Regulation of BRCA1 stability through the tandem UBX domains of isoleucyl-tRNA synthetase 1.

Aminoacyl-tRNA synthetases (ARSs) have evolved to acquire various additional domains. These domains allow ARSs to communicate with other cellular proteins in order to promote non-translational functions. Vertebrate cytoplasmic isoleucyl-tRNA synthetases (IARS1s) have an uncharacterized unique domain, UNE-I. Here, we present the crystal structure of the chicken IARS1 UNE-I complexed with glutamyl-tRNA synthetase 1 (EARS1). UNE-I consists of tandem ubiquitin regulatory X (UBX) domains that interact with a distinct hairpin loop on EARS1 and protect its neighboring proteins in the multi-synthetase complex from degradation. Phosphomimetic mutation of the two serine residues in the hairpin loop releases IARS1 from the complex. IARS1 interacts with BRCA1 in the nucleus, regulates its stability by inhibiting ubiquitylation via the UBX domains, and controls DNA repair function.

PARP1-MGMT complex underpins pathway crosstalk in O6-methylguanine repair.

DNA lesions induced by alkylating agents are repaired by two canonical mechanisms, base excision repair dependent on poly(ADP) ribose polymerase 1 (PARP1) and the other mediated by O6-methylguanine (O6meG)-DNA methyltransferase (MGMT) in a single-step catalysis of alkyl-group removal. O6meG is the most cytotoxic and mutagenic lesion among the methyl adducts induced by alkylating agents. Although it can accomplish the dealkylation reaction all by itself as a single protein without associating with other repair proteins, evidence is accumulating that MGMT can form complexes with repair proteins and is highly regulated by a variety of post-translational modifications, such as phosphorylation, ubiquitination, and others. Here, we show that PARP1 and MGMT proteins interact directly in a non-catalytic manner, that MGMT is subject to PARylation by PARP1 after DNA damage, and that the O6meG repair is enhanced upon MGMT PARylation. We provide the first evidence for the direct DNA-independent PARP1-MGMT interaction. Further, PARP1 and MGMT proteins also interact via PARylation of MGMT leading to formation of a novel DNA damage inducible PARP1-MGMT protein complex. This catalytic interaction activates O6meG repair underpinning the functional crosstalk between base excision and MGMT-mediated DNA repair mechanisms. Furthermore, clinically relevant 'chronic' temozolomide exposure induced PARylation of MGMT and increased binding of PARP1 and MGMT to chromatin in cells. Thus, we provide the first mechanistic description of physical interaction between PARP1 and MGMT and their functional cooperation through PARylation for activation of O6meG repair. Hence, the PARP1-MGMT protein complex could be targeted for the development of advanced and more effective cancer therapeutics, particularly for cancers sensitive to PARP1 and MGMT inhibition.

RAD51AP1 mediates RAD51 activity through nucleosome interaction.

RAD51-associated protein 1 (RAD51AP1) is a key protein in the homologous recombination (HR) DNA repair pathway. Loss of RAD51AP1 leads to defective HR, genome instability, and telomere erosion. RAD51AP1 physically interacts with the RAD51 recombinase and promotes RAD51-mediated capture of donor DNA, synaptic complex assembly, and displacement-loop formation when tested with nucleosome-free DNA substrates. In cells, however, DNA is packaged into chromatin, posing an additional barrier to the complexities of the HR reaction. In this study, we show that RAD51AP1 binds to nucleosome core particles (NCPs), the minimum basic unit of chromatin in which approximately two superhelical turns of 147 bp double-stranded DNA are wrapped around one histone octamer with no free DNA ends remaining. We identified a C-terminal region in RAD51AP1, including its previously mapped DNA-binding domain, as critical for mediating the association between RAD51AP1 and both the NCP and the histone octamer. Using in vitro surrogate assays of HR activity, we show that RAD51AP1 is capable of promoting duplex DNA capture and initiating joint-molecule formation with the NCP and chromatinized template DNA, respectively. Together, our results suggest that RAD51AP1 directly assists in the RAD51-mediated search for donor DNA in chromatin. We present a model, in which RAD51AP1 anchors the DNA template through affinity for its nucleosomes to the RAD51-ssDNA nucleoprotein filament.

Flavonoid-rich fruit and vegetables improve microvascular reactivity and inflammatory status in men at risk of cardiovascular disease--FLAVURS: a randomized controlled trial.

BACKGROUND: Observed associations between increased fruit and vegetable (F&V) consumption, particularly those F&Vs that are rich in flavonoids, and vascular health improvements require confirmation in adequately powered randomized controlled trials. OBJECTIVE: This study was designed to measure the dose-response relation between high-flavonoid (HF), low-flavonoid (LF), and habitual F&V intakes and vascular function and other cardiovascular disease (CVD) risk indicators. DESIGN: A single-blind, dose-dependent, parallel randomized controlled dietary intervention study was conducted. Male and female low-F&V consumers who had a ≥ 1.5-fold increased risk of CVD (n = 174) were randomly assigned to receive an HF F&V, an LF F&V, or a habitual diet, with HF and LF F&V amounts sequentially increasing by 2, 4, and 6 (+2, +4, and +6) portions/d every 6 wk over habitual intakes. Microvascular reactivity (laser Doppler imaging with iontophoresis), arterial stiffness [pulse wave velocity, pulse wave analysis (PWA)], 24-h ambulatory blood pressure, and biomarkers of nitric oxide (NO), vascular function, and inflammation were determined at baseline and at 6, 12, and 18 wk. RESULTS: In men, the HF F&V diet increased endothelium-dependent microvascular reactivity (P = 0.017) with +2 portions/d (at 6 wk) and reduced C-reactive protein (P = 0.001), E-selectin (P = 0.0005), and vascular cell adhesion molecule (P = 0.0468) with +4 portions/d (at 12 wk). HF F&Vs increased plasma NO (P = 0.0243) with +4 portions/d (at 12 wk) in the group as a whole. An increase in F&Vs, regardless of flavonoid content in the groups as a whole, mitigated increases in vascular stiffness measured by PWA (P = 0.0065) and reductions in NO (P = 0.0299) in the control group. CONCLUSION: These data support recommendations to increase F&V intake to ≥ 6 portions daily, with additional benefit from F&Vs that are rich in flavonoids, particularly in men with an increased risk of CVD.