MUS81 cleaves TOP1-derived lesions and other DNA-protein cross-links.

BACKGROUND: DNA-protein cross-links (DPCs) are one of the most deleterious DNA lesions, originating from various sources, including enzymatic activity. For instance, topoisomerases, which play a fundamental role in DNA metabolic processes such as replication and transcription, can be trapped and remain covalently bound to DNA in the presence of poisons or nearby DNA damage. Given the complexity of individual DPCs, numerous repair pathways have been described. The protein tyrosyl-DNA phosphodiesterase 1 (Tdp1) has been demonstrated to be responsible for removing topoisomerase 1 (Top1). Nevertheless, studies in budding yeast have indicated that alternative pathways involving Mus81, a structure-specific DNA endonuclease, could also remove Top1 and other DPCs. RESULTS: This study shows that MUS81 can efficiently cleave various DNA substrates modified by fluorescein, streptavidin or proteolytically processed topoisomerase. Furthermore, the inability of MUS81 to cleave substrates bearing native TOP1 suggests that TOP1 must be either dislodged or partially degraded prior to MUS81 cleavage. We demonstrated that MUS81 could cleave a model DPC in nuclear extracts and that depletion of TDP1 in MUS81-KO cells induces sensitivity to the TOP1 poison camptothecin (CPT) and affects cell proliferation. This sensitivity is only partially suppressed by TOP1 depletion, indicating that other DPCs might require the MUS81 activity for cell proliferation. CONCLUSIONS: Our data indicate that MUS81 and TDP1 play independent roles in the repair of CPT-induced lesions, thus representing new therapeutic targets for cancer cell sensitisation in combination with TOP1 inhibitors.

Imiquimod suppresses respiratory syncytial virus (RSV) replication via PKA pathway and reduces RSV induced-inflammation and viral load in mice lungs.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract disease and bronchiolitis in children, as well as an important cause of morbidity and mortality in elderly and immunocompromised individuals. However, there is no safe and efficacious RSV vaccine or antiviral treatment. Toll Like Receptors (TLR) are important molecular mediators linking innate and adaptive immunity, and their stimulation by cognate agonists has been explored as antiviral agents. Imiquimod is known as a TLR7 agonist, but additionally acts as an antagonist for adenosine receptors. In this study, we demonstrate that imiquimod, but not resiquimod, has direct anti-RSV activity via PKA pathway in HEp-2 and A549 cells, independently of an innate response. Imiquimod restricts RSV infection after viral entry into the host cell, interfering with viral RNA and protein synthesis. Probably as a consequence of these anti-RSV properties, imiquimod displays cytokine modulating activity in RSV infected epithelial cells. Moreover, in a murine model of RSV infection, imiquimod treatment improves the course of acute disease, evidenced by decreased weight loss, reduced RSV lung titers, and attenuated airway inflammation. Consequently, imiquimod represents a promising therapeutic alternative against RSV infection and may inform the development of novel therapeutic targets to control RSV pathogenesis.

RECQ5 Helicase Cooperates with MUS81 Endonuclease in Processing Stalled Replication Forks at Common Fragile Sites during Mitosis.

The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain, or phosphorylation site causes excessive binding of RAD51 to CFS loci and impairs CFS expression. This leads to defective chromosome segregation and accumulation of CFS-associated DNA damage in G1 cells. Biochemically, RECQ5 alleviates the inhibitory effect of RAD51 on 3'-flap DNA cleavage by MUS81-EME1 through its RAD51 filament disruption activity. These data suggest that RECQ5 removes RAD51 filaments stabilizing stalled replication forks at CFSs and hence facilitates CFS cleavage by MUS81-EME1.

The structure and DNA-binding properties of Mgm101 from a yeast with a linear mitochondrial genome.

To study the mechanisms involved in the maintenance of a linear mitochondrial genome we investigated the biochemical properties of the recombination protein Mgm101 from Candida parapsilosis. We show that CpMgm101 complements defects associated with the Saccharomyces cerevisiae mgm101-1(ts) mutation and that it is present in both the nucleus and mitochondrial nucleoids of C. parapsilosis. Unlike its S. cerevisiae counterpart, CpMgm101 is associated with the entire nucleoid population and is able to bind to a broad range of DNA substrates in a non-sequence specific manner. CpMgm101 is also able to catalyze strand annealing and D-loop formation. CpMgm101 forms a roughly C-shaped trimer in solution according to SAXS. Electron microscopy of a complex of CpMgm101 with a model mitochondrial telomere revealed homogeneous, ring-shaped structures at the telomeric single-stranded overhangs. The DNA-binding properties of CpMgm101, together with its DNA recombination properties, suggest that it can play a number of possible roles in the replication of the mitochondrial genome and the maintenance of its telomeres.

Yeast mitochondrial HMG proteins: DNA-binding properties of the most evolutionarily divergent component of mitochondrial nucleoids.

Yeast mtDNA is compacted into nucleoprotein structures called mitochondrial nucleoids (mt-nucleoids). The principal mediators of nucleoid formation are mitochondrial high-mobility group (HMG)-box containing (mtHMG) proteins. Although these proteins are some of the fastest evolving components of mt-nucleoids, it is not known whether the divergence of mtHMG proteins on the level of their amino acid sequences is accompanied by diversification of their biochemical properties. In the present study we performed a comparative biochemical analysis of yeast mtHMG proteins from Saccharomyces cerevisiae (ScAbf2p), Yarrowia lipolytica (YlMhb1p) and Candida parapsilosis (CpGcf1p). We found that all three proteins exhibit relatively weak binding to intact dsDNA. In fact, ScAbf2p and YlMhb1p bind quantitatively to this substrate only at very high protein to DNA ratios and CpGcf1p shows only negligible binding to dsDNA. In contrast, the proteins exhibit much higher preference for recombination intermediates such as Holliday junctions (HJ) and replication forks (RF). Therefore, we hypothesize that the roles of the yeast mtHMG proteins in maintenance and compaction of mtDNA in vivo are in large part mediated by their binding to recombination/replication intermediates. We also speculate that the distinct biochemical properties of CpGcf1p may represent one of the prerequisites for frequent evolutionary tinkering with the form of the mitochondrial genome in the CTG-clade of hemiascomycetous yeast species.

Srs2 promotes Mus81-Mms4-mediated resolution of recombination intermediates.

A variety of DNA lesions, secondary DNA structures or topological stress within the DNA template may lead to stalling of the replication fork. Recovery of such forks is essential for the maintenance of genomic stability. The structure-specific endonuclease Mus81-Mms4 has been implicated in processing DNA intermediates that arise from collapsed forks and homologous recombination. According to previous genetic studies, the Srs2 helicase may play a role in the repair of double-strand breaks and ssDNA gaps together with Mus81-Mms4. In this study, we show that the Srs2 and Mus81-Mms4 proteins physically interact in vitro and in vivo and we map the interaction domains within the Srs2 and Mus81 proteins. Further, we show that Srs2 plays a dual role in the stimulation of the Mus81-Mms4 nuclease activity on a variety of DNA substrates. First, Srs2 directly stimulates Mus81-Mms4 nuclease activity independent of its helicase activity. Second, Srs2 removes Rad51 from DNA to allow access of Mus81-Mms4 to cleave DNA. Concomitantly, Mus81-Mms4 inhibits the helicase activity of Srs2. Taken together, our data point to a coordinated role of Mus81-Mms4 and Srs2 in processing of recombination as well as replication intermediates.

Human Rap1 modulates TRF2 attraction to telomeric DNA.

More than two decades of genetic research have identified and assigned main biological functions of shelterin proteins that safeguard telomeres. However, a molecular mechanism of how each protein subunit contributes to the protecting function of the whole shelterin complex remains elusive. Human Repressor activator protein 1 (Rap1) forms a multifunctional complex with Telomeric Repeat binding Factor 2 (TRF2). Rap1-TRF2 complex is a critical part of shelterin as it suppresses homology-directed repair in Ku 70/80 heterodimer absence. To understand how Rap1 affects key functions of TRF2, we investigated full-length Rap1 binding to TRF2 and Rap1-TRF2 complex interactions with double-stranded DNA by quantitative biochemical approaches. We observed that Rap1 reduces the overall DNA duplex binding affinity of TRF2 but increases the selectivity of TRF2 to telomeric DNA. Additionally, we observed that Rap1 induces a partial release of TRF2 from DNA duplex. The improved TRF2 selectivity to telomeric DNA is caused by less pronounced electrostatic attractions between TRF2 and DNA in Rap1 presence. Thus, Rap1 prompts more accurate and selective TRF2 recognition of telomeric DNA and TRF2 localization on single/double-strand DNA junctions. These quantitative functional studies contribute to the understanding of the selective recognition of telomeric DNA by the whole shelterin complex.

The Role of Parental Distress in Moderating the Influence of Child Neglect on Maladjustment.

Despite pervasive evidence of the harmful impact of neglect on children's adjustment, individual differences in adaptation persist. This study examines parental distress as a contextual factor that may moderate the relation between neglect and child adjustment, while considering the specificity of the relation between neglect and internalizing versus externalizing problems. In a sample of 66 children (33 with a documented child protective services history of neglect prior to age six), neglect predicted internalizing, and to a lesser extent externalizing, problems as rated by teachers at age seven. Parental distress moderated the relation between neglect and internalizing, but not externalizing, problems. Specifically, higher levels of neglect predicted more internalizing problems only among children of distressed parents. These findings indicate that parent-level variables are important to consider in evaluating the consequences of neglect, and point to the importance of considering contextual factors when identifying those children most at risk following neglect.

The PCNA interaction protein box sequence in Rad54 is an integral part of its ATPase domain and is required for efficient DNA repair and recombination.

Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.

Srs2 mediates PCNA-SUMO-dependent inhibition of DNA repair synthesis.

Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability.

Externalizing problems in late childhood as a function of prenatal cocaine exposure and environmental risk.

OBJECTIVE: To examine whether prenatal cocaine exposure (PCE) predicts externalizing problems in late childhood. METHODS: Externalizing problems were assessed using caregiver, teacher, and child ratings and a laboratory task when children (N = 179; 74 cocaine exposed) were aged 8-10 years. PCE, environmental risk, sex, neonatal health, other prenatal exposures, and foster care history were examined as predictors of externalizing problems. RESULTS: Multiple regression analyses indicated that PCE, environmental risk, and male sex explained significant variance in externalizing problems in late childhood. Models varied by source of information. PCE predicted externalizing problems for child laboratory behavior and interacted with sex because males with PCE reported more externalizing problems. PCE did not predict caregiver or teacher ratings of externalizing problems. CONCLUSIONS: The effect of PCE on externalizing problems may persist into late childhood. The findings highlight the potential importance of including child-based measures of externalizing problems in studies of prenatal exposure.

Unwinding of synthetic replication and recombination substrates by Srs2.

The budding yeast Srs2 protein possesses 3' to 5' DNA helicase activity and channels untimely recombination to post-replication repair by removing Rad51 from ssDNA. However, it also promotes recombination via a synthesis-dependent strand-annealing pathway (SDSA). Furthermore, at the replication fork, Srs2 is required for fork progression and prevents the instability of trinucleotide repeats. To better understand the multiple roles of the Srs2 helicase during these processes, we analysed the ability of Srs2 to bind and unwind various DNA substrates that mimic structures present during DNA replication and recombination. While leading or lagging strands were efficiently unwound, the presence of ssDNA binding protein RPA presented an obstacle for Srs2 translocation. We also tested the preferred directionality of unwinding of various substrates and studied the effect of Rad51 and Mre11 proteins on Srs2 helicase activity. These biochemical results help us understand the possible role of Srs2 in the processing of stalled or blocked replication forks as a part of post-replication repair as well as homologous recombination (HR).

Gender differences in psychopathic traits, types, and correlates of aggression among adjudicated youth.

The current study investigated gender differences in types and correlates of aggression among 150 adjudicated youth (M age = 15.2, SD = 1.4). In cluster analysis, consistent with past studies, one aggressive group characterized by moderate levels of reactive aggression and one characterized by high levels of proactive and reactive aggression emerged and these patterns were consistent across gender. For both boys and girls, the combined proactive/reactive aggression cluster showed the greatest levels of aggression, impulsivity, and callous-unemotional traits, supporting a severity over a typology model of proactive and reactive aggression. Girls displayed significantly higher rates of physical and relational aggression than boys. Girls were highly aggressive toward both girls and boys, whereas boys were highly aggressive only toward other boys. Girls also showed multiple indications of severity and emotionality, indexed by higher rates of negative affect, anxiety, distress about social provocations, and empathy.

Srs2: the "Odd-Job Man" in DNA repair.

Homologous recombination plays a key role in the maintenance of genome integrity, especially during DNA replication and the repair of double-stranded DNA breaks (DSBs). Just a single un-repaired break can lead to aneuploidy, genetic aberrations or cell death. DSBs are caused by a vast number of both endogenous and exogenous agents including genotoxic chemicals or ionizing radiation, as well as through replication of a damaged template DNA or the replication fork collapse. It is essential for cell survival to recognise and process DSBs as well as other toxic intermediates and launch most appropriate repair mechanism. Many helicases have been implicated to play role in these processes, however their detail roles, specificities and co-operativity in the complex protein-protein interaction networks remain unclear. In this review we summarize the current knowledge about Saccharomyces cerevisiae helicase Srs2 and its effect on multiple DNA metabolic processes that generally affect genome stability. It would appear that Srs2 functions as an "Odd-Job Man" in these processes to make sure that the jobs proceed when and where they are needed.

Evidence for deficits in reward responsivity in antisocial youth with callous-unemotional traits.

This study investigated reward responsivity in youth with high levels of callous-unemotional (CU) traits using a cross-sectional design. Whereas deficits in responding to punishment cues are well established in youth with CU traits, it is unclear whether responsivity to rewarding stimuli is impaired as well. Participants were 148 predominantly Caucasian, adjudicated adolescents between the ages of 11 and 17 (M = 15.1, SD = 1.4) who completed the Balloon Analogue Risk Task as part of a larger battery investigating aggression and social information processing. A Reward Responsivity variable was created to capture changes in participants' responding after receiving a reward. A hierarchical regression analysis indicated that higher levels of CU traits significantly predicted less reward responsivity, above and beyond gender, sensation seeking, and impulsivity. Results support Blair's (2004) Integrated Emotion Systems model that proposes individuals with CU traits are impaired in their responsivity to both appetitive and aversive stimuli.

Cooperativity of Mus81.Mms4 with Rad54 in the resolution of recombination and replication intermediates.

The Saccharomyces cerevisiae Mus81.Mms4 protein complex, a DNA structure-specific endonuclease, helps preserve genomic integrity by resolving pathological DNA structures that arise from damaged or aborted replication forks and may also play a role in the resolution of DNA intermediates arising through homologous recombination. Previous yeast two-hybrid studies have found an interaction of the Mus81 protein with Rad54, a Swi2/Snf2-like factor that serves multiple roles in homologous recombination processes. However, the functional significance of this novel interaction remains unknown. Here, using highly purified S. cerevisiae proteins, we show that Rad54 strongly stimulates the Mus81.Mms4 nuclease activity on a broad range of DNA substrates. This nuclease enhancement does not require ATP binding nor its hydrolysis by Rad54. We present evidence that Rad54 acts by targeting the Mus81.Mms4 complex to its DNA substrates. In addition, we demonstrate that the Rad54-mediated enhancement of the Mus81.Mms4 (Eme1) nuclease function is evolutionarily conserved. We propose that Mus81.Mms4 together with Rad54 efficiently process perturbed replication forks to promote recovery and may constitute an alternative mechanism to the resolution/dissolution of the recombination intermediates by Sgs1.Top3. These findings provide functional insights into the biological importance of the higher order complex of Mus81.Mms4 or its orthologue with Rad54.

Biophysical studies on the stability of DNA intrastrand cross-links of transplatin.

Clinically ineffective transplatin [trans-diamminedichloridoplatinum(II)] is used in the studies of the structure-pharmacological activity relationship of platinum compounds. In addition, a number of transplatin analogs exhibit promising toxic effects in several tumor cell lines including those resistant to conventional antitumor cisplatin. Moreover, transplatin-modified oligonucleotides have been shown to be effective modulators of gene expression. Owing to these facts and because DNA is also considered the major pharmacological target of platinum complexes, interactions between transplatin and DNA are of great interest. We examined, using biophysical and biochemical methods, the stability of 1,3-GNG intrastrand cross-links (CLs) formed by transplatin in short synthetic oligodeoxyribonucleotide duplexes and natural double-helical DNA. We have found that transplatin forms in double-helical DNA 1,3-GNG intrastrand CLs, but their stability depends on the sequence context. In some sequences the 1,3-GNG intrastrand CLs formed by transplatin in double-helical DNA readily rearrange into interstrand CLs. On the other hand, in a number of other sequences these intrastrand CLs are relatively stable. We show that the stability of 1,3-GNG intrastrand CLs of transplatin correlates with the extent of conformational distortion and thermodynamic destabilization induced in double-helical DNA by this adduct.

Binding of mismatch repair protein MutS to mispaired DNA adducts of intercalating ruthenium(II) arene complexes.

The present study was performed to examine the affinity of Escherichia coli mismatch repair (MMR) protein MutS for DNA damaged by an intercalating compound. We examined the binding properties of this protein with various DNA substrates containing a single centrally located adduct of ruthenium(II) arene complexes [(eta(6)-arene)Ru(II)(en)Cl][PF(6)] [arene is tetrahydroanthracene (THA) or p-cymene (CYM); en is ethylenediamine]. These two complexes were chosen as representatives of two different classes of monofunctional ruthenium(II) arene compounds which differ in DNA-binding modes: one that involves combined coordination to G N7 along with noncovalent, hydrophobic interactions, such as partial arene intercalation (tricyclic-ring Ru-THA), and the other that binds to DNA only via coordination to G N7 and does not interact with double-helical DNA by intercalation (monoring Ru-CYM). Using electrophoretic mobility shift assays, we examined the binding properties of MutS protein with various DNA duplexes (homoduplexes or mismatched duplexes) containing a single centrally located adduct of ruthenium(II) arene compounds. We have shown that presence of the ruthenium(II) arene adducts decreases the affinity of MutS for ruthenated DNA duplexes that either have a regular sequence or contain a mismatch and that intercalation of the arene contributes considerably to this inhibitory effect. Since MutS initiates MMR by recognizing DNA lesions, the results of the present work support the view that DNA damage due to intercalation is removed from DNA by a mechanism(s) other than MMR.

Structural characterization and DNA interactions of new cytotoxic transplatin analogues containing one planar and one nonplanar heterocyclic amine ligand.

trans-Diaminedicholoroplatinum(II) complexes with one planar and one non-planar heterocyclic amine ligand were designed as new potential antitumor drugs. The X-ray crystallographic structures of trans-[PtCl2(4-picoline)(piperidine)] and trans-[PtCl2(4-picoline)(piperazine)].HCl revealed that the piperidine and piperazine ligands bind to the platinum through the equatorial position and that the ligands adopt the chair conformation. The nonplatinated amine of the piperazine can form hydrogen bonds with atoms that are approximately 7.5 A away from the Pt binding site. DNA is considered a major pharmacological target of platinum compounds. Hence, to expand the database correlating structural features of platinum compounds and DNA distortions induced by these compounds, which may facilitate identification of more effective anticancer platinum drugs, we describe the DNA binding mode in a cell-free medium of trans-[PtCl2(4-picoline)(piperidine)] and trans-[PtCl2(4-picoline)(piperazine)].HCl. Interestingly, the overall impact of the replacement of the second ammine group in transplatin by the heterocyclic ligands appears to change the character of the global conformational changes induced in DNA towards that induced by cisplatin. The clinical ineffectiveness of the parent transplatin has been proposed to be also associated with its reduced capability to form bifunctional adducts in double-helical DNA. The results of the present work support the view that replacement of both ammine groups of transplatin by heterocyclic ligands enhances cytotoxicity probably due to the marked enhancement of the stability of intrastrand cross-links in double-helical DNA.

Conformation, protein recognition and repair of DNA interstrand and intrastrand cross-links of antitumor trans-[PtCl2(NH3)(thiazole)].

Replacement of one ammine in clinically ineffective trans-[PtCl2(NH3)2] (transplatin) by a planar N-heterocycle, thiazole, results in significantly enhanced cytotoxicity. Unlike 'classical' cisplatin {cis-[PtCl2(NH3)2]} or transplatin, modification of DNA by this prototypical cytotoxic transplatinum complex trans-[PtCl2(NH3)(thiazole)] (trans-PtTz) leads to monofunctional and bifunctional intra or interstrand adducts in roughly equal proportions. DNA fragments containing site-specific bifunctional DNA adducts of trans-PtTz were prepared. The structural distortions induced in DNA by these adducts and their consequences for high-mobility group protein recognition, DNA polymerization and nucleotide excision repair were assessed in cell-free media by biochemical methods. Whereas monofunctional adducts of trans-PtTz behave similar to the major intrastrand adduct of cisplatin [J. Kasparkova, O. Novakova, N. Farrell and V. Brabec (2003) Biochemistry, 42, 792-800], bifunctional cross-links behave distinctly differently. The results suggest that the multiple DNA lesions available to trans-planaramine complexes may all contribute substantially to their cytotoxicity so that the overall drug cytotoxicity could be the sum of the contributions of each of these adducts. However, acquisition of drug resistance could be a relatively rare event, since it would have to entail resistance to or tolerance of multiple, structurally dissimilar DNA lesions.