Aidan J. Doherty

EMSY Links the BRCA2 Pathway to Sporadic Breast and Ovarian Cancer

The BRCA2 gene is mutated in familial breast and ovarian cancer, and its product is implicated in DNA repair and transcriptional regulation. Here we identify a protein, EMSY, which binds BRCA2 within a region (exon 3) deleted in cancer. EMSY is capable of silencing the activation potential of BRCA2 exon 3, associates with chromatin regulators HP1beta and BS69, and localizes to sites of repair following DNA damage. EMSY maps to chromosome 11q13.5, a region known to be involved in breast and ovarian cancer. We show that the EMSY gene is amplified almost exclusively in sporadic breast cancer (13%) and higher-grade ovarian cancer (17%). In addition, EMSY amplification is associated with worse survival, particularly in node-negative breast cancer, suggesting that it may be of prognostic value. The remarkable clinical overlap between sporadic EMSY amplification and familial BRCA2 deletion implicates a BRCA2 pathway in sporadic breast and ovarian cancer.

Identification of a defect in DNA ligase IV in a radiosensitive leukaemia patient

The major mechanism for the repair of DNA double-strand breaks (DSBs) in mammalian cells is non-homologous end-joining (NHEJ), a process that involves the DNA-dependent protein kinase [1] [2], XRCC4 and DNA ligase IV [3] [4] [5] [6]. Rodent cells and mice defective in these components are radiation-sensitive and defective in V(D)J-recombination, showing that NHEJ also functions to rejoin DSBs introduced during lymphocyte development [7] [8]. 180BR is a radiosensitive cell line defective in DSB repair, which was derived from a leukaemia patient who was highly sensitive to radiotherapy [9] [10] [11]. We have identified a mutation within a highly conserved motif encompassing the active site in DNA ligase IV from 180BR cells. The mutated protein is severely compromised in its ability to form a stable enzyme-adenylate complex, although residual activity can be detected at high ATP concentrations. Our results characterize the first patient with a defect in an NHEJ component and suggest that a significant defect in NHEJ that leads to pronounced radiosensitivity is compatible with normal human viability and does not cause any major immune dysfunction. The defect, however, may confer a predisposition to leukaemia.

A Heterotrimeric PCNA in the Hyperthermophilic Archaeon Sulfolobus solfataricus

The sliding clamp, PCNA, of the archaeon Sulfolobus solfataricus P2 is a heterotrimer of three distinct subunits (PCNA1, 2, and 3) that assembles in a defined manner. The PCNA heterotrimer, but not individual subunits, stimulates the activities of the DNA polymerase, DNA ligase I, and the flap endonuclease (FEN1) of S. solfataricus. Distinct PCNA subunits contact DNA polymerase, DNA ligase, or FEN1, imposing a defined architecture at the lagging strand fork and suggesting the existence of a preformed scanning complex at the fork. This provides a mechanism to tightly couple DNA synthesis and Okazaki fragment maturation. Additionally, unique subunit-specific interactions between components of the clamp loader, RFC, suggest a model for clamp loading of PCNA.

Crystal structure of human 53BP1 BRCT domains bound to p53 tumour suppressor.

The BRCT (BRCA1 C‐terminus) is an evolutionary conserved protein–protein interacting module found as single, tandem or multiple repeats in a diverse range of proteins known to play roles in the DNA‐damage response. The BRCT domains of 53BP1 bind to the tumour suppressor p53. To investigate the nature of this interaction, we have determined the crystal structure of the 53BP1 BRCT tandem repeat in complex with the DNA‐binding domain of p53. The structure of the 53BP1–p53 complex shows that the BRCT tandem repeats pack together through a conserved interface that also involves the inter‐domain linker. A comparison of the structure of the BRCT region of 53BP1 with the BRCA1 BRCT tandem repeat reveals that the interdomain interface and linker regions are remarkably well conserved. 53BP1 binds to p53 through contacts with the N‐terminal BRCT repeat and the inter‐BRCT linker. The p53 residues involved in this binding are mutated in cancer and are also important for DNA binding. We propose that BRCT domains bind to cellular target proteins through a conserved structural element termed the ‘BRCT recognition motif’.

Identification of bacterial homologues of the Ku DNA repair proteins

Double-strand breaks (DSB) arise in DNA as a result of damage by ionising radiation and radio-mimetic chemicals, and in mammalian cells, during the V(D)J chain recombination events [1]. Repair of DSB in eukaryotic cells can occur via a homologous recombination pathway involving RAD51 and RAD52, or via a non-homologous double-strand endjoining pathway (NHEJ), a form of DSB repair [1]. Although both pathways are present in all eukaryotes, in yeast, repair of DSB is primarily achieved by homologous recombination, whereas NHEJ repair is the predominant mechanism in vertebrates [1]. The NHEJ pathway in higher eukaryotes is dependent on a multiprotein complex, DNA-dependent protein kinase (DNA-PK) [1] which consists of a DNA-binding component, a heterodimer of two proteins, Ku70 and Ku80, and a catalytic subunit, DNA-PKcs. The main function of the Ku70/80 component of DNA-PK, is the primary recognition of DSB. Ku displays high a¤nity in vitro for a variety of DNA ends including blunt ends, 5P and 3P overhangs, and DNA hairpins which occur as intermediates in V(D)J chain recombination [2]. In mammalian cells, the Ku heterodimer recruits the catalytic subunit of DNA-PK, which is dependent on association with the Ku70/Ku80 heterodimer bound to DNA, for its protein kinase activity. When localised on damaged DNA by its Ku heterodimer targeting subunit, DNA-PK is an active protein kinase, preferentially phosphorylating proteins bound on the DNA. DNA-PK may also act as a binding site for recruiting other proteins, such as DNA ligases, directly involved in the repair of the double-stranded breaks. The presence of a protein kinase activity in DNA-PK suggests that it may also have a role in signaling the presence of DNA damage to cell-cycle checkpoint and apoptosis regulating systems. In yeast, in vivo, the end-binding activity of the Ku heterodimer appears to signi¢cantly stabilise DNA ends, and promotes e¤cient and accurate illegitimate end-joining repair. Mammalian cells that are de¢cient in Ku protein are sensitive to ionising radiation. The DNA end-binding activity of Ku may also be involved in protection of chromosomal ends from nucleases, as yeast de¢cient in Ku70 or Ku80 display marked telomere attrition [1,2]. Ku protein homologues have been identi¢ed in vertebrates, insects, Caenorhabditis elegans and yeast [2] but not, to date, in prokaryotes. It is important to understand the origins of these proteins because of the central role of Ku proteins in the double-strand DNA break repair pathway. The protein sequence database was searched for proteins with signi¢cant homology to the Ku80 protein (Arabidopsis thaliana) using iterative PSI-BLAST database searches [3] run to convergence with an E value of 0.01. In the third iteration of these searches a number of putative proteins were detected within several bacterial and archael genomes with signi¢cant homology to Ku70 and Ku80 (Fig. 1A). These include the proteins encoded by YkoV (Bacillus subtilis), BH2208 (Bacillus halodurans), SC6G9.24c, SCP1.285c, SCF55.25c (Streptomyces coelicolor), Rv0937c (Mycobacterium tuberculosis), PA2150 (Pseudomonas aeruginosa), Mlr9623, Mlr9624, Mll4607, Mll2074 (Mesorhizobium loti) and AFI1726 (Archaeoglobus fulgidus). Reverse PSI-blast searches of the protein sequence database using each of these bacterial Ku proteins retrieved not only each other, but also many of the eukaryotic Ku70 and Ku80 proteins with high accuracy. Our ¢ndings strongly suggest the existence of prokaryotic homologues of the Ku70 and Ku80 family of proteins. The bacterial Ku proteins are composed of approximately 270^350 amino acids. In contrast, the eukaryotic Ku proteins are much larger (70^80 kDa). The bacterial Ku homologues appear to represent a previously undetected conserved domain at the centre of the larger Ku proteins (Fig. 1B). This common core structure is conserved between amino acids 210 and 550 approximately in the eukaryotic Ku proteins. This is supported by the ¢ndings of a number of groups who have shown that this region of Ku70 and Ku80 is one of the major determinants for both heterodimerisation and DNA binding [2]. Recently, it has also been reported that this region of Ku also mediates interactions with other nuclear proteins. In the absence of any known structure for the Ku proteins the structure of the bacterial Ku proteins was analysed using the secondary structure prediction program, PSIPRED [4]. This is a simple and reliable secondary structure prediction method, incorporating two feed-forward neural networks which perform an analysis on output obtained from PSIBLAST. The N-terminal region (aa 1^80 approx.) is predicted to form a L-sheet sub-domain. This is followed by a much more highly conserved region (aa 80^170 approx.) with an K/L fold and ¢nally, a highly K-helical structure at the C-terminal end of the proteins. The eukaryotic Ku proteins have acquired additional domains, presumably to enhance their DNA repair role within the cell. The N-terminal region of Ku70 and Ku80 (aa 1^240 approx.) comprises a divergent member of the Von Willebrant factor A (VWA) domain (Fig. 1B) [5]. This domain is a protein^protein interacting module and has been implicated in the heterodimerisation of Ku70 and Ku80 [5]. It is likely that this domain also plays a role in sequestering other proteins to sites of DNA damage, forming larger protein complexes required for concerted DNA repair. Aravind and Koonin recently identi¢ed a SAP motif [6], a putative DNAbinding module, in a number of DNA-binding proteins including in the C-terminal region of eukaryotic Ku70 proteins. The SAP motif is composed of two amphipatic helices connected by a conserved loop of invariant length [6]. This motif appears to represent a new bi-helical DNA-binding motif that di¡ers from other motifs such as the Helix^hairpin^Helix (HhH), and Helix^turn^Helix (HtH). Do similar SAP motifs exist in the bacterial Ku proteins? Iterative PSI-BLAST searches identi¢ed signi¢cant homology between the C-terminal end of the S. coelicolor Ku protein, SCF55.25c and the

Making Ends Meet: Repairing Breaks in Bacterial DNA by Non-Homologous End-Joining

DNA double-strand breaks (DSBs) are one of the most dangerous forms of DNA lesion that can result in genomic instability and cell death. Therefore cells have developed elaborate DSB-repair pathways to maintain the integrity of genomic DNA. There are two major pathways for the repair of DSBs in eukaryotes: homologous recombination and non-homologous end-joining (NHEJ). Until very recently, the NHEJ pathway had been thought to be restricted to the eukarya. However, an evolutionarily related NHEJ apparatus has now been identified and characterized in the prokarya. Here we review the recent discoveries concerning bacterial NHEJ and discuss the possible origins of this repair system. We also examine the insights gained from the recent cellular and biochemical studies of this DSB-repair process and discuss the possible cellular roles of an NHEJ pathway in the life-cycle of prokaryotes and phages.

Role of DNA Repair by Nonhomologous-End Joining in Bacillus subtilis Spore Resistance to Extreme Dryness, Mono- and Polychromatic UV, and Ionizing Radiation

The role of DNA repair by nonhomologous-end joining (NHEJ) in spore resistance to UV, ionizing radiation, and ultrahigh vacuum was studied in wild-type and DNA repair mutants (recA, splB, ykoU, ykoV, and ykoU ykoV mutants) of Bacillus subtilis. NHEJ-defective spores with mutations in ykoU, ykoV, and ykoU ykoV were significantly more sensitive to UV, ionizing radiation, and ultrahigh vacuum than wild-type spores, indicating that NHEJ provides an important pathway during spore germination for repair of DNA double-strand breaks.

DNA repair: How Ku makes ends meet.

The recently determined crystal structure of the Ku heterodimer, in both DNA-bound and unbound forms, has shed new light on the mechanism by which this protein fulfills its key role in the repair of DNA double-strand breaks.

PrimPol Bypasses UV Photoproducts during Eukaryotic Chromosomal DNA Replication

Summary DNA damage can stall the DNA replication machinery, leading to genomic instability. Thus, numerous mechanisms exist to complete genome duplication in the absence of a pristine DNA template, but identification of the enzymes involved remains incomplete. Here, we establish that Primase-Polymerase (PrimPol; CCDC111), an archaeal-eukaryotic primase (AEP) in eukaryotic cells, is involved in chromosomal DNA replication. PrimPol is required for replication fork progression on ultraviolet (UV) light-damaged DNA templates, possibly mediated by its ability to catalyze translesion synthesis (TLS) of these lesions. This PrimPol UV lesion bypass pathway is not epistatic with the Pol η-dependent pathway and, as a consequence, protects xeroderma pigmentosum variant (XP-V) patient cells from UV-induced cytotoxicity. In addition, we establish that PrimPol is also required for efficient replication fork progression during an unperturbed S phase. These and other findings indicate that PrimPol is an important player in replication fork progression in eukaryotic cells.

Evolutionary and functional conservation of the DNA non-homologous end-joining protein, XLF/Cernunnos.

Non-homologous end-joining is a major pathway of DNA double-strand break repair in mammalian cells, deficiency in which confers radiosensitivity and immune deficiency at the whole organism level. A core protein complex comprising the Ku70/80 heterodimer together with a complex between DNA ligase IV and XRCC4 is conserved throughout eukaryotes and assembles at double-strand breaks to mediate ligation of broken DNA ends. In Saccharomyces cerevisiae an additional NHEJ protein, Nej1p, physically interacts with the ligase IV complex and is required in vivo for ligation of DNA double-strand breaks. Recent studies with cells derived from radiosensitive and immune-deficient patients have identified the human protein, XLF (also named Cernunnos), as a crucial NHEJ protein. Here we show that XLF and Nej1p are members of the same protein superfamily and that this family has members in diverse eukaryotes. Indeed, we show that a member of this family encoded by a previously uncharacterized open-reading frame in the Schizosaccharomyces pombe genome is required for NHEJ in this organism. Furthermore, our data reveal that XLF family proteins can bind to DNA and directly interact with the ligase IV-XRCC4 complex to promote DSB ligation. We therefore conclude that XLF family proteins interact with the ligase IV-XRCC4 complex to constitute the evolutionarily conserved enzymatic core of the NHEJ machinery.