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

Minna Mäkiniemi,Tomi Hillukkala,Jussi Tuusa,Kaarina Reini,Markku Vaara,Deqi Huang,Helmut Pospiech,Inkeri Majuri,Thomas Westerling,Tomi P Mäkelä,Juhani E Syväoja

doi:10.1074/jbc.m102245200

Minna Mäkiniemi, Tomi Hillukkala + Show 9 more

Open Access

https://doi.org/10.1074/jbc.m102245200

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Abstract

Topoisomerase IIbeta-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 epsilon. 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.

Highlights

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 ⑀
TopBP1 Is Related to Fission Yeast Cut5, Budding Yeast Dpb11, and Drosophila Mus101—We used homology searches to identify functional human homologs of budding yeast and fission yeast Cut5 and identified a human cDNA, KIAA0259 (40), predicted to encode a protein with eight BRCT domains

Summary

EXPERIMENTAL PROCEDURES

Cloning of TopBP1 and hRad9 —A human cDNA sequence KIAA0259 (GenBank accession D87448) was used to search for human EST clones from the expressed sequence tag data base. EST clones contained deletions, and nucleotides 308 –1560 and 1628 –2620 were amplified from human T-cell (HUT-78) and thymus cDNA libraries (CLONTECH). Specificity of antibodies was examined by Western analysis of human HeLa S3 cell extracts and by antigen block assay. For preparation of cell extract, IMR cells were washed twice with PBS and lysed for 5 min on ice with lysis buffer (50 mM Tris-Cl, pH 7.4, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, and 0.5% deoxycholic acid). To label nascent DNA, MCF-7 cells were incubated in growth medium containing 0.1 mM BrdUrd (Sigma) for 3 min at 37 °C. Yeast Two-hybrid Assay—The BRCT domains 4 and 5 (amino acids 534 –763) of human TopBP1 were fused to LexA DNA binding domain in pLex-a vector (38). Chromosomal Mapping—Chromosomal mapping of TopBP1 by fluorescence in situ hybridization (FISH) was carried out by SeeDNA Biotech Inc. (Canada) from lymphocytes isolated from human blood using a 2062-base pair cDNA probe, which was biotinylated with dATP

RESULTS

DISCUSSION

Syväoja