|TopBP1 in Human Breast. TopBP1 was detected in immersion fixed paraffin-embedded sections of human breast using Sheep Anti-Human TopBP1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF7120) at 3 µg/mL overnight at 4 °C. Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained using the Anti-Sheep HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS019) and counterstained with hematoxylin (blue). Specific staining was localized to epithelial cells. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.|
TopBP1 (DNA topoisomerase 2-binding protein 1) is a 175-180 kDa member of a critical DNA replication repair complex. It is widely expressed, and appears to serve as an intermediary in DNA replication. When a replication fork is stalled during S phase, DNA continues to unwind ahead of the stall. This becomes associated with RPA, which is essential for ATR and subsequent Chk1 activation. TopBP1 is key to ATR activation, and appears to first bind to DNA:pol-alpha, followed by a 9-1-1 complex and Rad17 interaction that leads to ATR stimulation. Human TopBP1 is 1522 amino acids (aa) in length. It contains eight BRCT domains (aa 101-1486) that interact with distinct binding partners. BRCT1 and 2 binds to Rad9 (aa 101-294), while BRCT 6 and 7 are involved in ATR activation. There are 12 utilized phosphorylation sites and two nuclear localization signals. Three isoform variants have been described. One contains an alternative start site at Met88, a second shows an 18 aa substitution for aa 1198-1522, and a third possesses a six aa substitution for aa 1292-1522. Over aa 88-194, human TopBP1 shares 88% aa identity with mouse TopBP1.
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