The BRCA1 C-terminal domain: structure and function

Mutation Research 460 Ž2000. 319–332 www.elsevier.comrlocaterdnarepair Community address: www.elsevier.comrlocatermutres

The BRCA1 C-terminal domain: structure and function Trevor Huyton a , Paul A. Bates b, Xiaodong Zhang a , Michael J.E. Sternberg b, Paul S. Freemont a,c,) a

Molecular Structure and Function, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London, WC2A 3PX, UK Biomolecular Modelling Laboratories, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London, WC2A 3PX, UK Centre for Structural Biology, Imperial College of Science, Technology and Medicine, South Kensington, London, SW7 2AY, UK b

c

Abstract The BRCA1 C-terminal region contains a duplicated globular domain termed BRCT that is found within many DNA damage repair and cell cycle checkpoint proteins. The unique diversity of this domain superfamily allows BRCT modules to interact forming homorhetero BRCT multimers, BRCT–non-BRCT interactions, and interactions with DNA strand breaks. The sequence and functional diversity of the BRCT superfamily suggests that BRCT domains are evolutionarily convenient interaction modules. q 2000 Published by Elsevier Science B.V. Keywords: BRCT; BRCA1; XRCC1; DNA damage repair; Protein interaction module

1. Introduction The integrity of an organisms genome is of primary importance and thus the effects of DNA damage mediated by radiation, carcinogens, and oxygen derived free radicals must be repaired to eliminate the chance of permanent genetic alterations. Repair of DNA damage is coordinated through cell cycle checkpoints in order to remove potential damage before DNA synthesis and mitosis w1x. Checkpoints therefore play two important roles, ensuring that essential cell cycle events are completed before the cell cycle can progress and allowing additional time for the DNA damage repair process to occur before DNA replication and mitosis w1x. ) Corresponding author. Molecular Structure and Function, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London, WC2A 3PX, UK. E-mail address: [email protected] ŽP.S. Freemont..

Checkpoint proteins are highly diverse structurally but many have been shown to contain a conserved globular domain, first identified in the breast cancer tumor suppressor protein BRCA1 and thus designated BRCT ŽBRCA1 C- terminus. w2,3x. From the initially identified BRCT domains of the human and mouse BRCA1 tumor suppressor proteins, p53 binding protein 1 Ž53BP1., and the yeast cell cycle checkpoint protein RAD9, BRCT domains have been detected in more than 50 other proteins w www.sanger.ac.ukrcgi-binrpfamx. Although some of these proteins have yet to be characterised and despite their structural diversity, the BRCT domain superfamily unites a functionally diverse group of proteins from animals, plants, and bacteria. Many of these proteins are known to play direct or indirect roles in DNA damage response and cell cycle checkpoint mediated repair w2–4x. In this review, we describe recent developments on understanding the structurerfunction of BRCT domains. In particular,

0921-8777r00r$ - see front matter q 2000 Published by Elsevier Science B.V. PII: S 0 9 2 1 - 8 7 7 7 Ž 0 0 . 0 0 0 3 4 - 3

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we discuss models of the BRCT domains from BRCA1 and describe structural insights into the effects of breast cancer pre-disposing mutations

2. The BRCT superfamily and sequence conservation BRCT domains are 85–95 amino acid domains that comprise several distinct clusters of conserved hydrophobic amino acids that together form the core of the BRCT fold. They can be found either as single modules or as multiple tandem repeats comprising two domains, and both can occur within individual proteins in a variety of different arrangements ŽFig. 1.. In tandem BRCT repeats, the single domains can be separated by variable linker regions, ranging between 0 and 24 residues in length, although lengths of 0 to 5 or 18 to 24 residues appear favoured. In addition to the BRCA1, 53BP1, and Rad9 proteins, some BRCT superfamily members have been functionally characterised, including the DNA repair proteins XRCC1, DNA ligases III and DNA ligase IV ŽFig. 1.. Many other BRCT-containing proteins have only been identified by sequence homology including putative proteins from humans, yeast, Caenorhabditis elegans and Arabidopsis thaliana. A sequence alignment of a number of BRCT domains is shown in Fig. 2A. It is surprising that there is not one invariant amino acid residue throughout the whole BRCT superfamily, even though they are predicted to contain a conserved fold. A distribution of sequence identity for a number of BRCT family members from all sources is shown in Fig. 2B. The average sequence ID is ; 14% and this lack of sequence identity within the BRCT superfamily makes recognition of distant family members difficult. Indeed, the BRCT family has been used as a test case for evaluating motif and profile detection software w5,6x. Based upon iterative sequence alignments as well as motif and profile analysis w4x, BRCT sequences contain two highly conserved motifs ŽFig. 2A.. Motif-1 consists of a Gly–GlyrGly–Ala pair prior to the b2-strand, corresponding to a sharp turn between a1 and b2 within the XRCC1 crystal structure Žsee later.. Motif-2 occurs within helix-a 3 near the C-terminus of the domain, and contains a Trp-X-X-X-CysrSer mo-

Fig. 1. A schematic showing selected BRCT domain family members. Examples of several BRCT containing proteins involved in DNA repair are illustrated. The variable number of BRCT domains, their organisation, and any additional functional domains, are shown for each protein with BRCT domains represented as red ellipsoids. Also represented are the DNA polymerase domain of terminal deoxynucleotidyl transferase ŽTdT. Ždark blue.; the DNA ligase domain Žyellow. and Helix-Hairpin-Helix ŽHHH. motif ŽPink. of the E. coli NADqdependent DNA ligase; the RING-finger domain of the breast cancer tumor suppressor protein BRCA1 Žbrown sphere.; the conserved N-terminal domain of XRCC1 Žcrimson.; the DNA ligase domains of DNA ligases III and IV Žgreen.; the and polyŽADP–ribose. polymerase ŽPARP. zinc finger motif Žlight blue. also found in DNA ligase III; the PARP catalytic region Žturquoise..

tif, where the two variable residues after the Trp are generally small hydrophobics. This motif is predominantly more conserved than Motif-1 in the vast majority of BRCT domains, but is significantly divergent in bacterial NAD q dependent DNA ligases, eukaryotic replication factor-C ŽRF-C. and polyŽADP–ribose. polymerase ŽPARP. w2x. Other interesting features of the BRCT domain superfamily concern regions of most variability. The region around and including helix-a 2 is particularly

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variable, in terms of both sequence length and amino acid composition. Proteins, such as the DNA ligase

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III, have deletions in this region suggesting that the degree of variability within helix-a 2 could be re-

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Fig. 2. Sequence conservation within the BRCT domain super family. ŽA. Multiple sequence alignment of selected BRCT family members was obtained from Pfam v3.0 w6x. Accession codes and residue numbering are shown for each BRCT sequence. The secondary structure from the XRCC1 BRCT crystal structure is shown at the top of the alignment ŽE, b-stand; H a-helix; C, coil. with individual secondary structure elements labelled Žsee text.. The conserved hydrophobic clusters are coloured gold while the two conserved motifs, Motif 1ŽGly–GlyrGly–Ala. and Motif 2 ŽTrp-X-X-X-CysrSer. are coloured red and labelled. The BRCA1 breast cancer predisposing missense mutations are coloured and underlined in cyan. ŽB. Profile of sequence identity between BRCT family members. From the full Pfam alignment wwww.sanger.ac.ukrcgi-binrpfamx, all hypothetical proteins were removed plus domains with greater than 40% sequence identity to each other Žsequence identity was calculated as the number of identical residues in each pairwise alignment compared to the number of residues equivalenced not including gaps.. These constraints created a list of 47 BRCT domains and thus a total of 1081 pairwise alignments. The profile is essentially that of a normal distribution with a mean of approx. 14% sequence identity and a standard deviation of 4.5.

sponsible for determining functional specificity, analogous to the complimentarity determining regions ŽCDR. in antibodies. There is also some variability in the lengths of the two loop regions C1 and C2 in a small number of family members which may also be specific to the functions of these proteins, e.g., Rad9 ŽFig. 2A..

3. XRCC1 BRCT domain structure The first structural information for a BRCT domain came from the crystal structure of the C-terminal BRCT domain of XRCC1 w7x. XRCC1 has no known enzymatic activity but apparently, acts as a scaffolding protein in the mammalian base excision repair ŽBER. pathway. XRCC1 contains two BRCT

domains separated by 125 residues w9x. The C-terminal BRCT domain mediates a direct BRCT–BRCT interaction with DNA ligase III, whereas the Nterminal domain interacts with a BRCT domain in polyŽADPP–ribose. polymerase ŽPARP. w9–12x. The non-BRCT N-terminal region of XRCC1 also mediates direct interactions with DNA polymerase b w13x. It is through these interactions that XRCC1 promotes the efficiency of the repair process. The XRCC1 BRCT structure shows a compact globular domain that is formed from a core fourstranded parallel b-sheet surrounded by three ahelices ŽFig. 3.. Helices a1 and a 3 form a helical bundle that contains conserved residues within the helical interface. This two-stranded helical bundle is an essential element of the BRCT domain fold and is likely to be conserved across the whole family Žsee

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Fig. 3. XRCC1 BRCT domain structure. A ribbon representation of the XRCC1 C-terminal BRCT domain structure. a-Helices are coloured blue, b-strands magenta and coil regions grey. The domain consists of a central four-stranded parallel b-sheet flanked by three a-helices which are numbered according to the overall babbaba topology of the domain. The C1 and C3 loops that from a flat surface are also labelled. Adapted from Ref. w7x.

Fig. 2A.. Several of the conserved hydrophobic clusters, that constitute the BRCT sequence motif, form the core b-sheet structure of the domain, whilst the conserved Gly–GlyrGly–Ala motif ŽMotif-1. provides the flexibility required for a sharp turn connecting a1 and b2. There is also a sequence preference for a SerrGly residue at the turn between a 2 and b4 suggesting that the geometry of both of these turns is maintained and essential to the BRCT fold. A specific interaction between the Cys and Trp residues of the conserved Trp-X-X-X-CysrSer motif ŽMotif 2. is also observed, positioning the C-terminal coil region across the hydrophobic core of the domain. The Trp residue of this motif is almost invariant in the whole BRCT family and is located within helix-a 3 near the C-termini in the majority of BRCT domains ŽFig. 2A.. Interestingly, mutation of this Trp in XRCC1 results in an abolishment of DNA ligase III interaction wDulic et al., personal communicationx, illustrating the critical nature of this residue in BRCT function. Residues that correspond to helixa 3 are among the most conserved in the BRCT superfamily, and together with conserved hydrophobic residues from the core b-sheet and C-terminal

coil region, play a critical role in forming the overall BRCT fold. It is these groups of conserved hydrophobic residues that constitute the BRCT sequence motif. An additional interesting feature of the XRCC1 structure concerns the C1rC3 loop regions that form a flat surface at one end of the domain ŽFig. 3.. In fact, data from a tetragonal crystal form of the XRCC1 BRCT domain shows an additional BRCT packing arrangement that utilises this surface Žsee later discussions..

4. DNA repair and BRCT domain proteins Interactions between different proteins via their BRCT domains have been demonstrated indicating that one of the functional roles for BRCT domains is to mediate protein–protein interactions. Many proteins containing BRCT domains have been functionally characterised and demonstrated to mediate either BRCT-nonBRCT or BRCT–BRCT interactions, some of which are discussed below in the context of their involvement in DNA repair.

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4.1. BRCA1 The highest profile BRCT superfamily member is the beast cancer susceptibility protein BRCA1, from which the BRCT motif was first described. Geneti-

cally inherited defects in BRCA1 predispose women to breast and ovarian cancer with ; 80% of known mutations resulting in a truncated form of the BRCA1 protein w14,15x. BRCA1 encodes a 220-kDa phosphoprotein w16x which acts as a tumor suppressor

Table 1 Missense mutations within the BRCA1 BRCT domains Residuermutation

Secondary structure, Coil ŽC., b-Strand ŽE., a-Helix ŽH.

Buried ŽB., Surface ŽS., Interface ŽIF.

Predicted effects, Fold Stability ŽFS., Disrupt Interface ŽDI., Protein Interaction ŽPI.

Sequence conservation in BRCT family

BRCA1 missense mutations within BRCT domain 1 (1642–1736) R1645M C S N1647K C S M1652I E B V1653M E B S1655F E S V1665M H B A1669S H B T1685I C B D1692N C IF F1695L C IF C1697R C S R1699WrQrL H IF G1706ArE H S A1708E C S V1713A E S S1715R E B T1720A H B

PI PI FS FS PI FS FS FS DI DI DI DI PI PI FS FS FS

Variable Variable Conserved Hydrophobic Conserved Hydrophobic Variable Variable Conserved Hydrophobic Variable Variable Variable Variable Variable Variable Variable Conserved Hydrophobic Variable Conserved Hydrophobic

BRCA1 point mutations within BRCT linker region (1737–1755) G1738E C S D1739G C S A1752P C S

DIrPI DIrPI DIrPI

– – –

BRCA1 missense mutations within BRCT domain 2 (1756–1855) L1764P E S P1771L C IF T1773S C IF M1775R H S M1783T H S C1787S H S G1788V C S G1803A C S V1804D C S P1806A C S V1808A E B V1810G E B Q1811R E S N1819S H S V1833M E B W1837R H B S1841N H B

PI DI DI PI PI PI PI PI PI PI FS FS PI PI FS FS FS

Conserved hydrophobic Variable Variable Variable Conserved Hydrophobic Variable Conserved Gly Partially conserved Gly Variable Variable Conserved Hydrophobic Conserved Hydrophobic Variable Variable Conserved Hydrophobic Conserved Trp Conserved CysrSer

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protein since overexpression leads to growth retardation of tumor cells in nude mice w17x. BRCA1 shows a distinct domain structure comprising an N-terminal RING finger motif, a predicted helical coiled-coil region and two BRCT domains at the C-terminus. The RING finger domain has been shown to mediate interactions with both BARD1 Žsee below. and a ubiquitin C-terminal hydrolase BAP1 w18x. The BRCT domains have also been shown to mediate a number of other protein–protein interactions Žsee below.. Interestingly a significant number of BRCA1 tumor-associated mutations are found within the RING finger and BRCT domains, emphasising the functional importance of these protein–protein interaction modules Žfor BRCT domain missense mutations see Table 1.. There are several pieces of evidence linking BRCA1 to DNA repair. BRCA1 colocalises with BRCA2 and RAD51, which mediates homologous recombinational repair of DNA double strand breaks w19x. BRCA1 has also been shown to disperse from nuclear foci and relocate to regions of damaged replicating DNA following UV or hydroxyurea-induced DNA damage w20x. The most direct evidence however is that mouse embryonic stem cells nullizygous for BRCA1 are defective in their ability to carry out transcriptionally coupled repair of oxidative DNA damage w21x. Interestingly, BRCA1 is also a component of the RNA polymerase II holoenzyme w22,23x forming direct associations with RNA helicase A through it’s C-terminal BRCT domains. These C-terminal BRCT domain can also activate transcription of reporter genes when fused to a yeast GAL4 DNA binding domain w24,25x. Although they are also implicated in transcriptional repression through interactions with the CtB interacting protein ŽCtlP., which is associated with the CtBP transcriptional co-repressor w26x. BRCA1 has many other protein partners including Bip1, Bap1, c-Myc, BRCA2, Rad51, and the universal tumor suppressor protein p53 w27x. Indeed there is a higher incidence of p53 mutations in BRCA1-associated breast cancers w28,29x. BRCA1 has been shown to associate with p53 and enhance p53-dependent transcription from the p21WAFrCIP1 and bax promoters w30,31x. BRCA1 has also been shown to affect expression of the cyclin-dependent kinase inhibitor p21 in a p53-independent manner w31x. Of

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particular interest is the recent observation that BRCA1 can bind to DNA strand breaks through the BRCT domains similar to TopBP1 w32x. This suggests a role for the BRCT domains of BRCA1 and possibly other BRCT domains, as sensors of DNA strand breaks which can sequester other repair pathway members to sites of DNA damage. 4.2. BARD1 A functionally and structurally related protein the BRCA1 associated Ring Domain ŽBARD1. protein was isolated via a yeast two-hybrid screen for BRCA1 interacting proteins w33x. BARD1 contains a very similar domain structure to BRCA1 possessing an N-terminal RING finger domain and two C-terminal BRCT domains. The expression pattern of BARD1 also mirrors that of BRCA1 w34x, thus implicating both proteins as partners in a common pathway of tumor suppression. The interaction of BARD1 and BRCA1 occurs, however, through interactions between their respective N-terminal RING finger domains w34x. Interactions between the BRCT domains of BARD1 and polyadenylation factor, CstF-50 Žcleavage stimulation factor., a non-BRCT partner, have also been recently characterised w35x. Based upon this interaction and subsequent functional studies w35x, BARD1 has been suggested to play a role in preventing premature or incorrect polyadenylation of nascent transcripts, potentially where polymerases stall at sites of DNA damage. 4.3. Terminal deoxynucleotidyl transferase (TdT) The terminal deoxynucleotidyl transferase protein ŽTdT. catalyses the addition of nucleotides at the junctions of rearranging immunoglobulin and T cell receptor gene segments, thus generating antigen receptor diversity w36x. Recently TdT has been shown to associate via an N-terminal BRCT domain to a non-BRCT partner, Ku, a heterodimeric protein composed of 70 and 86 kDa subunits. The Ku heterodimer binds DNA ends and is required for VŽD.J recombination and DNA double strand break repair w37x. Interestingly the association of full length TdT with Ku is specific and DNA dependent, with the N-terminal BRCT domain interacting only with Ku70 and not with Ku86 or the heterodimeric complex w38x.

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4.4. Topoisomerase II binding protein (TopBP1) The novel DNA topoisomerase II binding protein, designated TopBP1, due to interactions with the C-terminal region of DNA topoisomerase IIb w39x, has aroused much interest recently. TopB1, which possesses eight BRCT regions and is homologous to Rad4 Žcut5., is predicted to participate in DNA repair and cell cycle regulation through binding double strand DNA breaks w30x. An interesting feature of TopBP1 is the fact that several of its BRCT regions have demonstrated binding to both single and double stranded DNA fragments in a sequence independent manner w30x. The demonstration of comparative binding of DNA strand breaks by BRCA1 provides additional evidence that BRCT–non-BRCT interactions are not specific to proteins and that the BRCT domains of some proteins may have multiple roles, including binding to double strand DNA ends. 4.5. XRCC1, DNA ligase III and Poly(ADP–ribose) polymerase The biochemical characterisation of the BRCT– BRCT interaction between the X-ray Repair Cross Complementation 1 protein ŽXRCC1. and DNA ligase III from the base excision repair ŽBER. pathway provided the first clues to the function of BRCT domains w9–11,40x. XRCC1 has no apparent enzyme activity and appears to function as a scaffolding protein in the mammalian BER pathway thereby promoting the interaction of DNA polymerase b and DNA ligase III, as these enzymes do not interact directly w12x. The N-terminal BRCT domain of XRCC1 interacts with the BRCT domain of PolyŽADP–ribose. polymerase ŽPARP. w12,13x, while the XRCC1 C-terminal BRCT domain interacts with the BRCT domain of DNA ligase III forming a BRCT domain heterodimer w9–11x. Indeed the specific interaction between XRCC1 and DNA ligase III is required for ligase activity, and reduced ligase activity in XRCC1 mutants correlates with a deficiency in double strand break repair w41,42x.

recombination w43x. DNA ligase IV contains two C-terminal BRCT domains, similar to BARD1 and BRCA1, and binds to XRCC4. In human DNA ligase IV, the two BRCT domains are separated by linker region that is necessary and sufficient for the interaction with XRCC4 w44x. Indeed the association of these two proteins enhances the DNA end joining and binding activities of ligase IV w43,45x. Recently, the yeast homologue of XRCC4, Lif1p has been shown to be required for yeast Lig4p ŽDNA ligase IV. stability, with the Lig4p BRCT domains essential for NHEJ activity in vivo w46x. Furthermore, Lif1p stimulates the in vitro catalytic activity of Lig4p and is required in vivo for NHEJ activity w46x. There is evidence to suggest that Lif1p binds DNA and together with Ku targets Lig4p to DNA double strand breaks, an interaction that is dependent on the Lig4p BRCT domains w46x. 4.7. Rad9 The Rad9 cell cycle checkpoint protein from Saccharomyces ceriÕisiae is required for transient cellcycle arrest and the induction of DNA-repair genes in response to DNA damage w47x. Rad9 has been shown to homo-oligomerise via it’s C-terminal BRCT domains to form a stable RAD9 multimer w48x. The absence of further RAD9 BRCT –mediated interactions with other proteins from S. ceriÕisiae provides additional evidence for the specificity of the Rad9 BRCT–BRCT interaction w48x. The demonstration that the Rad9 BRCT domain preferentially interacts with hyperphosphorylated forms of Rad9 w48x, provides some evidence to suggest that phosphorylation could regulate BRCT domain interactions. BRCA1 has been recognised as being extensively phosphorylated and several sites lie close to the start of its first BRCT domain. There is therefore scope for regulating BRCT function via phosphorylation, at least in Rad9 and possibly in BRCA1. 5. Insights into possible BRCT packing arrangements from XRCC1 crystal structures

4.6. DNA ligase IV and XRCC4 DNA ligase IV plays a critical role in the non-homologous end-joining ŽNHEJ. pathway of DNA double strand break repair and site-specific VŽD.J

Functional interactions between different proteins via BRCT domains have been demonstrated Žsee above., suggesting that one functional role for BRCT domains is to mediate specific protein–protein inter-

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actions. Many proteins containing BRCT domains have been shown to interact with specific protein partners through either homorhetero-BRCT–BRCT or BRCT–non-BRCT interactions. The BRCT domain of XRCC1 forms a non-crystallographic homodimer in the crystal structure ŽFig. 4a. w5x. The interface is composed of symmetric interactions between helix a1 and N-terminal residues which form a predominantly electrostatic interface ŽFig. 4b.. The intermolecular interactions comprise a number of residues including two salt bridges between residues Arg23–Glu35 and Arg27–Asp4 of each molecule,

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respectively. Residues Asp4 and Arg23 from the XRCC1 non-crystallographic dimer interface are conserved in the DNA ligase III BRCT domain. A slight conservative rearrangement also occurs in DNA ligase III at the Glu35 and Arg27 positions. Glu35 is replaced by Asp in Ligase III and both Arg27 and Glu35 ŽAsp in ligase III. are shifted one residue towards the N-terminus. The buried surface area at ˚ 2 Ž750 A˚ 2 per subunit. w50x, a the interface is 1501 A value which is reported to be significant for a protein–protein interface w51x. This trigonal crystal structure therefore provides a model for a homorhet-

Fig. 4. XRCC1 BRCT homodimer structure. Ža. A ribbon representation of the XRCC1 a1–a1 non-crystallographic homodimer. The two monomers are twofold symmetrically related and interact primarily via interactions form a1 and the N-terminal region. a-helices are coloured blue, b-strands magenta and coil regions grey. Žb. A close up view of the a1–a1 interface showing the predominantly electrostatic interactions between both monomers. Two symmetric salt bridges, Arg23–Glu35 and Arg27–Asp4 and a hydrophobic interaction involving Thr30 are illustrated. Side chains at the dimer interface are shown and coloured by atom type.

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ero BRCT domain interface. Interestingly, the interface interactions between the a1 and N-terminal regions of the XRCC1 a1–a1 dimer correlate well with the corresponding sequence substitutions within the DNA ligase III BRCT domain, suggesting this to be representative of XRCC1-DNA ligase III BRCT packing. Indeed, relative to the low sequence identity between members of the BRCT family Žsee Fig. 2B, there is a significant sequence identity between the C-terminal XRCC1 BRCT domain and the DNA ligase III BRCT domain Žapprox. 30%; Fig. 5.. The finding that 20% of the conserved residues are located at the putative dimer interface may suggest additional evolutionary constraints on the fold in order to conserve this. In contrast, there is very little sequence identity between BRCT domains of DNA ligase III and PARP Žapprox. 5%., BRCT domains which do not interact with each other ŽFig. 5.. In the context of the BRCT superfamily, the high degree of sequence homology corresponding to a1 and a 3, could reflect the conservation of a protein– protein interaction interface. However, a second tetragonal crystal form of XRCC1, that contains two XRCC1–BRCT homodimers within the asymmetric unit, shows an additional close crystal packing arrangement between dimers, formed through hydrophobic interactions from the C1 and C3 loops Žour unpublished results.. Therefore, from the two

Fig. 5. Sequence relationships between BRCT domains within the BER pathway. Phenogram, constructed from percentage sequence identity Žcalculated as for Fig. 2B., showing the relationships between BRCT domains within the BER pathway. Domains are: DNL3 Žhuman DNA ligase III; 846–922., XRC1-N ŽN-terminal domain of human XRCC1; 315–403., XRC1-C ŽC-terminal domain of human XRCC1; 538–629. and PARP Žhuman PolyŽADP–ribose. polymerase; 384–476.. The link between DNL3 and XRC1-C has the highest sequence identity Žapprox. 30%., and between DNL3 and PARP the lowest Žapprox. 5%.. This diagram was created with the PHYLIP computer package w http:rrevolution.genetics.washington.edurphylib.htmlx.

crystal structures of the XRCC1 BRCT domain, we have identified two different packing arrangements for BRCT domains. Using these packing arrangements, our modelling studies of the XRCC1–Ligase III BRCT–BRCT interaction, suggests that the a1– a1 packing is characteristic of an inter-BRCT association while the C1rC3 interaction is a potential intra-BRCT association. This latter interface could therefore represent interactions between BRCT domains within BRCT duplicates and tandem repeats, for example BRCA1 Žsee below.. 6. Structural insights into BRCA1 breast cancer predisposing mutations and BRCA1 function The identification of mutations in the breast cancer susceptibility gene BRCA1 has provided the opportunity to help identify women who are at high risk of developing breast cancer. At present, known BRCA1 mutations are collated at the Breast Cancer Information Core Žwww.nhgri.nih.govrintramural – researchrlab – transferrbicr. and are freely available to researchers. Mutations are divided into several categories including Missense, Nonsense Polymorphisms, Frame Shift and Unclassified Variants and mutation data is provided for each exon separately. BRCA1 comprises 24 exons with the BRCT domains spanning exons 16–24. Of particular interest, is the significance to breast cancer risk associated with unclassified variants, where genetic penetrance studies are not available to assess their effects. In order therefore, to analyse the spatial distribution and structural consequences of BRCT mutations, we have constructed models for both BRCA1 BRCT domains from the crystal structure of XRCC1 w5x ŽFig. 6a.. Alignments for the models were initially taken from the Pfam database w8x, although some minor manual re-alignments were necessary. Loop modelling and side chain replacements were made via a modelling protocol described previously w49x. These comparative models have now allowed the first structural insights into the effects of BRCT mutations within BRCA1, the results of which are summarised in Table 1. The most frequently occurring mutation within both BRCT domains is the insertion of a cytosine base at position 5382 Žthe mutant 5382insC.. This mutation would result in a BRCA1 mutant protein

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Fig. 6. Comparative models of the BRCA1 BRCT domains. Ža. Ribbon representations of the BRCA1 BRCT domain models. The models are based on the XRCC1 BRCT crystal structure. Residues corresponding to some of the cancer predisposing missense mutations from the Breast Cancer Information Core Žwww.nhgri.nih.govrintramural – researchrlab – transferrbic. database have been mapped onto these models and are labelled. The C1rC3 interface is indicated on the individual models. Residues corresponding to missense mutations are coloured with potential interface residues coloured orange, surface residues green and buried residues yellow. a-helices are coloured blue, b-strands magenta, and coil regions grey. Žb. A surface representation of the BRCA1 BRCT C1rC3 interface model. The individual domain ˚ Missense models were superimposed on the XRCC1 C1rC3 interface and surface features calculated with a probe radius of 1.4 A. mutations are coloured as described in Ža..

that contains a nonsense coding region, comprising most of the second BRCT domain terminating at residue 1829. This would result in the effective loss of the most C-terminal BRCT domain. Within both BRCT domains, there are 38 other mutations that

result in either premature stop codons or nonsense coding regions. These would all have significant effects on the functioning of the BRCA1 BRCT domains, and most probably represent loss of function mutations. Equally deleterious are mutations that

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results in In Frame deletions, namely the removal of several amino acids from the coding frame. There are five such mutations within the BRCT domains, three of which occur in secondary structure elements. We would predict these to adversely effect the folding andror stability of the BRCT domain, and thus would probably be loss of function mutations. The third class of mutations are missense mutations, where individual amino acids become substituted ŽTable 1.. This class of mutations probably provides the greatest challenge to clinicians, in that they occur infrequently and their cancer predisposition-risk cannot at present be properly assessed. Of these mutations, ; 30% in the first BRCT domain are conserved BRCT family residues, whereas in the second BRCT domain, nearly 50% are at conserved positions ŽTable 1.. Mutations located within the conserved hydrophobic core of the BRCT domains would undoubtedly prevent correct folding or indeed destabilise the BRCT fold, depending on the nature of the mutation. These mutations are therefore denoted FS ŽFold Stability. in Table 1 and are predicted to lead to non-functional BRCT domains. Particularly deleterious examples could include the substitution of the absolutely conserved Trp in the BRCT family with Arg ŽW1837R. or the replacement of the conserved Gly between a1 and b2 to Val ŽG1788V.. Interestingly, a significant number of missense mutations result in alterations of variable residues within the BRCT family ŽTable 1.. In the BRCA1 BRCT models, these residues are found on the surface and thus could be involved in mediating BRCT protein–protein interactions including intra BRCT– BRCT interactions or BRCT–DNA interactions. Since there are limited data for many of the BRCA1 BRCT-mediated protein interactions, all missense mutations that map to the surface of the BRCT domains have been denoted PI ŽProtein Interaction. in Table 1, and could therefore disrupt potential BRCT–protein interactions. In order to address whether the two BRCA1 BRCT domains can form a specific intra-BRCT–BRCT complex, the XRCC1 BRCT packing arrangement was used as a basis for further modelling. Each individual BRCA1 BRCT domain was superimposed upon both XRCC1 packing arrangements observed in the two different crystal forms. The C1rC3 domain packing arrangement from the tetragonal crystal form appeared more suit-

able for an intra-BRCT–BRCT model, given that the internal linker could disrupt the a1–a1 packing arrangement observed in the trigonal form. Furthermore, the C1rC3 model required only slight adjustments within the two domains to remove steric clashes and improve the stereochemical quality of the final model. The interaction of the predominantly hydrophobic residues Leu1657, Phe1695, Pro1771, Thr1773, Phe1798, and Trp1815 provide a well packed BRCT–BRCT interface ŽFig. 6b.. For this ˚2 interface the total buried surface area is 1133 A 2 ˚ per subunit. w50x, a value which is reported Ž567 A to be significant for a protein–protein interface w51x. The distance representing the linker region between ˚ which would both domains was measured at 41 A, allow the 19 residue linker to span the two domains. Interestingly in our model, several of the missense mutations cluster at the interface between the two BRCT domains, at the base of a small cleft ŽFig. 6b. and have been labelled IF ŽInterface. residues in Table 1. It is plausible that the effects of these mutations could be to disrupt any intra-BRCT associations ŽDI; Disrupt Interface in Table 1.. As the M1775R mutation, previously shown to affect the binding of RNA helicase A, also lies at the base of this cleft, mutations in this region could lead to a decrease in binding of partner proteins. The other surface mutations are dispersed over the surface of the intra-BRCT model, however it is possible that these mutations could affect the association of some of the other partner proteins that associate with BRCA1, through its BRCT domains. It has previously been shown that the putative transcriptional activation domain of BRCA1 maps to a minimal region consisting of residues 1760–1863 from the second BRCT domain w21,22x. The transcriptional activity of this region alone is significantly less than for a construct that contains both BRCT domains, suggesting a potential requirement for a intra-BRCT–BRCT association. Interestingly, the predisposing mutations A1708E, P1749R, and M1775R also abolish any transcriptional transactivation activity. These mutations are located within the first BRCT domain, linker, and the second BRCT domain, respectively and map spatially close in our intra-BRCT model. The P1749R mutation lies within the linker residues Ž1737–1755. but potentially close to the intra-BRCT interface ŽFig. 6b..

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7. Future prospects Since the initial identification of the BRCT motif within BRCA1, a large number of studies have established several functional roles for BRCT domains. There is good evidence to support BRCT domains acting as protein–protein interaction modules. In particular BRCT domains have been shown to interact with other BRCT domains to form homo andror hetero BRCT–BRCT complexes, e.g., XRCC1 and DNA ligase III, Rad9. In addition, there are examples of BRCT domains interacting with non-BRCT proteins, e.g., p53 and p53BP1. Recently, tandem BRCT domains have been shown to bind directly to DNA strand breaks, e.g., TopBP1 and BRCA1. These latter observations are of particular interest, in that they support a multifunctional role for BRCT domains, which in the context of DNA repair, may involve binding to DNA strand breaks before sequestering other repair proteins to those sites of DNA damage. Clearly, the observed mutations in BRCA1 support a key functional role for BRCT domains in tumor suppression, which for BRCA1 probably involves an S-phase DNA repair mechanism. Other DNA repair activities including NHEJ and BER also require BRCT domains ŽDNA ligase IV and III, respectively, XRCC1. illustrating the importantressential functional roles that BRCT domains play in vivo. However, there still remains much to learn about the molecular functions of BRCT domains, which from a structural perspective, includes descriptions of homorhetero BRCT–BRCT and BRCT–non-BRCT complexes. These studies would allow a more detailed characterisation of the BRCT superfamily and may allow predictions as to the molecular roles of individual BRCT motifs.

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