ss DNA after binding ATP to form a conserved nucleoprotein filament complex structure.(2)Regarding the transmission of allosteric signals,the sensitive residues of each recombinase are conserved in the N-terminal or C-terminal region as well as in the L1 and L2 regions,which are responsible for sensing the signals from DNA,cofactors,and protein monomers,and the effective residues are conserved in the core catalytic region on the 8β-fold,which are responsible for transmitting allosteric signals.(3)As for hub residues,the key hub residues Leu47,Glu63 and Cys90 in RecA protein are also commonly found in other proteins,and are considered to be the key positions for maintaining the structural stability of proteins and transmitting ATP-induced conformational changes.The differences of RecA/Rad51 recombinase family members are manifested in the following aspects: The communication routes between ATPbinding region and DNA-binding region are different.The communication routes between RecA and UvsX are dense,while the communication routes between Rad A,Rad51 and DMC1 are sparse.The reason may be that ATP binding significantly increases the ss DNA affinity of RecA and UvsX,while it has no significant effect on the ss DNA affinity of Rad A,Rad51,and DMC1.The above findings provide a molecular level understanding of the functional conservation and uniqueness of the RecA/Rad51 family proteins,a theoretical basis for further exploration of the structural features and binding properties of RecA and UvsX in their binding state to ds DNA,and a necessary reference for the subsequent study of the mechanism of action of Rad51 inhibitors.2.UvsX proteins have efficient DNA strand exchange and recombination capabilities,making it an ideal choice for studying DNA repair mechanisms as well as in vitro applications.However,studies on the molecular level of UvsX are scarce.In order to continue to explore the structural features and binding characteristics of UvsX,a dimer model of UvsX binding ATP and ds DNA was constructed,and all-atom molecular dynamics simulations were carried out at ambient temperatures of 298 K and310 K,respectively,with RecA protein as a reference.The results showed that at 310 K,the dynamic cross-correlation between monomers in the RecA and UvsX protein systems would be enhanced,and the proteins could more easily reach a stable conformation,and at the same time,the binding ability of RecA and UvsX to ATP was stronger than that at 298 K.These phenomena were consistent with the enhanced coordination between the protein monomers,more stable protein conformation,and stronger binding ability of ATP of RecA and UvsX at the optimal temperature.However,the binding free energy of RecA to ds DNA fluctuates dramatically at 310 K,whereas the UvsX protein binds well to ds DNA at 310 K.This study also continued to explore the communication routes between the two binding sites of ATP and ds DNA in the current state,which showed significant differences compared with the communication route in 1.The allosteric key residues changed from Gln194 in RecA to Ile195 and His195 in UvsX to Thr196,indicating that the protein conformation changed in this state.Allosteric signals may thus be converted to RecA to release ds DNA,whereas UvsX does not catalyze ATP hydrolysis.The above research results have analyzed the properties and differences of UvsX and RecA from the molecular level,which is helpful to have a deeper understanding of UvsX and RecA recombinant enzymes and the development and application of these recombinases.The results of 1 and 2 provide strong guidance and inspiration for further understanding of the functional mechanism of RecA/Rad51 protein,drug design and bioengineering applications.3.Human Rad51 protein is an important target for cancer therapy,and the molecular mechanism of its presynaptic filament is elucidated in 1.In this study,three inhibitors targeting Rad51 monomer interface,B02,IBR2 and RI-1,were selected.Rad51-ATP-ss DNA,Rad51-ATP-ss DNA-B02,Rad51-ATP-ss DNA-IBR2,Rad51-ATP-ss DNA-RI-1 systems were simulated and enhanced with whole-atom molecular sampling.The mechanism of Rad51 inhibitor on Rad51 protein was explored at molecular level.The results showed that inhibitor binding can lead to the deterioration of protein skeleton flexibility,residue contact and protein conformation changes.These conditions are particularly significant in the N-terminal of monomer I,where the Nterminal of the inhibitor system is closer to the core catalytic region and forms a stable contact with the core catalytic region and a new communication network.At the same time,the effect of inhibitors also radiates the whole protein,which is reflected in the significantly weakened dynamic cross-correlation of Rad51 protein and the significantly reduced network centrality of the protein core region.Therefore,inhibitor binding has brought serious constraints on the cooperation ability and allosteric communication ability among protein residues,which may also contribute to the attenuation of the function of Rad51.The above study used molecular dynamics simulation to reveal the specific effects of Rad51 inhibitor binding to Rad51 at the molecular level,which is of great significance for the analysis of the inhibition mechanism and the development of small molecule inhibitors.4.Based on the above research,a combination of virtual screening and molecular dynamics simulation was used to further develop potential Rad51 inhibitors from the Chem BL database of small molecule compounds.It was shown that the key residues of Rad51 protein binding compounds are I: Arg193,I: Phe195,II: Arg95,II: Arg96,II:Phe92,II: Ile99,which mainly correspond to three types of residues: Arg,which is positively charged and possesses a hydrogen-bonding donor,Phe,which is aromatic,and Ile,which is hydrophobic.Therefore,most of the compounds screened structurally have aromatic rings,nitrogen-containing heterocycles,and hydroxyl or alkane groups.These results reveal the properties of Rad51 target binding site,as well as the structural features of the compounds with strong binding,providing important clues for the molecular design of Rad51 inhibitors.Among the screened compounds,Cmp-4 and Cmp-9 exhibited molecular dynamics properties very similar to those of the Rad51 inhibitors B02 and IBR2,which similarly adjusted the conformation of the N-terminal region to be closer to the core region and further attenuated the dynamic correlation and network centrality of the proteins.These results indicate that Cmp-4 and Cmp-9 have the characteristics of Rad51 inhibitors at the molecular level,and could be applied,modified and developed as potential inhibitors of Rad51.