BRCT Domain-containing Protein TopBP1 Functions in DNA Replication and Damage Response

Abstract

Topoisomerase II␤-binding protein (TopBP1), a human protein with eight BRCT domains, is similar to Saccharomyces cerevisiae Dpb11 and Schizosaccharomyces pombe Cut5 checkpoint proteins and closely related to Drosophila Mus101. We show that human TopBP1 is required for DNA replication and that it interacts with DNA polymerase ⑀. In S phase TopBP1 colocalizes with Brca1 to foci that do not represent sites of ongoing DNA replication. Inhibition of DNA synthesis leads to relocalization of TopBP1 together with Brca1 to replication forks, suggesting a role in rescue of stalled forks. DNA damage induces formation of distinct TopBP1 foci that colocalize with Brca1 in S phase, but not in G 1 phase. We also show that TopBP1 interacts with the checkpoint protein hRad9. Thus, these results implicate TopBP1 in replication and checkpoint functions. DNA polymerases (pol)1 play essential roles in chromosomal DNA replication and repair. In Saccharomyces cerevisiae three essential nuclear polymerases, ␣, ␦, and ⑀ have important functions in DNA replication. S. cerevisiae pol ⑀ is isolated as a complex of a catalytic subunit and three smaller subunits, Dpb2, 3, and 4 (1). This four-subunit structure is also conserved in the human enzyme, which consists of a catalytic subunit (2), a B subunit (3, 4), and two smaller subunits (5). Pol ⑀ is a proofreading DNA polymerase, which has been implicated in DNA replication, as temperature-sensitive mutants show defects in DNA replication in both S. cerevisiae and Schizosaccharomyces pombe (6 -8). Moreover, pol ⑀ is associated with origins of DNA replication and it proceeds along the replication fork (9). In human cells, pol ⑀ is associated with actively replicated cellular DNA (10) and has been shown to perform an important fraction of replicative DNA synthesis (11). Surprisingly, the catalytic domain of pol ⑀ is not essential for viability in S. cerevisiae. Instead, the C terminus, which interacts with Dpb2, exerts all of the essential functions (12).Pol ⑀ has been proposed to function in the repair of UVdamaged DNA because it is able to catalyze UV-induced DNA synthesis in vivo (13) and performs efficient gap-filling synthesis in the reconstituted nucleotide excision repair system (14). A role in base excision repair is suggested by the fact that pol ⑀ mutants fail to support repair synthesis in vitro, and repair activity can be restored by the addition of purified pol ⑀ (15). Pol ⑀ has also been proposed to function in a specialized replication process required to repair double strand breaks (16). In addition to replicative and repair roles, it has been suggested that pol ⑀ coordinates transcriptional and cell cycle responses to DNA damage and replication blocks (17).In S. cerevisiae, a BRCT domain-containing protein, Dpb11, interacts with the pol ⑀ complex and was originally identified as a suppressor of pol ⑀ catalytic and Dpb2 subunit mutants (18,19). DPB11 is an essential gene required for DNA replication (18). The inability of DPB11 mutants to restrain mitosis in the presence of inco...

The Rad9–Hus1–Rad1 (9–1–1) clamp activates checkpoint signaling via TopBP1

“…To identify its role in checkpoint signaling, we asked whether this site was important in binding other checkpoint proteins that might participate in Chk1 activation. Rad9 was previously reported to interact with TopBP1 (Makiniemi et al 2001;St. Onge et al 2003), a checkpoint protein that plays a role in Chk1 activation (Parrilla-Castellar and Karnitz 2003).…”

“…Several studies have demonstrated that Rad9 orthologs in Schizosaccharomyces pombe (Furuya et al 2004), Saccharomyces cerevisiae (Wang and Elledge 2002), and humans (Makiniemi et al 2001;St. Onge et al 2003) interact with their respective TopBP1 orthologs (Cut4, Dpb11, and TopBP1).…”

Expression of MCM10 and TopBP1 is regulated by cell proliferation and UV irradiation via the E2F transcription factor

“…Likewise, topoisomerase IIb-binding protein (TopBP1), a protein with eight BRCA1 C-terminus repeat domains, is also required for DNA replication (Makiniemi et al, 2001). In Xenopus, the TopBP1 protein is required for loading of Cdc45 onto the origin, and this it does in a manner distinct from MCM10 (Van Hatten et al, 2002).…”

“…TopBP1 association with chromatin is dependent on ORC, and independent of S-Cdk and MCM 2-7. TopBP1 is also involved in the cellular response to UV irradiation and DNA damage, indicating that it may be a functional homolog of yeast Cut5 (Yamamoto et al, 2000;Makiniemi et al, 2001;Yamane et al, 2002).…”

Mcm10 interacts with Rad4/Cut5TopBP1 and its association with origins of DNA replication is dependent on Rad4/Cut5TopBP1

“…This association is required for checkpointdependent Chk1 activation, as well as a number of other checkpoint proteins (29,31). Human 6 TopBP1 has also been shown to function in both DNA replication and the response to DNA damage and recent data mirror the requirement for interaction between hRad9 and TopBP1 for checkpoint signalling (32,33). In Xenopus, human and budding yeast systems TopBP1 has also been shown to directly interact with and activate the ATR-ATRIP protein kinase complex and in Xenopus regulation of this process is 9-1-1 dependent (34,35,36,37).…”