Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that persists as a multicopy episome in proliferating host cells. Episome maintenance is strictly dependent on EBNA1, a sequence-specific DNA-binding protein with no known enzymatic activities. Here, we show that EBNA1 forms a cell cycle-dependent DNA crosslink with the EBV origin of plasmid replication oriP. EBNA1 tyrosine 518 (Y518) is essential for crosslinking to oriP and functionally required for episome maintenance and generation of EBV-transformed lymphoblastoid cell lines (LCLs). Mechanistically, Y518 is required for replication fork termination at oriP in vivo and for formation of SDS-resistant complexes in vitro. EBNA1-DNA crosslinking corresponds to single-strand endonuclease activity specific to DNA structures enriched at replication-termination sites, such as 4-way junctions. These findings reveal that EBNA1 forms tyrosine-dependent DNA-protein crosslinks and single-strand cleavage at oriP required for replication termination and viral episome maintenance.

爱泼斯坦-巴尔病毒 (EBV) 是一种致癌的人类疱疹病毒，在增殖的宿主细胞中作为多拷贝附加体持续存在。附加体维持严格依赖于 EBNA1，这是一种序列特异性 DNA 结合蛋白，没有已知的酶活性。在这里，我们显示EBNA1 与质粒复制oriP 的EBV 起点形成细胞周期依赖性DNA 交联。EBNA1 酪氨酸 518 (Y518) 是与oriP交联所必​​需的，并且是附加体维持和产生 EBV 转化的淋巴母细胞系 (LCL) 所必需的。从机制上讲，Y518 是体内 oriP复制叉终止和体外形成 SDS 抗性复合物所必需的. EBNA1-DNA 交联对应于在复制终止位点富集的 DNA 结构特异性的单链核酸内切酶活性，例如 4 路连接点。这些发现表明，EBNA1 在复制终止和病毒附加体维持所需的oriP处形成酪氨酸依赖性 DNA 蛋白交联和单链切割。