Accumulation of FFA-1, the Xenopus homolog of Werner helicase, and DNA polymerase delta on chromatin in response to replication fork arrest.
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.
Sasakawa
Noriko
N
Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043.
Fukui
Tomoyuki
T
Waga
Shou
S
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000494:pharmacology; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism*; D004249:DNA Damage; D004265:DNA Helicases / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000494:pharmacology; D005260:Female; D064248:Geminin; D008297:Male; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D053484:RecQ Helicases; D051716:Replication Protein A / Q000378:metabolism; D013094:Spermatozoa / Q000378:metabolism; D000071657:Werner Syndrome Helicase; D014981:Xenopus; D029867:Xenopus Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D003845:Deoxycytosine Nucleotides; C577998:GMNN protein, Xenopus; D064248:Geminin; D018809:Proliferating Cell Nuclear Antigen; D051716:Replication Protein A; D029867:Xenopus Proteins; C024107:2'-deoxycytidine 5'-triphosphate; D016590:Aphidicolin; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D000251:Adenosine Triphosphatases; C097151:Bloom syndrome protein; D004265:DNA Helicases; D053484:RecQ Helicases; D000071657:Werner Syndrome Helicase; C507159:WRN protein, Xenopus