Structural And Functional Analysis Of HerA-NurA Complex In Deinococcus Radiodurans

Deinococcus radiodurans(DR)is a kind of extremophiles that is resistant to external stresses such as ionizing radiation and oxidative damage.Because of its powerful ability to repair DNA damage,it is a model organism for elucidating DNA repair system.Homologous recombination(HR)is an important pathway to repair DNA double-strand breaks(DSBs)in organisms with high-fidelity.In HR repair pathway,the first step is DNA end resection that the damaged DNA ends are processed by helicase and nuclease to form 3’single-stranded DNA(ss DNA),followed by strand invasion,exchange and recombination.In bacteria,the helicase-nuclease complex,Rec BCD/Add AB,initiates HR repair by degrading both DNA ends.Given that the 3’-5’exonuclease activity is inhibited in the presence of the Chi sequence,the enzyme primarily generates 3’ss DNA by its 5’-3’nuclease activity.The Rec FOR pathway is also involved in bacterial end resection,in which the helicase Rec Q cooperates with the5’-3’exonuclease Rec J to produce a 3’ss DNA.Archaeal HerA-NurA complex is also found in bacteria,which is considered to be a minimal end-resection apparatus and could efficiently process DSB ends.However,the high resolution of HerA-NurA complex and how it mediates DNA end resection are still unclear.In this thesis,we firstly determined the structure of HerA-NurA complex consisting of helicase HerA and nuclease NurA from Deinococcus radiodurans(DR)by single-particle cryo-electron microscopy with a resolution of 3.9(?),and the main conclusions were drawn:1.The interaction interface between helicase Dr HerA and nuclease Dr NurA show how three HerA monomers interact with one NurA monomer.The interaction interface is mainly concentrated between the C-terminal region,β2-β3 loop,a long linking loop protruding from Dr NurA(termed L_P loop for brevity)and the HAS domain in Dr HerA.2.The barrel-like hexamer Dr HerA with weak helicase activity contains HAS(HerA and ATP synthase)domain,Rec A-like domain and Four-helix bundle domain.The diameter of the channel is about 25(?),which can accommodate ds DNA.In the Rec A-like domain,the exceptional long linker betweenβ10 andβ11 folds into two helices(α7-α8)and embraces the ATP binding pocket,andα7 also interacts with the C terminus.While the ATP binding pocket is solvent-exposed in Sso HerA(Sulfolobus solfataricus HerA).Therefore,Dr HerA may require greater conformational change forATP binding.3.The nuclease Dr NurA folds into a dimer with an RNase H-like domain.The extended N-terminal regions(ENRs)divide the channel into two parts and interact with each other.The ENR also interacts with the L_P loop,β1-β2 loop and the C terminus of the other subunit in dimerization,which the L_P loop and the C terminus interact with Dr HerA.Biochemical experiments found that the ENRs located above the interaction interface modulate the nuclease activity of the Dr HerA-Dr NurA complex.Taken together,we revealed a possible mechanism for the activation of NurA nuclease activity by helicase HerA in bacterial DNA end resection.When DSBs occur in vivo,Dr HerA transmits the conformational change to the ENR of Dr NurA through the interaction interface,and the nuclease activity of Dr NurA is regulated by the ENR.The thesis provides new clues and insight into the interaction mechanism between helicase and nuclease during HR repair.