SUMMARY Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as ‘ciliopathies’. However, disease mechanisms remain poorly understood. Here we identify by whole exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway, hitherto not implicated in ciliopathies. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164 and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents, and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. We identify TTBK2, CCDC92, NPHP3 and DVL3 as novel CEP164 interaction partners. Our findings link degenerative diseases of kidney and retina, disorders of increasing prevalence, to mechanisms of DDR.

Summary Kinetoplast DNA (kDNA), the trypanosome mitochondrial DNA, contains thousands of minicircles and dozens of maxicircles interlocked in a giant network. Remarkably, Trypanosoma brucei's genome encodes eight PIF1-like helicases, six of which are mitochondrial. We now show that TbPIF2 is essential for maxicircle replication. Maxicircle abundance is controlled by TbPIF2 level, as RNAi of this helicase caused maxicircle loss and its overexpression caused a 3- to 6-fold increase in maxicircle abundance. This regulation of maxicircle level is mediated by the TbHslVU protease. Previous experiments demonstrated that RNAi knockdown of TbHslVU dramatically increased abundance of minicircles and maxicircles, presumably because a positive regulator of their synthesis escaped proteolysis and allowed synthesis to continue. Here we found that TbPIF2 level increases following RNAi of the protease. Therefore this helicase is a TbHslVU substrate and the first example of a positive regulator, thus providing a molecular mechanism for controlling maxicircle replication.

Expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene in T cells is activated by the combination of phorbol ester (phorbol myristate acetate) and calcium ionophore (A23187), which mimic antigen stimulation through the T-cell receptor. We have previously shown that a fragment containing bp -95 to +27 of the mouse GM-CSF promoter can confer inducibility to reporter genes in the human Jurkat T-cell line. Here we use an in vitro transcription system to demonstrate that a cis-acting element (positions -54 to -40), referred to as CLEO, is a target for the induction signals. We observed induction with templates containing intact CLEO but not with templates with deleted or mutated CLEO. We also observed that two distinct signals were required for the stimulation through CLEO, since only extracts from cells treated with both phorbol myristate acetate and A23187 supported optimal induction. Stimulation probably was mediated by CLEO-binding proteins because depletion of these proteins specifically reduced GM-CSF transcription. One of the binding factors possessed biochemical and immunological features identical to those of the transcription factor APi. Another factor resembled the T-cell-specific factor NFAT. The characteristics of these two factors are consistent with their involvement in GM-CSF induction. The presence of CLEO-like elements in the promoters of interleukin-3 (IL-3), IL-4, IL.5, GM-CSF, and NFAT sites in the IL-2 promoter suggests that the factors we detected, or related factors that recognize these sites, may account for the coordinate induction of these genes during T-cell activation.

Replication of kinetoplast DNA minicircles of trypanosomatids initiates at a conserved 12-nucleotide sequence, termed the universal minicircle sequence (UMS, 5-GGGGTTGGTGTA-3). A single-stranded nucleic acid binding protein that binds specifically to this origin-associated sequence was purified to apparent homogeneity from Crithidia fasciculata cell extracts. This UMS-binding protein (UMSBP) is a dimer of 27.4 kDa with a 13.7-kDa protomer. UMSBP binds single-stranded DNA as well as single-stranded RNA but not doublestranded or four-stranded DNA structures. Stoichiometry analysis indicates the binding of UMSBP as a protein dimer to the UMS site. The five CCHC-type zinc finger motifs of UMSBP, predicted from its cDNA sequence, are similar to the CCHC motifs found in retroviral Gag polyproteins. The remarkable conservation of this motif in a family of proteins found in eukaryotic organisms from yeast and protozoa to mammals is discussed.Kinetoplast DNA (kDNA) 1 is a unique extrachromosomal DNA network found in the single mitochondrion of parasitic flagellated protozoa of the family Trypanosomatidae. In Crithidia fasciculata, kDNA consists of about 5,000 DNA minicircles (2.5 kilobase pairs each) and about 50 DNA maxicircles (37 kilobase pairs each) interlocked topologically to form a huge DNA network (for review, see Refs. 1-3). Minicircles are heterogeneous in their nucleotide sequence but contain two short sequences, 70 -100 base pairs apart, that are conserved in all the species studied so far: the dodecamer sequence known as universal minicircle sequence (UMS), 5Ј-GGGGTTGGTGTA-3Ј, and the hexamer sequence 5Ј-ACGCCC-3Ј.On the basis of in vivo observations, Englund and co-workers (4 -8) have described the replication of kDNA minicircles as a process in which individual minicircles are detached from the central zone of the disc-shaped network, replicated, and reattached to the periphery of the disc. The network increases in size until it doubles and then divides and segregates into two daughter networks. Extensive studies of minicircle replication intermediates (9 -16) have suggested that replication begins at the UMS site with synthesis of an RNA primer and proceeds by continuous elongation of the leading light strand (L-strand). A single gap of 6 -10 nucleotides remains in the newly synthesized light strand at the UMS site (13) and is repaired only after replication of the minicircles and their reattachment to the network have been completed (8). Discontinuous synthesis of the lagging heavy strand (H-strand) starts when its origin, containing the conserved hexamer sequence, is exposed by the advancing replication fork. Highly gapped and nicked nascent H-strands are generated.We have previously reported on the recognition of UMS by a unique sequence-specific single-stranded DNA binding protein from C. fasciculata (17) and on the isolation and analysis of the UMSBP-encoding cDNA (18). The amino acid sequence of the polypeptide, predicted from the cDNA, is 116 residues long and contains five Cys-X 2 -Cys-X 4 -His-...

Protein n', a prepriming DNA replication enzyme of Escherichia coli, is a 46X174 DNA-dependent ATPase DNA, by primase (4, 5). However, SSB-coated OX DNA, although primed by the same E. coli primase, must first be activated in a prepriming stage. In this prepriming reaction, the E. coli proteins n', n and n", i, dnaB, and dnaC form an activated complex with OX DNA (2, 6-8).The distinctions in primer synthesis on these three phage templates are probably due to structural differences in "promoter"-like sites recognized by the distinctive priming systems. Although the origin of complementary DNA strand replication is unique and well characterized for M13 (9) and G4 (10-14), it does not appear to be at a unique site for OX, as judged by in vivo and in vitro studies (7, 9, 11, 15).We will describe elsewhere the purification of protein n' to near homogeneity, its 4X DNA-dependent ATPase activity, and its capacity to destabilize an SSB-qX DNA complex. In this paper we report the recognition by protein n' of a specific sequence in qX DNA located at an intergenic region and suggest that it may be the signal that leads to the initiation of OX complementary DNA strand replication. MATERIALS AND METHODSNucleic Acids, Enzymes, Resins, and Nucleotides. OX, OX replicative form (RF)I, G4, and M13 DNAs were prepared as described (16) [a-32P]ATP and could also be applied to the production of 32p; from ['y-32P]ATP. Standard assays were carried out in 25-1ul reaction mixtures containing 10 mM KC1, 1 mM MgCl2, 1 mM labeled ATP or dATP, 120 pmol of XX DNA (as nucleotide), 50mM Tris-HCl (pH 7.5), 6% (wt/vol) sucrose, and bovine serum albumin at 0.2 mg/mi. Samples to be assayed were diluted in 50 mM imidazole.HCI, pH 6.8/25% glycerol/100 mM ammonium sulfate/bovine serum albumin at 0.2 mg per mI/i mM EDTA.When SSB was used, 1 Atg was added, unless otherwise noted.Reactions were carried out at 30'C unless otherwise noted.Aliquots of 2 Ml were applied to polyethyleneimine-cellulose strips (0.6 X 6 cm) together with unlabeled ATP, ADP, and AMP markers. The strips were developed with 1 M formic acid/0.5 M LiCI at room temperature, dried, and examined with UV light to locate and cut out the ATP and ADP spots. Radioactivity was determined in scintillation fluid without Abbreviations: OX, kX174; SS, single-stranded circular DNA; RF, double-stranded DNA of circular replicative form; SSB, singlestranded-DNA binding protein; DBM, diazobenzyloxymethyl.