Structural Analysis Of Pifl Helicas Gomplexed With DNA/RNA

Pifl is a member of the5’-3’ATP dependent helicase family protein which includes ScPifl, mPifl, hPifl, SpPfhl. It can bind and unwind various DNA/RNA substrates, such as stranded DNA, G4structure, DNA-RNA hybrid and thus is involved in the regulation of chromosome stability, telomerase function, genomic replication, Okazaki fragment maturation and ribosomal DNA replication etc. To obtain the structure of Pifl complexed with DNA/RNA, I cloned and expressed Pifl family members from mouse, human, S. cerevisiae and S. pombe in E.coli. After large-scale expression and purification, mPifl1-200, hPifl200-641, SpPfhl305-805, ScPfhl305-790, ScPIFl40-859and ScPIFl40-859-K264A were obtained. Among the6proteins, hPifl200-641showed high purity, stability and homogeneity. After testing the binding affinities between the hPifl200-641and DNA/RNA substrate, I chose DNA-H and RNAb+DNAc as the best substrates of hPifl200-641which showed strongest binding affinities. After crystallization and optimization of hPifl200-641alone and its complex with DNA/RNA, rod-like crystals were obtained. Crystals of hPifl200-641diffract to3.5A resolution and belong space group of P321, with unit-cell parameters a=b=204.4A, c=77.93A, a=90°, β=900,γ=120°. The result reported here provides a basis for further structural and functional studies of Pifl protein and Pifl-DNA/RNA complex.