Mechanistic Insights Into DHX36-mediated G-quadruplex Unfolding

Helicases are molecular motor proteins present in viruses,bacteria,and eukaryotes and participate in almost all processes involving nucleic acids such as replication,recombination,repair,transcription,translation,splicing,mRNA editing.Mutations of helicases that associated in DNA repair processes may lead to a variety of human diseases such as genomic instability,immune deficiency,mental retardation,premature aging and susceptibility to cancer.DHX36 helicase(also known as RHAU,G4R1),originally isolated from HeLa cell lysates as part of a protein complex that associated with the AU-rich element(ARE)of urokinase plasminogen activator mRNA,belongs to the DEAH-box family of RNA helicases.Previous studies have shown that DHX36 are involved in different biological processes,such as hematopoiesis,cardiac development,spermatogonial differentiation and dendritic localization of neuronal precursor microRNAs.Furthermore,DHX36 in plasmacytoid dendric cells(pDCs)specifically recognizes and binds a G-rich DNA(CpG-A)via a DEAH box motif to trigger differential cytokine responses.Thus,DHX36 is also involved in anti-viral immunity.DHX36 is considered to provide the main source of G-quadruplex-resolving activity in cell lysates.A large number of in vitro experimental data have confirmed that DHX36 also has a G-quadruplex unfolding activity.DHX36 consists of a helicase core domain which responsible for ATP binding,hydrolysis and unwinding activity,an extended N-terminal and a C-terminal extension structure.Although the biological function of the C-terminal domain is still unknown,it has been shown that the N-terminal domain extends specifically to bind the G-quadruplex.Due to the lack of atomic-level structure information,the molecular mechanism of DHX36 is still unclear.In this paper,we combine biochemical with biophysical technique such as Size Exclusion Chromatography,Stop-Flow,smFRET,X-ray Crystallography and Bio-SAXS to illustrate the mechanism of DmDHX36.The main achievements are as follows:Firstly,we get the high resolution crystal structure of DmDHX36-DNA/RNA complex,thus explaining the molecular basis of how DmDHX36 recognize DNA/RNA substrates.Secondly,we obtained crystal structures of DmDHX36 in complex with different DNA substrates.By in-depth analysis of those structures and other biochemical results,we concluded that DmDHX36 had a strong preference for G-rich DNA sequence and this preference was not only in the strong binding affinity for G-rich DNA but also the strength of dsDNA unwinding and ssDNA translocation.The strong affinity for G-rich DNA sequences may play important roles in DHX36-mediated innate immunity.Thirdly,by comparing DmDHX36-RNA complex with MLE-RNA-ADPAlF4 complex and Prp43p-ADP complex,we first proposed a possible translocation model of DHX36.Finally,through the combination of biological small angle diffraction and single molecule technique and the above structures,we first propose a new G4-resolving model,a two-step unwinding model(passive-active model).In the first step,DmDHX36 recognized Gquadruplex through the N-terminal specific sequence and the positive pocket and G-quadruplex will become unstable.In the second step,G-quadruplex will be resolved completely by ATPdependent translocation.Due to G-rich preference of Dm DHX36,this step will be very strong.