Enzymatic Properties And Structure Investigations Of CaPif1 Helicase

Helicase is a kind of motor protein,which is widely distributed in prokaryotes,eukaryotes,and viruses.It plays an important role in the process of nucleic acid metabolism.Its deletion or mutation can lead to many genetic diseases.Pif1 helicase is an important branch of the helicase family.Experiments have shown that Pif1 helicase is involved in DNA replication and repair,gene transcription,maintenance of mitochondrial DNA stability,inhibition of telomerase activity and promotion of Okazaki fragment maturation.Although some of the commonalities and characteristics of Pif1 helicase have been found in previous studies,there are still many shortcomings in the current situation.Firstly,in the conservation analysis of the amino acid sequence of Pif1 helicase,it is found that the conserved amino acid sequence of Pif1 helicase is concentrated in the helicase core region,the NTD and CTD contain a large number of non-conserved sequences,and the role of non-conserved sequences is still not very clear;Secondly,due to the technical limitations,most of the previous studies on helicase were limited to the cellular level,but did not go deep into the molecular level and the atomic level,resulting in insufficient understanding of many issues.In view of this,this research project uses Candida albicans'Pif1 helicase as experimental material,combined with stopped-flow FRET technology,sm FRET technology,fluorescence polarization technology,gel filtration chromatography technology,dynamic laser scattering technology,small angle X-ray scattering technology,and the crystallographic experiments of biological macromolecules,has done a comprehensive and detailed study on its structure and function.The main conclusions obtained as follows:(1)The unwinding direction of CaPif1 protein is 5?-3?,and the protein has no activity to anneal double-stranded DNA,the global kinetic step size of unwinding the double-stranded chain is 1 bp,the unwinding rate is about 110 bp/s,and the continuous unwinding distance is about 11 bp;CaPif1 Protein tends to unwind substrates with 5?single-stranded DNA when facing a variety of substrates,and the unwinding rate is higher than that of Bs Pif1 and h Pif1 helicase when unwinding the forked substrate,which indicates that it may play an important role in the process of DNA replication;CaPif1 protein can also efficiently unwind G4 structure and DNA-RNA hybridization chain,and the unwinding rate is higher than the rate of unwinding double-strand DNA,indicating that it plays an important role in inhibiting telomerase activity and regulating transcription.(2)The sm FRET experiment found that there are two kinds of unwinding states when the low concentration of CaPif1 protein unwinds the double-stranded substrate,one is direct unwinding,the other is the unwinding of retention,and the unwinding of the retention is about the 10%of total reaction,this paused unwinding may be related to the allosteric structure of the protein;When the low concentration of CaPif1 protein unwinds the substrate with the G4 structure,the G4 structure is not opened at one time,but is performed step by step.When unwinding the G4 structure,the G4 structure is first unwound into the G3 or G2 structure,but since the G4 structure is relatively stable,the G3or G2 structure is likely to be annealed to the G4 structure again,resulting in repeated unwinding.(3)The alone NTD of CaPif1 protein has no affinity for single-stranded DNA,but it can promote the recognition and binding of the protein to the substrate,and improve the protein's ability to unwind the nucleic acid substrates;CaPif1^91-906 protein is a dimer,which controls the dimerized peptide of CaPif1^91-906 protein is located between amino acids 91 to138.Unlike Sc Pif1 protein,the dimerization of CaPif1 protein does not depend on the induction of DNA strands,and its dimerization site may be related to the recognition between proteins;The CTD of CaPif1 protein has an impact on the binding activity and stability of the protein.If the CTD is completely removed,the stability of the protein will decrease,and the ability to bind the single-stranded DNA will also decrease.(4)Structural biology studies have shown that the Q-motif(Q373)exists in CaPif1protein,which can specifically recognize and bind ATP;The glutamate at position 473 and the glutamine at position 512 of CaPif1 protein are related to the hydrolysis of ATP,and their mutation will result in a loss of unwinding activity of the CaPif1 protein;Many amino acids of CaPif1 protein are involved in the binding of nucleic acid strand,and the histidines at positions 434 and 818 can hinder the binding of CaPif1 protein to single-stranded RNA.When the CaPif1 protein unwinds the hybrid strand of DNA and RNA,it can specifically recognize and bind the DNA strand,while removing the RNA strand;After the CaPif1protein binds to single-stranded DNA,the twisting angle of the 2B domain is different from other Pif1 proteins whose structures have been resolved.The angle that the 2B domain of CaPif1 protein can be twisted is smaller than that of Bs Pif1 protein and Sc Pif1 protein.This feature makes it difficult for CaPif1 protein to recognize and bind nucleic acid substrates with a larger spatial structure;There is a pair of disulfide bonds between the 1A domain and the 2B domain of CaPif1 protein.This pair of disulfide bonds will lock the 1A domain and the 2B domain,which restricts the movement of the 2B domain and ultimately affects the unwinding of the protein;The 2C domain of CaPif1 protein plays an important role in protein folding and maintaining protein stability.G4 DNA is widely present in the genome of organisms and involved in DNA replication,gene transcription,and telomere regulation.There has been a lot of evidence that Pif1 helicase is related to G4 structure,but so far,people have not obtained the crystal structure of the complex of Pif1 protein and G4 DNA.This subject has done a preliminary study on the structure of the complex of CaPif1 protein and G4 DNA.The main conclusions obtained are as follows:The structural model of the complex of CaPif1 protein and G4 DNA with a 5?single-stranded tail simulated by Py MOL software shows that the single-stranded DNA is in the positively charged groove formed by the two Rec A-like domains of the CaPif1 protein,the G4 structure located outside the 1A domain and 2B domain,and the G4 structure mainly interacts with the 1A domain with denser positive charges on the surface;CaPif1 protein can form a dimer complex with G4 DNA with single-stranded tails at both ends,small angle X-ray scattering experiments found that two CaPif1 proteins are distributed on both sides of the G4 structure in a centrosymmetric manner.The dimer complex is a dense sphere.Through preliminary crystallization experiments,the crystals of the complex can be obtained and the diffraction resolution of the crystal is up to 5?.The above are the main enzymatic and structural characteristics of the CaPif1 protein discovered in this study.From this,we can find some properties that are similar to other helicases,and we can also find some unique properties of the protein.These results can provide some reference and basis for people to understand the structure and function of Pif1 protein more comprehensively.