A DNA‐Topoisomerase‐11–Binding Protein with Eight Repeating Regions Similar to DNA‐repair Enzymes and to a Cell‐Cycle Regulator

Abstract

A two-hybrid system was used to isolate factors that interact with the C-terminal region of DNA topoisomerase IIP. A positive clone isolated from a HeLa cDNA library encoded 1.522 amino acid residues (molecular mass 170670). The protein, designated topoisomerase-ITP-binding protein 1 (TopBPl), interacted with the C-terminal region of topoisomerase IIP synthesized in vitw. A database search indicated that TopBPl possessed eight regions similar to regions of Rad4, Cut.5, Ect2, Revl and X-ray repair cross-complementing 1 (XRCC1) proteins and a region similar to auto-modification sites of poly(ADPribose) polymerase, suggesting that TopBPl supported catalytic reactions of topoisomerase I1 through transient breakages of DNA strands. . Therefore, a decrease in the number of cleavable complexes, including a reduction in enzyme quantity and activity, could confer drug resistance. Solid tumors that are naturally under stress conditions (e.g. hypoxia and glucose starvation) show a low amount of topoisomerase 11, which suggests that the resistance of solid tumors to chemotherapy is partly due to the decreased amount of topoisomerase I1 as the molecular target of major antitumor reagents [6, 71. To identify the factors involved in the regulatory mechanisms of topoisomerase I1 and to investigate the resistance mechanisms of solid tumors to chemotherapy, we adopted a twohybrid system [S], using the C-terminal region of human topoisomerase IIP. An isolated clone encoded eight repeating regions that are similar to the regions of Rad4, Cuts, Revl and X-ray repair cross-complementing (XRCC1) proteins, which are involved in DNA repair or regulation of the cell cycle. The protein, designated topoisomerase-binding protein 1 (TopBPl) contained a region similar to automodification sites of poly(ADPribose) polymerase, and may be involved in cell-cycle regulation and DNA repair, caused by failure of catalytic reactions of topoisomerase IT through DNA strand breaks. Keywords MATERIALS AND METHODSPlasmid construction. The C-terminal region of the human topoisomerase IlP gene [2] was amplified by PCR. The templates were cDNAs prepared from human kidney (Clontech, Quick-clone). Oligonucleotides used were 5'-GGATCCGGT-CAAAGAAAGATTTGAT-TCAAATG-3' and 5'-GATTTGTT-GAAAAATGTTTGTGCTC-3'. The region corresponding to amino acid 1143-1621 of topoisomerase Ilg was cloned into the BumH1-XhoI site of pEG202 [S], carrying the Zed gene to construct pK83.5 (a bait plasmid).Yeast two-hybrid system. The system was a kind gift of Dr Roger Brent, Harvard Medical School. Screening was performed according to his procedures [S]. Saccharumyces cer-evi.r.iue, ECY48 was transformed with pSH18-34 (a reporter) and pK83.5 (a bait). A HeLa cDNA expression library 181 was introduced into the transformants. After 2 days, approximately 10' independent clones which contained pSH18-34, pK835 and a cDNA plasmid, were replicated onto plates that lacked leucine but contained 5-broino-4-chloro-3-~ndoyl-~-~-galactopyranos~de and Dgalactose. A positive cDNA clone (termed pK835-5...

Retinoblastoma susceptibility protein, Rb, possesses multiple BRCT-Ws, BRCA1 carboxyl-terminus-related W regions with DNA break-binding activity

“…&: hydrophilic residues. (b) Small parts of original BRCT regions in TopBP1 (Yamane et al, 1997) and BRCA1 (Miki et al, 1994) were presented for comparison. (c) Another subfamily, designated BRCT-G region whose G box is characteristic and conserved, was also presented for comparison.…”

“…TopBP1 possessed eight BRCT regions (Yamane et al, 1997), three BRCTrelated regions, and was most similar to Cut5/Rad4, which regulates G1/S and G2/M checkpoints in ®ssion yeast (Saka et al, 1994). The DNA break binding activity of these BRCTs suggests linkages to cell cycle checkpoints and to DNA repair.…”

TopBP1 and ATR Colocalization at Meiotic Chromosomes: Role of TopBP1/Cut5 in the Meiotic Recombination Checkpoint

“…Human BRCT domain-containing protein TopBP1 was first identified through its association with topoisomerase II␤ (Yamane et al, 1997), and its function seems to be linked to replication and DNA damage checkpoint processes (Mä-kiniemi et al, 2001;Yamane et al, 2003). Indeed, TopBP1 localizes to the sites of DNA damage in response to DSBs and replication blocks (Honda et al, 2002;Yamane et al, 2002).…”

Cut5 Is Required for the Binding of Atr and DNA Polymerase α to Genotoxin-damaged Chromatin

“…In higher eukaryotes, orthologs of Cut5 and Dpb11 have also been identified. Drosophila Mus101, human TopBP1, and Xenopus Cut5 (also known as Mus101) are highly related to one another and more distantly related to Cut5/Dpb11 (15)(16)(17)(18). In Drosophila, hypomorphic alleles of the Mus101 gene confer sensitivity to genotoxins (19).…”