Assuntos
Histonas , Histonas/metabolismo , Histonas/química , Ligação Proteica , Modelos Moleculares , Domínios Proteicos , Humanos , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/genética , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 2 do Complexo de Manutenção de Minicromossomo/química , Componente 2 do Complexo de Manutenção de Minicromossomo/genéticaRESUMO
How parental histones, the carriers of epigenetic modifications, are deposited onto replicating DNA remains poorly understood. Here, we describe the eSPAN method (enrichment and sequencing of protein-associated nascent DNA) in mouse embryonic stem (ES) cells and use it to detect histone deposition onto replicating DNA strands with a relatively small number of cells. We show that DNA polymerase α (Pol α), which synthesizes short primers for DNA synthesis, binds histone H3-H4 preferentially. A Pol α mutant defective in histone binding in vitro impairs the transfer of parental H3-H4 to lagging strands in both yeast and mouse ES cells. Last, dysregulation of both coding genes and noncoding endogenous retroviruses is detected in mutant ES cells defective in parental histone transfer. Together, we report an efficient eSPAN method for analysis of DNA replication-linked processes in mouse ES cells and reveal the mechanism of Pol α in parental histone transfer.
Assuntos
DNA Polimerase I , Histonas , Animais , DNA/genética , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , Replicação do DNA , Histonas/genética , Histonas/metabolismo , Camundongos , Nucleossomos/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
How parental histone (H3-H4)2 tetramers, the primary carriers of epigenetic modifications, are transferred onto leading and lagging strands of DNA replication forks for epigenetic inheritance remains elusive. Here we show that parental (H3-H4)2 tetramers are assembled into nucleosomes onto both leading and lagging strands, with a slight preference for lagging strands. The lagging-strand preference increases markedly in budding yeast cells lacking Dpb3 and Dpb4, two subunits of the leading strand DNA polymerase, Pol ε, owing to the impairment of parental (H3-H4)2 transfer to leading strands. Dpb3-Dpb4 binds H3-H4 in vitro and participates in the inheritance of heterochromatin. These results indicate that different proteins facilitate the transfer of parental (H3-H4)2 onto leading versus lagging strands and that Dbp3-Dpb4 plays an important role in this poorly understood process.