Accumulation of FFA-1, the Xenopus Homolog of Werner Helicase, and DNA Polymerase δ on Chromatin in Response to Replication Fork Arrest

Abstract

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.

RecQ helicases: guardian angels of the DNA replication fork

“…Upon relief of the blockade, this tighter association is diminished. The increased association of pol δ and RPA upon replication arrest, however, occurs independently of the presence of FFA-1 (Sasakawa et al 2006). Taken together, these data would suggest a role for FFA-1 in the assembly of replication foci, perhaps by assisting in the unwinding of the replication origin.…”

DNA Polymerase ε: A Polymerase of Unusual Size (and Complexity)

“…A role was proposed for WRN in unwinding dsDNA byproducts generated during lagging strand synthesis (202). Consistent both with Pol ε carrying out leading strand synthesis and with Pol ε being involved in stalled replication fork signaling, xWRN/FFA-1 and Pol δ accumulate on chromatin after aphidicolin treatment, while the chromatin-bound level of Pol ε does not increase (203). Claspin, the Xenopus homolog of the mediator protein Mrc1, associates with replication forks during S phase (204) in a xCdc45-dependent manner.…”

Eukaryotic Translesion DNA Synthesis on the Leading and Lagging Strands: Unique Detours around the Same Obstacle

“…In cellular studies on X. laevis , only pol δ accumulated on replication-arrested chromatin in response to UV or Aph treatment while the level of chromatin-bound pol ε did not change 88, 159 . Furthermore, the level of pol δ on chromatin gradually decreased after release from Aph-induced fork arrest 159 .…”

“…Furthermore, the level of pol δ on chromatin gradually decreased after release from Aph-induced fork arrest 159 . These observations are similar to those from later studies on human cells that analyzed proteins on daughter DNAs in relation to progressing (active) or stalled replication forks.…”