Abstract

CENP-A incorporation is critical for centromere specification and is mediated by its chaperone HJURP. The CENP-A-targeting-domain (CATD) of CENP-A specifically binds to HJURP and this binding itself is conserved. However, critical residues of CENP-A vary between species, and the binding interface of CENP-A–HJURP is yet to be understood. Here, we identified the critical residues for chicken CENP-A or HJURP. Interestingly, the A59Q mutation in the α1-helix of chicken CENP-A caused CENP-A mis-incorporation and subsequent cell death, while the corresponding mutation in human CENP-A (S68Q) did not. We also found that W53 of HJURP, which is a contact site of A59 in CENP-A, is also essential in chicken cells. Our comprehensive analyses revealed that the affinity of human HJURP to CATD is strong enough even with the CENP-A α1-helix mutation, but this is not the case in chicken. However, introduction of two arginine residues to the chicken HJURP αA-helix suppressed the CENP-A mis-incorporation in chicken cells expressing CENP-A^A59Q. Our data explain the mechanisms and evolution of CENP-A essentiality via CENP-A–HJURP interaction.