Ring-shaped Rad51 paralog protein complexes bind Holliday junctions and replication forks as visualized by electron microscopy.
In mammals, there are five Rad51 paralogs that form two distinct complexes in vivo. One complex is composed of Rad51B-Rad51C-Rad51D-Xrcc2 (BCDX2) and the other Rad51C-Xrcc3 (CX3). We co-expressed and purified human BCDX2 and CX3 protein complexes from insect cells and investigated their binding preferences and structure using transmission electron microscopy (TEM). We visualized the binding of BCDX2 and CX3 to DNA templates containing replication forks and Holliday junctions, intermediates observed during DNA replication and recombination, respectively. We show that both complexes bind with exceptionally high specificity to the DNA junctions with little binding observed elsewhere on the DNAs. Further analysis of the structure of free or DNA-bound BCDX2 and CX3 complexes revealed a multimeric ring structure whose subunits are arranged into a flat disc around a central channel. This work provides the first EM visualization of BCDX2 and CX3 binding to Holliday junctions and forked DNAs and suggests the complexes form ring-shaped structures.
Compton
Sarah A
SA
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA. compton@med.unc.edu
Ozgür
Sezgin
S
Griffith
Jack D
JD
D000818:Animals; D004261:DNA Replication / Q000502:physiology; D045566:DNA, Cruciform / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D046529:Microscopy, Electron, Transmission; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera
D045566:DNA, Cruciform; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; C448305:RAD51C protein, human; D011994:Recombinant Proteins; C110901:X-ray repair cross complementing protein 3; C113187:XRCC2 protein, human