A 330 kb CENP-A binding domain and altered replication timing at a human neocentromere

Centromere protein A (CENP-A) is an essential centromere-specific histone H 3 homologue, Using combined chromatin immunoprecipitation and DNA array ana lysis, we have defined a 330 kb CENP-A binding domain of a 10q25.3 neocentr omere found on the human marker chromosome mardel (10), This domain is situ ated adjacent to the 80 kb region identified previously as the neocentromer e site through lower-resolution immunofluorescence/FISH analysis of metapha se chromosomes. The 330 kb CENP-A binding domain shows a depletion of histo ne H3, providing evidence for the replacement of histone H3 by CENP-A withi n centromere-specific nucleosomes. The DNA within this domain has a high AT -content comparable to that of alpha -satellite, a high prevalence of LINEs and tandem repeats, and fewer SINEs and potential genes than the surroundi ng region. FISH analysis indicates that the normal 10q25.3 genomic region r eplicates around mid-S phase, Neoceutromere formation is accompanied by a r eplication time lag around but not within the CENP-A binding region, with t his lag being significantly more prominent to one side. The availability of fully sequenced genomic markers makes human neocentromeres a powerful mode l for dissecting the functional domains of complex higher eukaryotic centro meres.