SummaryClustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5Ј-flaps. The crystal structure of YgbT and sitedirected mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.

Background:The human SAMHD1 protein has dNTP triphosphatase activity and is involved in HIV-1 restriction and autoimmune syndrome. Results: Purified SAMHD1 exhibits nuclease activity against single-stranded DNA and RNA. Conclusion: The nuclease activity of SAMHD1 is associated with its HD domain. Significance: Identification of nuclease activity in SAMHD1 provides novel insight into the mechanisms of HIV-1 restriction and regulation of autoimmune response.

decreasing the levels of intracellular dNTPs 14,15 , which apparently compete with the 47 thymidine analog triphosphates for incorporation into HIV-1 cDNA during reverse 48 transcription 16 . We postulated that SAMHD1 could have a similar effect on nucleoside 49analog-based therapy in leukemia 6 . 50To investigate whether SAMHD1 expression enhances Ara-C cytotoxicity in AML 51 cells, we tested whether Ara-C sensitivity in 13 AML cell lines, determined by the half 52 maximal inhibitory concentration (IC 50 ), is correlated with SAMHD1 protein and mRNA 53 levels. Both SAMHD1 expression (Fig. 1a and Supplementary Fig. 1) and Ara-C sensitivity 54 (Supplementary Table 1) varied considerably among these cell lines. Unexpectedly, 55 SAMHD1 levels inversely correlated with Ara-C cytotoxicity (p=0.0037, Fig. 1b and 56 Supplementary Fig. 2a,b), as well as with the levels of early (Caspase 3 and 7 activity, 57 p=0.02, Supplementary Fig. 3a,b) and late (sub-G1 cells, apoptotic DNA fragmentation, 58 p=0.029, Supplementary Fig. 3c,d) markers of apoptosis. In contrast, no significant 59 correlation could be established between Ara-C IC 50 values and the expression of cellular 60 4 proteins previously implicated in Ara-C uptake or its conversion to Ara-CTP 1 , including 61 equilibrative nucleoside transporter (ENT1/SLC29A1), deoxycytidine kinase (DCK), cytidine 62 deaminase (CDA), deoxycytidilate deaminase (DCTD), or 5'-nucleotidase (NT5C2) (Fig. 63 1a,c-g). 64To further investigate its role in Ara-C resistance, we tested the effects of SAMHD1 65 deficiency by a number of approaches: (i) depletion of SAMHD1 in AML cell lines 66 expressing high endogenous SAMHD1 levels using either lentiviral vectors encoding 67 SAMHD1-specific shRNA or transfection with SAMHD1-specific siRNA; (ii) CRISPR/Cas9-68 mediated disruption of the SAMHD1 gene; and (iii) targeted degradation of SAMHD1 using 69 virus-like particles (VLPs) which shuttle the SAMHD1-interacting lentiviral Vpx protein 70 (Vpx-VLPs) into cells 7,8,17 (Fig. 2a and Supplementary Fig. 4). Vpx recruits SAMHD1 to a 71 cullin4A-RING E3 ubiquitin ligase (CRL4 DCAF1 ), which targets the enzyme for proteasomal 72 degradation 7,8 . 73SAMHD1 depletion in AML cell lines by RNA interference (OCI-AML3, THP-1), 74 SAMHD1 knockout (THP-1 -/-), or transduction with Vpx-VLPs (MonoMac6 cells, THP-1) 75 markedly sensitized AML cell lines to Ara-C toxicity relative to the respective controls (Fig. 76 2a,b and Supplementary Fig. 4). In contrast, SAMHD1 siRNA had only a marginal effect on 77 Ara-C toxicity in low SAMHD1-expressing HEL cells (Fig. 2a,b). Interestingly, we observed 78 SAMHD1 dependency, although less pronounced, for the purine analog fludarabine 79 ( Supplementary Fig. 5a); however, the IC 50 values for the topoisomerase II inhibitors 80 etoposide and daunorubicin, as well as for dFdC (2',2'-difluorodeoxycytidine; gemcitabine), 81were not consistently affected by SAMHD1 down-modulation ( Supplementary Fig. 5b-d), 82 indicating a certain degree of drug specificity. 83 5In HEL...

Cyclic diguanylate (c-di-GMP) is a ubiquitous second messenger regulating diverse cellular functions including motility, biofilm formation, cell cycle progression and virulence in bacteria. In the cell, degradation of c-di-GMP is catalyzed by highly specific EAL domain phosphodiesterases whose catalytic mechanism is still unclear. Here, we purified 13 EAL domain proteins from various organisms and demonstrated that their catalytic activity is associated with the presence of 10 conserved EAL domain residues. The crystal structure of the TDB1265 EAL domain was determined in a free state (1.8 Å) and in complex with c-di-GMP (2.35 Å) and unveiled the role of the conserved residues in substrate binding and catalysis. The structure revealed the presence of two metal ions directly coordinated by six conserved residues, two oxygens of the c-di-GMP phosphate, and potential catalytic water molecule. Our results support a two-metal-ion catalytic mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.

SUMMARY Parkinson’s disease (PD) is a common neurodegenerative disorder caused by loss of midbrain dopaminergic neurons, the pathogenetic mechanisms of which remain unclear. Mitochondrial dysfunction, which has long been implicated in sporadic PD, has recently been highlighted as a key pathological cause, particularly with the identification of mutations in the PTEN-induced putative kinase (pink1), parkin and htrA2 (also known as omi) genes that are linked to PD. Studies in Drosophila melanogaster have shown that pink1 and parkin act in a common genetic pathway that maintains mitochondrial integrity, but other upstream or downstream components of this pathway are currently unknown. Using ectopic expression in the Drosophila eye as an assay, we have investigated the involvement of the mitochondrial protease encoded by omi in the Pink1/Parkin pathway and found that it acts genetically downstream of pink1 but functions independently of Parkin. Using the same approach, we also found that Rhomboid-7, a mitochondrial protease not previously implicated in PD, acts as an upstream component of this pathway, and showed that it is required to cleave the precursor forms of both Pink1 and Omi. These data further elucidate the composition of the Pink1 pathway and suggest that regulated intramembrane proteolysis is involved in its regulation.