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From BRCA1 to RAP1: a Widespread BRCT Module Closely Associated

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From BRCA1 to RAP1: a Widespread BRCT Module Closely Associated FEBS 17918 FEBS Letters 400 (1997) 25-30 From BRCAl to RAPl: a widespread BRCT module closely associated with DNA repair Isabelle Callebaut*, Jean-Paul Mornon Systemes Moleculaires & Biologie Structurale, Laboratoire de Mineralogie-Cristallographie, CNRS URA09, Universites Paris 6 and Paris 7, case 115, T.16, 4 place Jussieu, 75252 Paris Cedex 05, France Received 26 September 1996 pears to be tumor-type specific as BRCAl cannot inhibit the Abstract Inherited mutations in BRCAl predispose to breast and ovarian cancer, but the biological function of the BRCAl growth of some other cancer cell lines, nor can it inhibit the protein has remained largely elusive. The recent correspondence growth of normal fibroblasts [8]. of Koonin et al. [Koonin, E.V., Altschul, S.F. and Bork, P. BRCAl encodes a predicted protein of 1863 amino acids (1996) Nature Genet. 13, 266-267] has emphasized the potential containing in its NH2-terminus a single CsHQ-type zinc fin- importance of the BRCAl C-terminal region for BRCAl- ger domain, also referred to as the RING finger or A-box and mediated breast cancer suppression, as this domain shows found in various proteins showing transactivation activity for similarities with the C-terminal regions of a p53-binding protein a number of viral and cellular genes [1,9]. The rest of the (53BP1), the yeast RAD9 protein involved in DNA repair, and protein was initially reported to contain no significant similar- two uncharacterized, hypothetical proteins (KIAA0170 and ities to any known genes. A recent report paradoxically sug- SPAC19G10.7). The highlighted domain has been suggested to gests that the BRCAl protein, whose sequence is found to be the result of an internal duplication, each of the tandem domains being designated as a 'BRCT domain' (for BRCAl C- match with a 'granin' consensus, might be secreted and so terminus). Sequence analysis using Hydrophobie cluster analysis would function by a mechanism so far undescribed for tumor reveals here the presence of 50 copies of the BRCT domain in 23 suppressor gene products [10]. On the other hand, several lines different proteins, including, in addition to BRCAl, 53BP1 and of evidence suggest that the C-terminal end of BRCAl is RAD9, XRCC1, RAD4, Ect2, REVI, Crb2, RAPl, terminal essential to the normal function of the protein in breast deoxynucleotidyltransferases (TdT) and three eukaryotic DNA epithelial cells. Patients inheriting 1853Stop were shown to ligases. Most of these proteins are known to be involved in DNA develop very early onset breast cancer [11]. Moreover, trunca- repair. The BRCT domain is not limited to the C-termini of tions of the BRCAl C-terminal region were shown to sup- protein sequences and can be found in multiple copies or in a press the ability of BRCAl to inhibit breast cancer cell growth single copy as in RAPl and TdT, suggesting that it could well [8]. Finally, this region of BRCAl has recently been reported constitute an autonomous folding unit of approx. 90-100 amino acids. to act as a transcriptional transactivator when fused to the GAL4 DNA-binding domain [12]. Key words: Hydrophobie cluster analysis; Sequence analysis; In order to gain more insight into the structural and func- BRCAl; DNA repair; Cancer; RAPl; DNA polymerase; tional features of this essential region, we have used hydro- DNA ligase phobic cluster analysis (HCA) [13,14] in combination with well-established linear methods of sequence analysis. HCA is indeed able to detect three-dimensional similarities between 1. Introduction proteins showing very limited sequence relatedness. Its sensi- tivity at low levels of sequence identity (typically below the so- The cloning of the familial breast and ovarian cancer sus- called twilight zone (25-30%)) stems from its ability to detect ceptibility gene BRCAl [1] was an important milestone in significantly secondary structure elements [15]. The effective- cancer research. Cancer-predisposing alíeles of BRCAl, which ness of the HCA method has been widely demonstrated (see, generally behave as recessive alíeles in somatic cells, typically among others [16-20]). carry mutations that cause loss or reduction of the gene func- The use of this method has led us to identify within the tion and the wild-type alíele is lost in tumor tissue [1,2], sug- BRCAl C-terminus a repeated motif which is widespread in gesting that BRCAl, like many other genes involved in famil- several nuclear proteins closely related to cell cycle regulation ial cancer, is a tumor suppressor gene. In sporadic tumors, and DNA repair. These findings complement the recent cor- somatic point mutations are very rare; complete somatic dele- respondence of Koonin and colleagues [21] in which they re- tion of one alíele of BRCAl is often observed, with a decrease port the presence of this motif, which they named BRCT in BRCAl mRNA expression [3-6]. Evidence of a role in (BRCAl C-terminus), in the repair protein RAD9 and in a tumor suppression is further supported by the observations p53-binding protein. Here we extend the retrieval of this mod- of growth acceleration of both normal and malignant breast ule to XRCC1, RAD4, Ect2, REVI, Crb2, RAPl, terminal epithelial cells following inhibition of BRCAl expression [7] deoxynucleotidyltransferases (TdT) and three eukaryotic and growth inhibition of tumor cell Unes after transfection DNA ligases and emphasize its potential role in cell cycle with wild-type, but not mutant, BRCAl [8]. This activity ap- control. »Corresponding author. Fax: (33) (1) 44 27 37 85. E-mail: [email protected] 2. Materials and methods Abbreviations: HCA, hydrophobic cluster analysis; TdT, terminal Systematic searches of databanks [22,23] allow detection of se- deoxynucleotidyltransferase. quences which could belong to the same functional and/or structural 0014-5793/97/S17.00 © 1997 Federation of European Biochemical Societies. All rights reserved. P//S0014-5 79 3(9 6)01312-9 26 /. Callebaut, J.-P. MomonlFEBS Letters 400 (1997) 25-30 family. However, at the low levels of sequence identity (< 25-30%) RING often observed, these automatic methods are unable to distinguish BKCA1_Q -QQ m BRCT module similarities due to structural relationships from background noise. 53BP1 -00 100 aa The 'hydrophobic cluster analysis' method [13,14] is helpful in this XHCC1 0-Ö regard insofar as it allows comparison of not only the sequences but TDT also the protein secondary structures statistically centered on hydro- -Q[w KIAA0170 . -00 phobic clusters, as well as their distribution [15]. Similar plots could "fl&iM!— YOR005c . ■OO therefore indicate similar three-dimensional folds. Guidelines to the RAD9 , YM8021.03 . use of this method are given in [13,14]. 00 REVI ^_A_gDcL. YGR103W . o- The HCA score is proportional to the hydrophobic amino acids o- which are topologically conserved (often not chemically identical), YHV4Q0-0 and therefore reflects the degree of conservation of the hydrophobic Y,J0 core. High HCA scores are associated with low root mean squares (XH3 values between three-dimensional structures [14]. The accuracy of the ϋΑ04ΑΑ-ΠΠ—- SPAC19G10.7 /WV\—ΛΑ alignments can be assessed by computing identity, similarity or HCA PARPz ra'B«.i ΡΥΛ-ΓνΠη ñ— 4Γ LjQ L_f\ human III scores, as well as the corresponding Z scores as initially suggested by ZK675.2 A G—hQ-Q i™»iiv Doolittle [24]; these represent differences between the alignment score T19E10.1 —ΓΨ) under consideration and the mean score of a distribution computed LIG hf)— CMlbwam for alignment of sequence 1 versus a large number of randomly shuffled versions of sequence 2 (here 1000). These differences are ex- Fig. 1. Position of the BRCT domains within BRCT domain-con- pressed relative to the standard deviation (SD) of the random distri- taining proteins. Abbreviations, correspondences and sequence refer- bution. Scores that are 3.0 or more standard deviation above the ences are given in the legend to Fig. 2. Additional modules showing scrambled mean scores can reasonably be expected to represent similarities with other proteins: RING, ring finger domain; POLß, authentic relationships. region similar to polymerase ß; GEF, region similar to GTP-ex- changing factor; UMUC, region similar to the bacterial DNA re- pair protein UmuC; DBD, DNA binding domain; LIG, region 3. Results showing similarities with human DNA ligase I and corresponding to the minimal size of ATP-dependent bacterial Hgases (Callebaut et al., in preparation); PARPz, region similar to the Zn fingers of hu- The BRCT family members listed in Fig. 1 have been iden- man poly(ADP-ribose) polymerase (PARP). tified by first searching the sequence databases using standard ID methods such as BlastP [23] and Fast A [22] and then sorting and assessing the putative 3D relationships to the Motif C corresponds to a continuous stretch of three or family through HCA [13,14] (see Section 2). four hydrophobic amino acids (a vertical shape in the HCA Conserved motifs (similar hydrophobic motifs often asso- plots) or, in several domains, to the sequence ΤΗΦΦ where Φ ciated with sequence conservation) define five regions, desig- is a hydrophobic amino acid (V, I or L). This motif most nated A-E, which can be used to decipher the main features probably corresponds to an internal ß-strand. Two other ß- of the BRCT module (Figs. 2 and 3). The most highly con- strands probably constitute the motifs A and E, which begins served motif, motif D, is organized around a conserved aro- and ends the domain, respectively, and whose shapes are also matic residue (W, F or Y). The residue following it is always well retrieved within the family. hydrophobic, as are usually the fourth and fifth residues pre- The limits of the domain can be well defined, especially ceding it. The conservation of this hydrophobic pattern can since in some proteins such as RAP1, it is surrounded by easily be visualized on the HCA plots (Fig.
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