A zinc site in the C-terminal domain of RAG1 is essential for DNA cleavage activity.
The recombination-activating protein, RAG1, a key component of the V(D)J recombinase, binds multiple Zn(2+) ions in its catalytically required core region. However, the role of zinc in the DNA cleavage activity of RAG1 is not well resolved. To address this issue, we determined the stoichiometry of Zn(2+) ions bound to the catalytically active core region of RAG1 under various conditions. Using metal quantitation methods, we determined that core RAG1 can bind up to four Zn(2+) ions. Stripping the full complement of bound Zn(2+) ions to produce apoprotein abrogated DNA cleavage activity. Moreover, even partial removal of zinc-binding equivalents resulted in a significant diminishment of DNA cleavage activity, as compared to holo-Zn(2+) core RAG1. Mutants of the intact core RAG1 and the isolated core RAG1 domains were studied to identify the location of zinc-binding sites. Significantly, the C-terminal domain in core RAG1 binds at least two Zn(2+) ions, with one zinc-binding site containing C902 and C907 as ligands (termed the CC zinc site) and H937 and H942 coordinating a Zn(2+) ion in a separate site (HH zinc site). The latter zinc-binding site is essential for DNA cleavage activity, given that the H937A and H942A mutants were defective in both in vitro DNA cleavage assays and cellular recombination assays. Furthermore, as mutation of the active-site residue E962 reduces Zn(2+) coordination, we propose that the HH zinc site is located in close proximity to the DDE active site. Overall, these results demonstrate that Zn(2+) serves an important auxiliary role for RAG1 DNA cleavage activity. Furthermore, we propose that one of the zinc-binding sites is linked to the active site of core RAG1 directly or indirectly by E962.
Gwyn
Lori M
LM
Department of Biochemistry and Molecular Biology, The University of Oklahoma Health Sciences Center, Oklahoma City, 73190, USA.
Peak
Mandy M
MM
De
Pallabi
P
Rahman
Negar S
NS
Rodgers
Karla K
KK
D001665:Binding Sites; D055162:Biocatalysis; D002850:Chromatography, Gel; D017124:Conserved Sequence; D003545:Cysteine / Q000378:metabolism; D053837:DNA Cleavage; D006639:Histidine / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000378:metabolism*; D007477:Ions; D008024:Ligands; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic / Q000235:genetics; D015032:Zinc / Q000378:metabolism*
D018398:Homeodomain Proteins; D007477:Ions; D008024:Ligands; C064658:RAG-1 protein; D006639:Histidine; D015032:Zinc; D003545:Cysteine