(A) HeLa CENP-A-SNAP cells were transfected with indicated constructs, and synchronized in mitosis by an overnight treatment with Eg5 inhibitor (DMEIII). Newly synthesized CENP-A pool was quenched in mitosis, followed by 5h of release in early G1 when nascent CENP-A-SNAP was labeled with TMR (G1 specific pool).
(B) Schematic of relevant domains in centromere targeted M18BP1.
(C) GFP positive cells were selected and CENP-A TMR fluorescent intensities were determined using CRaQ, with the exception of the untransfected control where all cell were analyzed.
(D) Model summarizing the key molecular steps that are sufficient to restrict CENP-A assembly to G1 phase. CD: HJURP vertebrate conserved domain.