Structural And Functional Study Of Chloroplast Resolvase MOC1

Genetic recombination plays an important role in the genetic diversity and genome stability.As a key DNA intermediate in the recombination process,the Holliday junction needs to be timely eliminated in the late stage of recombination.Otherwise,the two covalently linked ds DNA cannot be separated normally,which will cause severe consequences including chromosomal aberration,nuclear fusion,cell death,and cancers.Resolvase,a type of recombinases widely existing in various organisms,resolves Holliday junctions.Thus far,several resolvases have been identified,including T4 endonuclease VII,T7 endonuclease I,bacterial Ruv C,archaeal Hjc/Hje,eukaryotic GEN1,mitochondrial CCE1/Ydc2 and chloroplast MOC1.Some of the resolvases can resolve Holliday junction in a sequence-dependent manner,as exemplified by Ruv C（5’-A/TTT↓G/C-3’）,Cce1（5’-ACT↓A-3’）,Ydc2（5’-C/TT↓-3’）and MOC1（5’-C↓C-3’）.Among them,the chloroplast resolvase MOC1 was the latest one to be identified,whose discovery bridges the gap in chloroplast Holliday junction resolution.The chloroplast resolvase MOC1 is essential for the development of plant chloroplasts.Malfunction of MOC1 leads to the unequal segregation of chloroplast nucleoids after replication,which will in turn lead to impaired chloroplast biogenesis and ultimately affect crop production.However,there is still very limited understanding of the molecular mechanisms underlying the sequence-dependent resolution of Holliday junction mediated by chloroplast resolvase MOC1.Utilizing integrated biochemistry and structural biology methods,this study focused on the chloroplast resolvase MOC1 and systematically analyzed the structural basis of the specific binding of the MOC1 to Holliday junction.The outcome of this study revealed the molecular mechanism of MOC1’s sequence-dependent resolution activity.The main results are listed below:1.Solved the high-resolution crystal structure of MOC1 and MOC1 in complex with Holliday junctionTo reveal the Holliday junction resolution mechanism of MOC1,we first recombinantly expressed MOC1 homologous from six species using E.coli protein expression system and verified their activity through in vitro biochemical experiments.After numerous optimization trials in proteins engineering and crystallization conditions,high-resolution protein crystal diffraction data were obtained and the crystal phase was determined by single-wavelength anomalous scattering.Finally,we determined the dimeric structures of Zm MOC1 and NtMOC1 at 2.5?and 2.0?resolutions,respectively.We also solved the complex structures of wild-type NtMOC1with a Holliday junction variant（2.5?）and an inactive NtMOC1^mut with a Holliday junction（2.55?）.2.Revealed the structural basis of the specific binding of MOC1 dimer to the Holliday junctionBased on the monomer and complex structures of MOC1,we analyzed the MOC1dimer interface and the MOC1-Holliday junction interface.Through the mutational analyses of the residues at the interface followed by biochemical experiments,it was confirmed that the MOC1 functions in the form of a dimer.The dimeric MOC1 binds to the Holliday junction through the positively charged groove on the surface,which fixes the Holliday junction into an open planar structure for subsequent DNA cleavage.3.Elucidated the molecular mechanism of MOC1 cytosine-dependent Holliday junction resolutionAfter analyzing the complex structure,we found a base recognition loop（BR loop）protruding into the center of Holliday junction.This BR loop can disrupt the C-G base pair at the center of Holliday junction,and the D138 residue in it recognizes the unpaired cytosine base.Thus,this BR loop provides the molecular basis for the sequence-dependent resolution of Holliday junction.In summary,this study determined the structures of chloroplast resolvase MOC1and MOC1 in complex with a Holliday junction.Combined with biochemical analyses,this study revealed the molecular mechanism underlying MOC1’s sequence-dependent Holliday junction resolution activity and in the meantime provides a reference for understanding the sequence-dependent resolution of other resolvases.