A conserved loop-wedge motif moderates reaction site search and recognition by FEN1

DNA replication and repair frequently involve intermediate two-way junction structures with overhangs, or flaps, that must be promptly removed; a task performed by the essential enzyme flap endonuclease 1 (FEN1). We demonstrate a functional relationship between two intrinsically disordered regions of the FEN1 protein, which recognise opposing sides of the junction and order in response to the requisite substrate. Our results inform a model in which short-range translocation of FEN1 on DNA facilitates search for the annealed 3′‑terminus of a primer strand, which is recognised by breaking the terminal base pair to generate a substrate with a single nucleotide 3′‑flap. This recognition event allosterically signals hydrolytic removal of the 5′-flap through reaction in the opposing junction duplex, by controlling access of the scissile phosphate diester to the active site. The recognition process relies on a highly-conserved ‘wedge’ residue located on a mobile loop that orders to bind the newly-unpaired base. The unanticipated ‘loop–wedge’ mechanism exerts control over substrate selection, rate of reaction and reaction site precision, and shares features with other enzymes that recognise irregular DNA structures. These new findings reveal how FEN1 precisely couples 3′-flap verification to function

DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1 Catalyzed Reaction.

Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5'-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5'-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5'-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5'-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Y40, D181 and R100 and a reacting duplex 5'-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound, 5'-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage

Adjacent single-stranded regions mediate processing of tRNA precursors by RNase E direct entry

The RNase E family is renowned for being central to the processing and decay of all types of RNA in many species of bacteria, as well as providing the first examples of endonucleases that can recognize 50 -monophosphorylated ends thereby increasing the efficiency of cleavage. However, there is increasing evidence that some transcripts can be cleaved efficiently by Escherichia coli RNase E via direct entry, i.e. in the absence of the recognition of a 50 -monophosphorylated end. Here, we provide biochemical evidence that direct entry is central to the processing of transfer RNA (tRNA) in E. coli, one of the core functions of RNase E, and show that it is mediated by specific unpaired regions that are adjacent, but not contiguous to segments cleaved by RNase E. In addition, we find that direct entry at a site on the 50 side of a tRNA precursor triggers a series of 50 -monophosphate-dependent cleavages. Consistent with a major role for direct entry in tRNA processing, we provide additional evidence that a 50 -monophosphate is not required to activate the catalysis step in cleavage. Other examples of tRNA precursors processed via direct entry are also provided. Thus, it appears increasingly that direct entry by RNase E has a major role in bacterial RNA metabolism

Cellular Active N-Hydroxyurea FEN1 Inhibitors Block Substrate Entry to the Active Site

The structure-specific nuclease human flap endonuclease-1 (hFEN1) plays a key role in DNA replication and repair and may be of interest as an oncology target. We present the first crystal structure of inhibitor-bound hFEN1 and show a cyclic N-hydroxyurea bound in the active site coordinated to two magnesium ions. Three such compounds had similar IC50 values but differed subtly in mode of action. One had comparable affinity for protein and protein– substrate complex and prevented reaction by binding to active site catalytic metal ions, blocking the unpairing of substrate DNA necessary for reaction. Other compounds were more competitive with substrate. Cellular thermal shift data showed engagement of both inhibitor types with hFEN1 in cells with activation of the DNA damage response evident upon treatment. However, cellular EC50s were significantly higher than in vitro inhibition constants and the implications of this for exploitation of hFEN1 as a drug target are discussed

Associations Between Physical Fitness and Brain Structure in Young Adulthood

A comprehensive analysis of associations between physical fitness and brain structure in young adulthood is lacking, and further, it is unclear the degree to which associations between physical fitness and brain health can be attributed to a common genetic pathway or to environmental factors that jointly influences physical fitness and brain health. This study examined genotype-confirmed monozygotic and dizygotic twins, along with non-twin full-siblings to estimate the contribution of genetic and environmental factors to variation within, and covariation between, physical fitness and brain structure. Participants were 1,065 young adults between the ages of 22 and 36 from open-access Young Adult Human Connectome Project (YA-HCP). Physical fitness was assessed by submaximal endurance (2-min walk test), grip strength, and body mass index. Brain structure was assessed using magnetic resonance imaging on a Siemens 3T customized ‘Connectome Skyra’ at Washington University in St. Louis, using a 32-channel Siemens head coil. Acquired T1-weighted images provided measures of cortical surface area and thickness, and subcortical volume following processing by the YA-HCP structural FreeSurfer pipeline. Diffusion weighted imaging was acquired to assess white matter tract integrity, as measured by fractional anisotropy, following processing by the YA-HCP diffusion pipeline and tensor fit. Following correction for multiple testing, body mass index was negatively associated with fractional anisotropy in various white matter regions of interest (all | z| statistics > 3.9) and positively associated with cortical thickness within the right superior parietal lobe (z statistic = 4.6). Performance-based measures of fitness (i.e., endurance and grip strength) were not associated with any structural neuroimaging markers. Behavioral genetic analysis suggested that heritability of white matter integrity varied by region, but consistently explained >50% of the phenotypic variation. Heritability of right superior parietal thickness was large (∼75% variation). Heritability of body mass index was also fairly large (∼60% variation). Generally, 12 to 23 of the correlation between brain structure and body mass index could be attributed to heritability effects. Overall, this study suggests that greater body mass index is associated with lower white matter integrity, which may be due to common genetic effects that impact body composition and white matter integrity

The synthesis and uses of modified oligodeoxynucleotides

A brief review of oligodeoxynucleotide synthesis is presented with special reference to the synthesis of modified oligodeoxynucleotides. The uses of 5'-phosphorylated, 5'-thiol containing and phosphorothioate containing oligodeoxynucleotides are discussed. One method for the synthesis of thiol containing oligodeoxynucleotides utilises S-trityl-O-cyanoethyl-N,N-diisopropylphosphityl-3-mercaptopropan-1-ol. Occasionally the trityl group is labile during DNA synthesis producing previously unidentified products. Additionally it has been reported that disulphide formation is the result of iodine detritylation of trityl protected thiols. However, detritylation of trityl protected mercaptopropanol containing oligodeoxynucleotides with iodine results in the formation of several additional products. The major product of iodine detritylation has been found to be the sulphonic acid. A second product produced in high yield is dimeric possibly a thiolsulphonate or thiolsulphinate. The phosphite described above is a viscous oil. Significant advantages would be gained if a solid reagent was available. Described here are attempts to synthesise such a reagent by varying the protecting groups employed for the thiol moiety in attempt to induce crystallinity or make the molecule sufficiently polar to be precipitated from pentane. The syntheses of S-dimethoxytrityl- and S-(9-phenylxanthenyl)- O-cynaoethyl-N,N-diisopropylphosphityl-2-mercaptoethanol are described. These reagents can be used for the 5'-phosphorylation of synthetic oligodeoxynucleotides with one slight modification to the deprotection procedures namely the addition of DTT prior to ammonia deprotection. The protecting group is labile during iodine treatment and therefore compatible with any type of exocyclic amino group protection. The synthesis and resolution of the diastereoisomers of d(GACunderline GATsATCGTC) is described. This oligodeoxynucleotide contains the EcoRV restriction site (underlined) with a phosphorothioate group replacing the scissile phosphodiester bond. The Rp diastereoisomer is a substrate for the enzyme and performing the reaction in H218O results in the formation of a chiral product. In this way the stereochemical course of the EcoRV catalysed hydrolysis of DNA has been monitored and determined to proceed with inversion of configuration at phosphorus. This result implies that the reaction proceeds via direct in-line attack by H2O without the intermediacy of a covalent enzyme intermediate.</p

Preconditioning of Spatial and Auditory Cues: Roles of the Hippocampus, Frontal Cortex, and Cue-Directed Attention

Loss of function of the hippocampus or frontal cortex is associated with reduced performance on memory tasks, in which subjects are incidentally exposed to cues at specific places in the environment and are subsequently asked to recollect the location at which the cue was experienced. Here, we examined the roles of the rodent hippocampus and frontal cortex in cue-directed attention during encoding of memory for the location of a single incidentally experienced cue. During a spatial sensory preconditioning task, rats explored an elevated platform while an auditory cue was incidentally presented at one corner. The opposite corner acted as an unpaired control location. The rats demonstrated recollection of location by avoiding the paired corner after the auditory cue was in turn paired with shock. Damage to either the dorsal hippocampus or the frontal cortex impaired this memory ability. However, we also found that hippocampal lesions enhanced attention directed towards the cue during the encoding phase, while frontal cortical lesions reduced cue-directed attention. These results suggest that the deficit in spatial sensory preconditioning caused by frontal cortical damage may be mediated by inattention to the location of cues during the latent encoding phase, while deficits following hippocampal damage must be related to other mechanisms such as generation of neural plasticity