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1.  DNA Repair Pathways in Trypanosomatids: from DNA Repair to Drug Resistance 
All living organisms are continuously faced with endogenous or exogenous stress conditions affecting genome stability. DNA repair pathways act as a defense mechanism, which is essential to maintain DNA integrity. There is much to learn about the regulation and functions of these mechanisms, not only in human cells but also equally in divergent organisms. In trypanosomatids, DNA repair pathways protect the genome against mutations but also act as an adaptive mechanism to promote drug resistance. In this review, we scrutinize the molecular mechanisms and DNA repair pathways which are conserved in trypanosomatids. The recent advances made by the genome consortiums reveal the complete genomic sequences of several pathogens. Therefore, using bioinformatics and genomic sequences, we analyze the conservation of DNA repair proteins and their key protein motifs in trypanosomatids. We thus present a comprehensive view of DNA repair processes in trypanosomatids at the crossroads of DNA repair and drug resistance.
PMCID: PMC3957735  PMID: 24600040
2.  Roles of Rad51 paralogs for promoting homologous recombination in Leishmania infantum 
Nucleic Acids Research  2015;43(5):2701-2715.
To achieve drug resistance Leishmania parasite alters gene copy number by using its repeated sequences widely distributed through the genome. Even though homologous recombination (HR) is ascribed to maintain genome stability, this eukaryote exploits this potent mechanism driven by the Rad51 recombinase to form beneficial extrachromosomal circular amplicons. Here, we provide insights on the formation of these circular amplicons by analyzing the functions of the Rad51 paralogs. We purified three Leishmania infantum Rad51 paralogs homologs (LiRad51-3, LiRad51-4 and LiRad51-6) all of which directly interact with LiRad51. LiRad51-3, LiRad51-4 and LiRad51-6 show differences in DNA binding and annealing capacities. Moreover, it is also noteworthy that LiRad51-3 and LiRad51-4 are able to stimulate Rad51-mediated D-loop formation. In addition, we succeed to inactivate the LiRad51-4 gene and report a decrease of circular amplicons in this mutant. The LiRad51-3 gene was found to be essential for cell viability. Thus, we propose that the LiRad51 paralogs play crucial functions in extrachromosomal circular DNA amplification to circumvent drug actions and preserve survival.
PMCID: PMC4357719  PMID: 25712090
3.  Breast cancer proteins PALB2 and BRCA2 stimulate polymerase η in recombination-associated DNA synthesis at blocked replication forks 
Cell reports  2014;6(3):553-564.
One envisioned function of homologous recombination (HR) is to find a template for DNA synthesis from the resected 3′-OH molecules that occur during double-strand break (DSB) repair at broken or stalled replication forks. However, the interplay between DNA synthesis and HR remains poorly understood in higher eukaryotic cells. Here, we reveal new functions for breast cancer proteins BRCA2 and PALB2 at blocked replication forks and show a role for these proteins in stimulating polymerase eta (Polη) to initiate DNA synthesis. PALB2, BRCA2 and Polη co-localize at stalled or collapsed replication forks after hydroxyurea treatment. Moreover, PALB2 and BRCA2 interact with Polη, and are required to sustain the recruitment of Polη at blocked replication forks. PALB2 and BRCA2 stimulate Polη-dependent DNA synthesis on D-loop substrates. We conclude that PALB2 and BRCA2, in addition to their functions in D-loop formation, play crucial roles in the initiation of recombination-associated DNA synthesis by Polη-mediated DNA repair.
PMCID: PMC4162405  PMID: 24485656
PALB2; BRCA2; polymerase η; DNA replication and homologous recombination
4.  Functional and Structural Basis for a Bacteriophage Homolog of Human RAD52 
Current biology : CB  2008;18(15):1142-1146.
In eukaryotes, homologous recombination proteins such as RAD51 and RAD52 play crucial roles in DNA repair and genome stability. Human RAD52 is a member of a large single-strand annealing protein (SSAP) family [1] and stimulates Rad51-dependent recombination [2, 3]. In prokaryotes and phages, it has been difficult to establish the presence of RAD52 homologs with conserved sequences. Putative SSAPs were recently found in several phages that infect strains of Lactococcus lactis [4]. One of these SSAPs was identified as Sak and was found in the virulent L. lactis phage ul36, which belongs to the Siphoviridae family [4, 5]. In this study, we show that Sak is homologous to the N terminus of human RAD52. Purified Sak binds single-stranded DNA (ssDNA) preferentially over double-stranded DNA (dsDNA) and promotes the renaturation of long complementary ssDNAs. Sak also binds RecA and stimulates homologous recombination reactions. Mutations shown to modulate RAD52 DNA binding [6] affect Sak similarly. Remarkably, electron-microscopic reconstruction of Sak reveals an undecameric (11) subunit ring, similar to the crystal structure of the N-terminal fragment of human RAD52 [7, 8]. For the first time, we propose a viral homolog of RAD52 at the amino acid, phylogenic, functional, and structural levels.
PMCID: PMC4149281  PMID: 18656357
5.  Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination 
Nature structural & molecular biology  2010;17(10):1247-1254.
Inherited mutations in human PALB2 are associated with a predisposition to breast and pancreatic cancers. The tumor-suppressing capability of PALB2 is thought to be based on its ability to enable BRCA2 function in homologous recombination. However, the biochemical properties of PALB2 are unknown. Here we show that human PALB2 binds DNA, preferentially D-loop structures, and directly interacts with the RAD51 recombinase to strongly stimulates strand invasion, a vital step of homologous recombination. Such stimulation occur by reinforcing biochemical mechanisms as PALB2 alleviates the inhibitory role of RPA and stabilizes the RAD51 filament. Moreover, PALB2 can function synergistically with a BRCA2 chimera (termed piccolo) to further promote strand invasion. Finally, we show that PALB2-deficient cells are sensitive to PARP inhibitors. Collectively, our studies provide the first biochemical insights into the homologous recombination mediator functions of PALB2 with piBRCA2 in DNA double-strand break repair.
PMCID: PMC4094107  PMID: 20871615
RAD51; PALB2; BRCA2; homologous recombination
6.  The microRNA pathway controls germ cell proliferation and differentiation in C. elegans 
Cell Research  2012;22(6):1034-1045.
The discovery of the miRNA pathway revealed a new layer of molecular control of biological processes. To uncover new functions of this gene regulatory pathway, we undertook the characterization of the two miRNA-specific Argonaute proteins in Caenorhabditis elegans, ALG-1 and ALG-2. We first observed that the loss-of-function of alg-1 and alg-2 genes resulted in reduced progeny number. An extensive analysis of the germline of these mutants revealed a reduced mitotic region, indicating fewer proliferating germ cells. We also observed an early entry into meiosis in alg-1 and alg-2 mutant animals. We detected ALG-1 and ALG-2 protein expressions in the distal tip cell (DTC), a specialized cell located at the tip of both C. elegans gonadal arms that regulates mitosis-meiosis transition. Re-establishing the expression of alg-1 specifically in the DTC of mutant animals partially rescued the observed germline defects. Further analyses also support the implication of the miRNA pathway in gametogenesis. Interestingly, we observed that disruption of five miRNAs expressed in the DTC led to similar phenotypes. Finally, gene expression analysis of alg-1 mutant gonads suggests that the miRNA pathway is involved in the regulation of different pathways important for germline proliferation and differentiation. Collectively, our data indicate that the miRNA pathway plays a crucial role in the control of germ cell biogenesis in C. elegans.
PMCID: PMC3367518  PMID: 22370633
argonaute; miRNA; germline
7.  PARP activation regulates the RNA-binding protein NONO in the DNA damage response to DNA double-strand breaks 
Nucleic Acids Research  2012;40(20):10287-10301.
After the generation of DNA double-strand breaks (DSBs), poly(ADP-ribose) polymerase-1 (PARP-1) is one of the first proteins to be recruited and activated through its binding to the free DNA ends. Upon activation, PARP-1 uses NAD+ to generate large amounts of poly(ADP-ribose) (PAR), which facilitates the recruitment of DNA repair factors. Here, we identify the RNA-binding protein NONO, a partner protein of SFPQ, as a novel PAR-binding protein. The protein motif being primarily responsible for PAR-binding is the RNA recognition motif 1 (RRM1), which is also crucial for RNA-binding, highlighting a competition between RNA and PAR as they share the same binding site. Strikingly, the in vivo recruitment of NONO to DNA damage sites completely depends on PAR, generated by activated PARP-1. Furthermore, we show that upon PAR-dependent recruitment, NONO stimulates nonhomologous end joining (NHEJ) and represses homologous recombination (HR) in vivo. Our results therefore place NONO after PARP activation in the context of DNA DSB repair pathway decision. Understanding the mechanism of action of proteins that act in the same pathway as PARP-1 is crucial to shed more light onto the effect of interference on PAR-mediated pathways with PARP inhibitors, which have already reached phase III clinical trials but are until date poorly understood.
PMCID: PMC3488241  PMID: 22941645
8.  PALB2 self-interaction controls homologous recombination 
Nucleic Acids Research  2012;40(20):10312-10323.
PALB2 is essential for BRCA2 anchorage to nuclear structures and for homologous recombinational repair of DNA double-strand breaks. Here, we report that the N-terminal coiled-coil motif of PALB2 regulates its self-association and homologous recombination. Monomeric PALB2 shows higher efficiency to bind DNA and promotes RAD51 filament formation with or without the inhibitory effect of Replication Protein A. Moreover, overexpression of the PALB2 coiled-coil domain severely affects RAD51 loading to DNA damage sites suggesting a competition between PALB2 self-interaction and PALB2–BRCA1 interaction. In the presence of DNA damage, the switch between PALB2–PALB2 and PALB2–BRCA1 interactions allows the activation of HR. Controlling HR via PALB2 self-interactions could be important to prevent aberrant recombination in normal conditions and activate DNA repair when required.
PMCID: PMC3488246  PMID: 22941656
9.  Interactions between BRCA2 and RAD51 for promoting homologous recombination in Leishmania infantum 
Nucleic Acids Research  2012;40(14):6570-6584.
In most organisms, the primary function of homologous recombination (HR) is to allow genome protection by the faithful repair of DNA double-strand breaks. The vital step of HR is the search for sequence homology, mediated by the RAD51 recombinase, which is stimulated further by proteins mediators such as the tumor suppressor BRCA2. The biochemical interplay between RAD51 and BRCA2 is unknown in Leishmania or Trypanosoma. Here we show that the Leishmania infantum BRCA2 protein possesses several critical features important for the regulation of DNA recombination at the genetic and biochemical level. A BRCA2 null mutant, generated by gene disruption, displayed genomic instability and gene-targeting defects. Furthermore, cytological studies show that LiRAD51 can no longer localize to the nucleus in this mutant. The Leishmania RAD51 and BRCA2 interact together and the purified proteins bind single-strand DNA. Remarkably, LiBRCA2 is a recombination mediator that stimulates the invasion of a resected DNA double-strand break in an undamaged template by LiRAD51 to form a D-loop structure. Collectively, our data show that LiBRCA2 and LiRAD51 promote HR at the genetic and biochemical level in L. infantum, the causative agent of visceral leishmaniasis.
PMCID: PMC3413117  PMID: 22505581
10.  A Glycine-Arginine Domain in Control of the Human MRE11 DNA Repair Protein▿ †  
Molecular and Cellular Biology  2008;28(9):3058-3069.
Human MRE11 is a key enzyme in DNA double-strand break repair and genome stability. Human MRE11 bears a glycine-arginine-rich (GAR) motif that is conserved among multicellular eukaryotic species. We investigated how this motif influences MRE11 function. Human MRE11 alone or a complex of MRE11, RAD50, and NBS1 (MRN) was methylated in insect cells, suggesting that this modification is conserved during evolution. We demonstrate that PRMT1 interacts with MRE11 but not with the MRN complex, suggesting that MRE11 arginine methylation occurs prior to the binding of NBS1 and RAD50. Moreover, the first six methylated arginines are essential for the regulation of MRE11 DNA binding and nuclease activity. The inhibition of arginine methylation leads to a reduction in MRE11 and RAD51 focus formation on a unique double-strand break in vivo. Furthermore, the MRE11-methylated GAR domain is sufficient for its targeting to DNA damage foci and colocalization with γ-H2AX. These studies highlight an important role for the GAR domain in regulating MRE11 function at the biochemical and cellular levels during DNA double-strand break repair.
PMCID: PMC2293076  PMID: 18285453
11.  Stimulation of fission yeast and mouse Hop2-Mnd1 of the Dmc1 and Rad51 recombinases 
Nucleic Acids Research  2007;35(8):2719-2733.
Genetic analysis of fission yeast suggests a role for the spHop2–Mnd1 proteins in the Rad51 and Dmc1-dependent meiotic recombination pathways. In order to gain biochemical insights into this process, we purified Schizosaccharomyces pombe Hop2-Mnd1 to homogeneity. spHop2 and spMnd1 interact by co-immunoprecipitation and two-hybrid analysis. Electron microscopy reveals that S. pombe Hop2–Mnd1 binds single-strand DNA ends of 3′-tailed DNA. Interestingly, spHop2-Mnd1 promotes the renaturation of complementary single-strand DNA and catalyses strand exchange reactions with short oligonucleotides. Importantly, we show that spHop2-Mnd1 stimulates spDmc1-dependent strand exchange and strand invasion. Ca2+ alleviate the requirement for the order of addition of the proteins on DNA. We also demonstrate that while spHop2-Mnd1 affects spDmc1 specifically, mHop2 or mHop2-Mnd1 stimulates both the hRad51 and hDmc1 recombinases in strand exchange assays. Thus, our results suggest a crucial role for S. pombe and mouse Hop2-Mnd1 in homologous pairing and strand exchange and reveal evolutionary divergence in their specificity for the Dmc1 and Rad51 recombinases.
PMCID: PMC1885673  PMID: 17426123
12.  Fission Yeast Rad51 and Dmc1, Two Efficient DNA Recombinases Forming Helical Nucleoprotein Filaments 
Molecular and Cellular Biology  2005;25(11):4377-4387.
Homologous recombination is important for the repair of double-strand breaks during meiosis. Eukaryotic cells require two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, for meiotic recombination. To date, it is not clear, at the biochemical level, why two homologs of RecA are necessary during meiosis. To gain insight into this, we purified Schizosaccharomyces pombe Rad51 and Dmc1 to homogeneity. Purified Rad51 and Dmc1 form homo-oligomers, bind single-stranded DNA preferentially, and exhibit DNA-stimulated ATPase activity. Both Rad51 and Dmc1 promote the renaturation of complementary single-stranded DNA. Importantly, Rad51 and Dmc1 proteins catalyze ATP-dependent strand exchange reactions with homologous duplex DNA. Electron microscopy reveals that both S. pombe Rad51 and Dmc1 form nucleoprotein filaments. Rad51 formed helical nucleoprotein filaments on single-stranded DNA, whereas Dmc1 was found in two forms, as helical filaments and also as stacked rings. These results demonstrate that Rad51 and Dmc1 are both efficient recombinases in lower eukaryotes and reveal closer functional and structural similarities between the meiotic recombinase Dmc1 and Rad51. The DNA strand exchange activity of both Rad51 and Dmc1 is most likely critical for proper meiotic DNA double-strand break repair in lower eukaryotes.
PMCID: PMC1140613  PMID: 15899844
13.  Arginine methylation of hnRNPUL1 regulates interaction with NBS1 and recruitment to sites of DNA damage 
Scientific Reports  2015;5:10475.
Arginine methylation is a post-translational modification required for the maintenance of genomic integrity. Cells deficient in protein arginine methyltransferase 1 (PRMT1) have DNA damage signaling defects, defective checkpoint activation and extensive genomic instability. Herein we identify the DNA damage protein and RNA binding protein, hnRNPUL1, to be a substrate of PRMT1. We identify the dimethylation of R584, R618, R620, R645, and R656, as well as the monomethylation of R661 R685 and R690 within hnRNPUL1 in U2OS cells by mass spectrometry. Moreover, we define the arginines within the RGG/RG motifs as the site of methylation by PRMT1 both in vitro and in vivo. The arginines 612, 618, 620, 639, 645, 656 and 661 within the human hnRNPUL1 RGG/RG motifs were substituted with lysines to generate hnRNPUL1RK. hnRNPUL1RK was hypomethylated and lacked the ability to interact with PRMT1, unlike wild type hnRNPUL1. Co-immunoprecipitation studies showed that hnRNPUL1RK had impaired ability to associate with the DNA damage protein NBS1. Moreover, hnRNPUL1RK was not recruited to sites of DNA damage, unlike wild type hnRNPUL1, in the presence of transcriptional inhibitors. These findings define a role for arginine methylation during the DNA damage response to regulate protein-protein interactions for the recruitment at sites of damage.
PMCID: PMC4447065  PMID: 26020839
14.  DNA Double Strand Break Repair Pathway Choice Is Directed by Distinct MRE11 Nuclease Activities 
Molecular cell  2013;53(1):7-18.
MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection and signaling; yet, how its endo- and exonuclease activities regulate DSB repair by non-homologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors we examined repair pathway choice at DSBs generated in G2 following radiation exposure. Whilst endo- or exonuclease inhibition impairs radiation-induced RPA chromatin binding, suggesting diminished resection, the inhibitors surprisingly direct different repair outcomes. Endonuclease inhibition promotes NHEJ in lieu of HR, whilst exonuclease inhibition confers a repair defect. Collectively, the results describe nuclease-specific MRE11 inhibitors, define distinct nuclease roles in DSB repair, and support a mechanism whereby MRE11 endonuclease initiates resection, thereby licensing HR followed by MRE11 exo and EXO1/BLM bidirectional resection towards and away from the DNA end, which commits to HR.
PMCID: PMC3909494  PMID: 24316220
15.  Formation of Linear Amplicons with Inverted Duplications in Leishmania Requires the MRE11 Nuclease 
PLoS Genetics  2014;10(12):e1004805.
Extrachromosomal DNA amplification is frequent in the protozoan parasite Leishmania selected for drug resistance. The extrachromosomal amplified DNA is either circular or linear, and is formed at the level of direct or inverted homologous repeated sequences that abound in the Leishmania genome. The RAD51 recombinase plays an important role in circular amplicons formation, but the mechanism by which linear amplicons are formed is unknown. We hypothesized that the Leishmania infantum DNA repair protein MRE11 is required for linear amplicons following rearrangements at the level of inverted repeats. The purified LiMRE11 protein showed both DNA binding and exonuclease activities. Inactivation of the LiMRE11 gene led to parasites with enhanced sensitivity to DNA damaging agents. The MRE11−/− parasites had a reduced capacity to form linear amplicons after drug selection, and the reintroduction of an MRE11 allele led to parasites regaining their capacity to generate linear amplicons, but only when MRE11 had an active nuclease activity. These results highlight a novel MRE11-dependent pathway used by Leishmania to amplify portions of its genome to respond to a changing environment.
Author Summary
Extrachromosomal DNA amplification is frequent in the human protozoan parasite Leishmania when challenged with drug or other stressful conditions. DNA amplicons, either circular or linear, are formed by recombination between direct or inverted repeats spread throughout the genome of the parasite. The recombinase RAD51 is involved in the formation of circular amplicons, but the mechanism by which linear amplicons are formed is still unknown in this parasite. Studies in other organisms have provided some evidence that a DNA break is required for linear amplifications, and that the DNA repair protein MRE11 can be involved in this process. In this work, we present our biochemical, cellular and molecular characterization of the Leishmania infantum MRE11 orthologue and provide evidence that this nuclease is involved in the formation of linear amplicons in Leishmania. Our results highlight a novel MRE11-dependent pathway used by Leishmania to amplify portions of its genome to respond to a changing environment.
PMCID: PMC4256157  PMID: 25474106
16.  Reprogramming cellular events by poly(ADP-ribose)-binding proteins 
Molecular aspects of medicine  2012;34(6):10.1016/j.mam.2012.12.005.
Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions.
PMCID: PMC3812366  PMID: 23268355
PARP; PARG; Poly(ADP-ribose); WWE; PBZ; Macro domain
17.  Fanconi Anemia Group J Helicase and MRE11 Nuclease Interact To Facilitate the DNA Damage Response 
Molecular and Cellular Biology  2013;33(11):2212-2227.
FANCJ mutations are linked to Fanconi anemia (FA) and increase breast cancer risk. FANCJ encodes a DNA helicase implicated in homologous recombination (HR) repair of double-strand breaks (DSBs) and interstrand cross-links (ICLs), but its mechanism of action is not well understood. Here we show with live-cell imaging that FANCJ recruitment to laser-induced DSBs but not psoralen-induced ICLs is dependent on nuclease-active MRE11. FANCJ interacts directly with MRE11 and inhibits its exonuclease activity in a specific manner, suggesting that FANCJ regulates the MRE11 nuclease to facilitate DSB processing and appropriate end resection. Cells deficient in FANCJ and MRE11 show increased ionizing radiation (IR) resistance, reduced numbers of γH2AX and RAD51 foci, and elevated numbers of DNA-dependent protein kinase catalytic subunit foci, suggesting that HR is compromised and the nonhomologous end-joining (NHEJ) pathway is elicited to help cells cope with IR-induced strand breaks. Interplay between FANCJ and MRE11 ensures a normal response to IR-induced DSBs, whereas FANCJ involvement in ICL repair is regulated by MLH1 and the FA pathway. Our findings are discussed in light of the current model for HR repair.
PMCID: PMC3648079  PMID: 23530059
18.  Detection of the HIV-1 Minus-Strand-Encoded Antisense Protein and Its Association with Autophagy 
Journal of Virology  2013;87(9):5089-5105.
HIV-1 proteins are synthesized from a single transcript in an unspliced form or following splicing, but the existence of an antisense protein (ASP) expressed from an antisense polyadenylated transcript has been suggested. Difficulties linked to the detection of this protein in mammalian cells led us to codon optimize its cDNA. Codon-optimized ASP was indeed efficiently detected in various transfected cell lines following flow cytometry and confocal microscopy analyses. Western blot analyses also led to the detection of optimized ASP in transfected cells but also provided evidence of its instability and high multimerization potential. ASP was mainly distributed in the cytoplasm in a punctate manner, which was reminiscent of autophagosomes. In agreement with this observation, a significant increase in ASP-positive cells and loss of its punctate distribution was observed in transfected cells when autophagy was inhibited at early steps. Induction of autophagy was confirmed by Western blot analyses that showed an ASP-mediated increase in levels of LC3b-II and Beclin 1, as well as colocalization and interaction between ASP and LC3. Interestingly, Myc-tagged ASP was detected in the context of proviral DNA following autophagy inhibition with a concomitant increase in the level and punctate distribution of LC3b-II. Finally, 3-methyladenine treatment of transfected or infected U937 cells decreased extracellular p24 levels in wild-type proviral DNA and to a much lesser extent in ASP-mutated proviral DNA. This study provides the first detection of ASP in mammalian cells by Western blotting. ASP-induced autophagy might explain the inherent difficulty in detecting this viral protein and might justify its presumed low abundance in infected cells.
PMCID: PMC3624327  PMID: 23427159
20.  The MRE11 GAR motif regulates DNA double-strand break processing and ATR activation 
Cell Research  2011;22(2):305-320.
The MRE11/RAD50/NBS1 complex is the primary sensor rapidly recruited to DNA double-strand breaks (DSBs). MRE11 is known to be arginine methylated by PRMT1 within its glycine-arginine-rich (GAR) motif. In this study, we report a mouse knock-in allele of Mre11 that substitutes the arginines with lysines in the GAR motif and generates the MRE11RK protein devoid of methylated arginines. The Mre11RK/RK mice were hypersensitive to γ-irradiation (IR) and the cells from these mice displayed cell cycle checkpoint defects and chromosome instability. Moreover, the Mre11RK/RK MEFs exhibited ATR/CHK1 signaling defects and impairment in the recruitment of RPA and RAD51 to the damaged sites. The MRKRN complex formed and localized to the sites of DNA damage and normally activated the ATM pathway in response to IR. The MRKRN complex exhibited exonuclease and DNA-binding defects in vitro responsible for the impaired DNA end resection and ATR activation observed in vivo in response to IR. Our findings provide genetic evidence for the critical role of the MRE11 GAR motif in DSB repair, and demonstrate a mechanistic link between post-translational modifications at the MRE11 GAR motif and DSB processing, as well as the ATR/CHK1 checkpoint signaling.
PMCID: PMC3271587  PMID: 21826105
DNA damage signaling; arginine methylation; GAR motif; MRE11; ATR
21.  CBX4-mediated SUMO modification regulates BMI1 recruitment at sites of DNA damage 
Nucleic Acids Research  2012;40(12):5497-5510.
Polycomb group (PcG) proteins are involved in epigenetic silencing where they function as major determinants of cell identity, stem cell pluripotency and the epigenetic gene silencing involved in cancer development. Recently numerous PcG proteins, including CBX4, have been shown to accumulate at sites of DNA damage. However, it remains unclear whether or not CBX4 or its E3 sumo ligase activity is directly involved in the DNA damage response (DDR). Here we define a novel role for CBX4 as an early DDR protein that mediates SUMO conjugation at sites of DNA lesions. DNA damage stimulates sumoylation of BMI1 by CBX4 at lysine 88, which is required for the accumulation of BMI1 at DNA damage sites. Moreover, we establish that CBX4 recruitment to the sites of laser micro-irradiation-induced DNA damage requires PARP activity but does not require H2AX, RNF8, BMI1 nor PI-3-related kinases. The importance of CBX4 in the DDR was confirmed by the depletion of CBX4, which resulted in decreased cellular resistance to ionizing radiation. Our results reveal a direct role for CBX4 in the DDR pathway.
PMCID: PMC3384338  PMID: 22402492
22.  The Effect of a DNA Repair Gene on Cellular Invasiveness: Xrcc3 Over-Expression in Breast Cancer Cells 
PLoS ONE  2011;6(1):e16394.
Over-expression of DNA repair genes has been associated with resistance to radiation and DNA-damage induced by chemotherapeutic agents such as cisplatin. More recently, based on the analysis of genome expression profiling, it was proposed that over-expression of DNA repair genes enhances the invasive behaviour of tumour cells. In this study we present experimental evidence utilizing functional assays to test this hypothesis. We assessed the effect of the DNA repair proteins known as X-ray complementing protein 3 (XRCC3) and RAD51, to the invasive behavior of the MCF-7 luminal epithelial-like and BT20 basal-like triple negative human breast cancer cell lines. We report that stable or transient over-expression of XRCC3 but not RAD51 increased invasiveness in both cell lines in vitro. Moreover, XRCC3 over-expressing MCF-7 cells also showed a higher tumorigenesis in vivo and this phenotype was associated with increased activity of the metalloproteinase MMP-9 and the expression of known modulators of cell-cell adhesion and metastasis such as CD44, ID-1, DDR1 and TFF1. Our results suggest that in addition to its' role in facilitating repair of DNA damage, XRCC3 affects invasiveness of breast cancer cell lines and the expression of genes associated with cell adhesion and invasion.
PMCID: PMC3025979  PMID: 21283680
23.  A Key Role for Poly(ADP-Ribose) Polymerase 3 in Ectodermal Specification and Neural Crest Development 
PLoS ONE  2011;6(1):e15834.
The PARP family member poly(ADP-ribose) polymerase 3 (PARP3) is structurally related to the well characterized PARP1 that orchestrates cellular responses to DNA strand breaks and cell death by the synthesis of poly(ADP-ribose). In contrast to PARP1 and PARP2, the functions of PARP3 are undefined. Here, we reveal critical functions for PARP3 during vertebrate development.
Principal Findings
We have used several in vitro and in vivo approaches to examine the possible functions of PARP3 as a transcriptional regulator, a function suggested from its previously reported association with several Polycomb group (PcG) proteins. We demonstrate that PARP3 gene occupancy in the human neuroblastoma cell line SK-N-SH occurs preferentially with developmental genes regulating cell fate specification, tissue patterning, craniofacial development and neurogenesis. Addressing the significance of this association during zebrafish development, we show that morpholino oligonucleotide-directed inhibition of parp3 expression in zebrafish impairs the expression of the neural crest cell specifier sox9a and of dlx3b/dlx4b, the formation of cranial sensory placodes, inner ears and pectoral fins. It delays pigmentation and severely impedes the development of the median fin fold and tail bud.
Our findings demonstrate that Parp3 is crucial in the early stages of zebrafish development, possibly by exerting its transcriptional regulatory functions as early as during the specification of the neural plate border.
PMCID: PMC3022025  PMID: 21264220
24.  SMN Protects cells against mutant SOD1 toxicity by increasing chaperone activity 
Deletion or mutation of the survival of motor neuron (SMN1) gene causes Spinal Muscular Atrophy (SMA), a motor neuron degenerative disease. To study the SMN function, we co-transfected mouse NSC34 cells with SMN and mutant superoxide dismutase 1 (SOD1) constructs. We demonstrated that SMN protected NSC34 cells against cell death induced by mutant SOD1 under oxidative stress. Further studies indicated that over-expression of wild-type SMN up regulated chaperone activity. In contrast, chaperone activity was decreased in cells expressing SMN mutant Y272C or in cells with SMN suppressed by shRNA. In vitro assays using bacteria lysates expressing GST-SMN or purified GST-SMN protein showed that the GST-SMN reduced catalase aggregation, indicating that SMN may possess chaperone activity. We conclude that SMN plays a protective role in motor neurons by its chaperone activity. Our results provide support for the potential development of therapy for SMA and amyotrophic lateral sclerosis (ALS).
PMCID: PMC2169267  PMID: 17964281
Spinal muscular atrophy (SMA); the survival of motor neuron (SMN); superoxide dismutase 1 (SOD1); Amyotrophic lateral sclerosis (ALS); M Chaperone
25.  The Transcriptional Histone Acetyltransferase Cofactor TRRAP Associates with the MRN Repair Complex and Plays a Role in DNA Double-Strand Break Repair†  
Molecular and Cellular Biology  2006;26(2):402-412.
Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, we describe the stable association of TRRAP with the MRN complex. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling.
PMCID: PMC1346889  PMID: 16382133

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