After DNA replication, chromosomal processes including DNA repair and
transcription take place in the context of sister chromatids. While cell cycle regulation
can guide these processes globally, mechanisms to distinguish pre- and postreplicative
states locally remain unknown. Here, we reveal that new histones
incorporated during DNA replication provide a signature of post-replicative
chromatin, read by the TONSL–MMS22L1-4 homologous recombination (HR)
complex. We identify the TONSL Ankyrin Repeat Domain (ARD) as a reader of
histone H4 tails unmethylated at K20 (H4K20me0), which are specific to new
histones incorporated during DNA replication and mark post-replicative chromatin
until G2/M. Accordingly, TONSL–MMS22L binds new histones H3–H4 both prior to
and after incorporation into nucleosomes, remaining on replicated chromatin until
late G2/M. H4K20me0 recognition is required for TONSL–MMS22L binding to
chromatin and accumulation at challenged replication forks and DNA lesions.
Consequently, TONSL ARD mutants are toxic, compromising genome stability, cell
viability and resistance to replication stress. Together, this reveals a histone reader
based mechanism to recognize the post-replicative state, offering a new approach
and opportunity to understand DNA repair with potential for targeted cancer therapy.