Werner syndrome is a genetic disorder characterized by premature aging and cancer-prone symptoms, and is caused by mutation of the WRN gene. WRN is a member of the RecQ helicase family and is thought to function in processes implicated in DNA replication and repair to maintain genome stability; however, its precise function is still unclear. We found that replication fork arrest markedly enhances chromatin binding of focus-forming activity 1 (FFA-1), a Xenopus WRN homolog, in Xenopus egg extracts. In addition to FFA-1, DNA polymerase delta (Poldelta) and replication protein A, but not DNA polymerase epsilon and proliferating cell nuclear antigen, accumulated increasingly on replication-arrested chromatin. Elevated accumulation of these proteins was dependent on formation of pre-replicative complexes (pre-RCs). Double-strand break (DSB) formation also enhanced chromatin binding of FFA-1, but not Poldelta, independently of pre-RC formation. In contrast to FFA-1, chromatin binding of Xenopus Bloom syndrome helicase (xBLM) only slightly increased after replication arrest or DSB formation. Thus, WRN-specific, distinct processes can be reproduced in the in vitro system in egg extracts, and this system is useful for biochemical analysis of WRN functions during DNA metabolism.