To restore chromatin on new DNA during replication, recycling of histones evicted ahead of the fork is combined with new histone deposition. The Asf1 histone chaperone, which buffers excess histones under stress, is a key player in this process. Yet how histones handled by human Asf1 are modified remains unclear. Here we identify marks on histones H3-H4 bound to Asf1 and changes induced upon replication stress. In S phase, distinct cytosolic and nuclear Asf1b complexes show ubiquitous H4K5K12diAc and heterogeneous H3 marks, including K9me1, K14ac, K18ac, and K56ac. Upon acute replication arrest, the predeposition mark H3K9me1 and modifications typical of chromatin accumulate in Asf1 complexes. In parallel, ssDNA is generated at replication sites, consistent with evicted histones being trapped with Asf1. During recovery, histones stored with Asf1 are rapidly used as replication resumes. This shows that replication stress interferes with predeposition marking and histone recycling with potential impact on epigenetic stability.

Budding yeast Mms22 is required for homologous recombination (HR)-mediated repair of stalled or broken DNA replication forks. Here we identify a human Mms22-like protein (MMS22L) and an MMS22L-interacting protein, NFκBIL2/TONSL. Depletion of MMS22L or TONSL from human cells causes a high level of double-strand breaks (DSBs) during DNA replication. Both proteins accumulate at stressed replication forks, and depletion of MMS22L or TONSL from cells causes hypersensitivity to agents that cause S phase-associated DSBs, such as topoisomerase (TOP) inhibitors. In this light, MMS22L and TONSL are required for the HR-mediated repair of replication fork-associated DSBs. In cells depleted of either protein, DSBs induced by the TOP1 inhibitor camptothecin are resected normally, but the loading of the RAD51 recombinase is defective. Therefore, MMS22L and TONSL are required for the maintenance of genome stability when unscheduled DSBs occur in the vicinity of DNA replication forks.

During DNA replication, nucleosomes are rapidly assembled on newly synthesized DNA to restore chromatin organization. Asf1, a key histone H3-H4 chaperone required for this process, is phosphorylated by Tousled-Like Kinases (TLKs). Here, we identify TLK phosphorylation sites by mass spectrometry and dissect how phosphorylation impacts on human Asf1 function. The divergent C-terminal tail of Asf1a is phosphorylated at several sites and this is required for timely progression through S phase. Consistent with this, biochemical analysis of wild-type and phosphomimetic Asf1a shows that phosphorylation enhances binding to histones and the downstream chaperones CAF-1 and HIRA. Moreover, we find that TLK phosphorylation of Asf1a is induced in cells experiencing deficiency of new histones and that TLK interaction with Asf1a involves its histone-binding pocket. We thus propose that TLK signaling promotes histone supply in S phase by targeting histone-free Asf1 and stimulating its ability to shuttle histones to sites of chromatin assembly.

Efficient supply of new histones during DNA replication is critical to restore chromatin organization and maintain genome function. The histone chaperone anti‐silencing function 1 (Asf1) serves a key function in providing H3.1‐H4 to CAF‐1 for replication‐coupled nucleosome assembly. We identify Codanin‐1 as a novel interaction partner of Asf1 regulating S‐phase histone supply. Mutations in Codanin‐1 can cause congenital dyserythropoietic anaemia type I (CDAI), characterized by chromatin abnormalities in bone marrow erythroblasts. Codanin‐1 is part of a cytosolic Asf1–H3.1‐H4–Importin‐4 complex and binds directly to Asf1 via a conserved B‐domain, implying a mutually exclusive interaction with the chaperones CAF‐1 and HIRA. Codanin‐1 depletion accelerates the rate of DNA replication and increases the level of chromatin‐bound Asf1, suggesting that Codanin‐1 guards a limiting step in chromatin replication. Consistently, ectopic Codanin‐1 expression arrests S‐phase progression by sequestering Asf1 in the cytoplasm, blocking histone delivery. We propose that Codanin‐1 acts as a negative regulator of Asf1 function in chromatin assembly. This function is compromised by two CDAI mutations that impair complex formation with Asf1, providing insight into the molecular basis for CDAI disease.

The authors have noticed that the sequence provided in Materials and methods for siRNA#1 targeting CDAN1 is incorrect, the correct sequence is 5 0 -GGAAAGAAUUCGUACCGUU-3 0 (sense strand). The conclusions drawn from the paper remain unchanged.The authors apologize for any inconvenience caused.