Mammalian DNA replication starts at distinct chromosomal sites in a 
tissue-specific pattern coordinated with transcription, but previous studies 
have not yet identified a chromatin modification that correlates with the 
initiation of DNA replication at particular genomic locations. Here we report 
that a distinct fraction of replication initiation sites in the human genome are 
associated with a high frequency of dimethylation of histone H3 lysine K79 
(H3K79Me2). H3K79Me2-containing chromatin exhibited the highest genome-wide 
enrichment for replication initiation events observed for any chromatin 
modification examined thus far (23.39% of H3K79Me2 peaks were detected in 
regions adjacent to replication initiation events). The association of H3K79Me2 
with replication initiation sites was independent and not synergistic with other 
chromatin modifications. H3K79 dimethylation exhibited wider distribution on 
chromatin during S-phase, but only regions with H3K79 methylation in G1 and G2 
were enriched in replication initiation events. H3K79 was dimethylated in a 
region containing a functional replicator (a DNA sequence capable of initiating 
DNA replication), but the methylation was not evident in a mutant replicator 
that could not initiate replication. Depletion of DOT1L, the sole enzyme 
responsible for H3K79 methylation, triggered limited genomic over-replication 
although most cells could continue to proliferate and replicate DNA in the 
absence of methylated H3K79. Thus, prevention of H3K79 methylation might affect 
regulatory processes that modulate the order and timing of DNA replication. 
These data are consistent with the hypothesis that dimethylated H3K79 associates 
with some replication origins and marks replicated chromatin during S-phase to 
prevent re-replication and preserve genomic stability.