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Biochemical Mechanism for Recruitment of the Checkpoint Activator TOPBP1 to Stalled Replication Forks
Author(s) -
Michael Matthew
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.560.12
Subject(s) - microbiology and biotechnology , chek1 , dna replication , g2 m dna damage checkpoint , cell cycle checkpoint , dna damage , dna replication factor cdt1 , eukaryotic dna replication , biology , chemistry , cell cycle , dna , genetics , cell
During a replication stress response, the ATR protein kinase is activated on stalled replication forks to allow a delay in cell cycle progression, a block to further origin firing, and stabilization of the stalled fork. ATR is recruited to stalled forks via an interaction between its binding partner ATRIP and RPA‐coated single‐stranded DNA (ssDNA). However, binding of ATR‐ATRIP to RPA‐coated ssDNA is not sufficient for ATR activation as both the 9‐1‐1 clamp protein and the checkpoint activator TOPBP1 must also be recruited so that TOPBP1 may activate ATR. The biochemical mechanism for assembly of the checkpoint‐signaling complex has been an important unanswered question in the cell cycle field for many years. Previous work from our laboratory, and other groups, has shown that ATR/ATRIP and TOPBP1/9‐1‐1 are recruited independently to stalled forks, and then come together on the DNA to form an active checkpoint‐signaling complex. Furthermore, we have previously shown that TOPBP1 is required for loading of the 9‐1‐1 clamp, however the means by which TOPBP1 senses the stalled fork to allow checkpoint complex assembly was not previously known. We will present data, obtained using Xenopus egg extracts, showing that direct interaction between TOPBP1 BRCT repeat 2 and RPA‐coated ssDNA is the mechanism for TOPBP1 recruitment. In addition, we present evidence that direct interaction with RPA‐coated ssDNA is a general feature of a subclass of BRCT domains from other proteins, including PTIP and REV1.