Mutations can be beneficial under conditions where genetic diversity is advantageous, such as somatic hypermutation and antibody generation, but they can also be lethal when they disrupt basic cellular processes or cause uncontrolled proliferation and cancer. Mutations arise from inaccurate processing of lesions generated by endogenous and exogenous DNA damaging agents, and the genome is particularly vulnerable to such damage during S phase. In this phase of the cell cycle, many lesions in the DNA template block replication. Such lesions must be bypassed in order to preserve fork stability and to ensure completion of DNA replication. Lesion bypass is carried out by a set of error-prone and error-free processes collectively referred to as DNA damage tolerance mechanisms. Here, we discuss how two types of DNA damage tolerance, translesion synthesis and template switching, are regulated at stalled replication forks by ubiquitination of PCNA, and the conditions under which they occur.

Proliferating cell nuclear antigen (PCNA) plays an important role in eukaryotic genomic maintenance by topologically binding DNA and recruiting replication and repair proteins. The ring-shaped protein forms a closed circle around doublestranded DNA and is able to move along the DNA in a random walk. The molecular nature of this diffusion process is poorly understood. We use single-molecule imaging to visualize the movement of individual, fluorescently labeled PCNA molecules along stretched DNA. Measurements of diffusional properties as a function of viscosity and protein size suggest that PCNA moves along DNA using two different sliding modes. Most of the time, the clamp moves while rotationally tracking the helical pitch of the DNA duplex. In a less frequently used second mode of diffusion, the movement of the protein is uncoupled from the helical pitch, and the clamp diffuses at much higher rates.The proliferating cell nuclear antigen (PCNA) 3 is a homotrimeric, ring-shaped protein that forms a closed circle around double-stranded DNA. The protein serves as a processivity factor for the eukaryotic replicative polymerases ␦ and ⑀ by tethering them to the DNA (1). Additionally, PCNA interacts with a large number of replication, repair, and signaling factors to coordinate enzymatic processes at sites of replication and repair (2). This recruitment of nucleic-acid enzymes to a topological clamp around the DNA is a strategy employed in organisms ranging from bacteriophage to humans. The remarkable similarity of the ring-shaped structures of the Escherichia coli and bacteriophage T4 sliding clamps to PCNA underscores the evolutionary success of this molecular approach.PCNA is a homotrimer consisting of 37-kDa subunits, each of which comprises two similar globular domains. The PCNA monomers are arrange in head-to-tail fashion, forming a ring with pseudo 6-fold symmetry. The central channel has a diameter of 34 Å, large enough to accommodate double-stranded DNA (3, 4). PCNA forms stable ring-shaped trimers in solution (5) that need to be opened to load onto DNA (6). The clamp loader, replication factor C (RFC), mediates the assembly of PCNA onto DNA at primer-template junctions (7) or at nicks in the DNA backbone (8) in a process that is dependent on ATP. The PCNA⅐DNA complex is very stable, exhibiting a half-life of tens of minutes (9, 10).Although the interactions of various replication and repair proteins with PCNA are well studied (recently reviewed in Ref.2), the interactions between PCNA and DNA are less well understood. Structural studies reveal that the central channel of the clamp is lined with highly conserved, positively charged residues (3, 4). Mutational analysis indicates that these residues may be more important for PCNA loading onto DNA than for sliding along DNA (11). Molecular dynamics simulations suggest that the positively charged residues interact with the phosphodiester backbone but that these interactions are highly dynamic and are frequently displaced by ions from solution (12).The ring struc...

Cyclin-dependent kinases (CDKs) play a central role in cell cycle control, apoptosis, transcription and neuronal functions. They are important targets for the design of drugs with antimitotic or antineurodegenerative effects. CDK4 and CDK6 form a subfamily among the CDKs in mammalian cells, as defined by sequence similarities. Compared to CDK2 and CDK5, structural information on CDK4 and CDK6 is sparse. We describe here the complex structure of human CDK6 in complex with a viral cyclin and a flavonol inhibitor, fisetin. Fisetin binds to the active form of CDK6, forming hydrogen bonds with the side chains of residues in the binding pocket that undergo large conformational changes during CDK activation by cyclin binding. The 4-keto group and the 3-hydroxyl group of fisetin are hydrogen bonded with the backbone in the hinge region between the N-terminal and C-terminal kinase domain, as has been observed for many CDK inhibitors. However, CDK2 and HCK kinase in complex with other flavonol inhibitors such as quercetine and flavopiridol showed a different binding mode with the inhibitor rotated by about 180°. The structural information of the CDK6-fisetin complex is correlated with the binding affinities of different flavonol inhibitors for CDK6. This complex structure is the first description of an inhibitor complex with a kinase from the CDK4/6 subfamily and can provide a basis for selecting and designing inhibitor compounds with higher affinities and specificities. Values in parentheses refer to the highest resolution shell. 1 R cryst = ∑h||Fobs(h)|-|Fc(h)||/∑h|Fobs(h)| for all data, R free was calculated from 7.5% of structure factor amplitudes that were excluded from refinement; 2 Overall/outer shell; 3 R sym = ∑hkl∑i |I-|/∑hkl∑I; 4 Most favored region in Ramachandran plot as defined in PROCHECK. Overall Structure of the Complex• • Fisetin (3,7,3',4'-Tetrahydroxyflavone) is a relatively small flavonol inhibitor with a hydroxyl-group in the 3 position of the benzopyran ring. This distinguishes it from several other flavonol inhibitors whose inhibitory activity on CDK6-Vcyclin was determined in this study as well ( Figure 1& 5). • • The complex structure of CDK6-Vcyclin with the inhibitor fisetin is the first inhibitor complex structures with a kinase from the CDK subfamily consisting of CDK4 and CDK6 ( Figure 2 & Table 1). • • Fisetin was found to bind in the ATP binding pocket which is located between the two kinase domains (Figure 2). Correlation of Structural Results with Binding

Proliferating cell nuclear antigen (PCNA) is a homotrimeric, ring-shaped protein complex that functions as a processivity factor for DNA polymerases. Following genotoxic stress, PCNA is modified at a conserved site by either a single ubiquitin moiety or a polyubiquitin chain. These modifications are required to coordinate DNA damage tolerance processes with ongoing replication. The molecular mechanisms responsible for inducing PCNA ubiquitination are not well understood. Using Xenopus egg extracts, we show that ultraviolet radiation and aphidicolin treatment induce the mono-and diubiquitination of PCNA. PCNA ubiquitination is replication-dependent and coincides with activation of the ataxia telangiectasia mutated and Rad3-related (ATR)-dependent DNA damage checkpoint pathway. However, loss of ATR signaling by depletion of the ATR-interacting protein (ATRIP) or Rad1, a component of the 911 checkpoint clamp, does not impair PCNA ubiquitination. Primed single-stranded DNA generated by uncoupling of mini-chromosome maintenance helicase and DNA polymerase activities has been shown previously to be necessary for ATR activation. Here we show that PCNA ubiquitination also requires uncoupling of helicase and polymerase activities. We further demonstrate that replicating single-stranded DNA, which mimics the structure produced upon uncoupling, is sufficient to induce PCNA monoubiquitination. Our results suggest that PCNA ubiquitination and ATR activation are two independent events that occur in response to a common single-stranded DNA intermediate generated by functional uncoupling of mini-chromosome maintenance (MCM) helicase and DNA polymerase activities.Bulky lesions in the DNA cause arrest of replicative polymerases, halting cell cycle progression and activating DNA damage checkpoints. DNA damage tolerance mechanisms allow the cell to continue replication under these conditions and permit repair of the damage at a later time (1). Translesion synthesis (TLS) 2 is one type of DNA damage tolerance mechanism that allows specialized, low fidelity DNA polymerases to bypass DNA damage in an error-prone fashion. A second mechanism of DNA damage tolerance involves error-free strategies of bypass that include post-replication recombinational repair and replication fork regression. How these DNA damage tolerance mechanisms are coordinated with replication at stalled forks is largely unknown.One protein that plays a critical role in linking these processes is proliferating cell nuclear antigen (PCNA). PCNA is a homotrimeric ring-shaped protein complex that functions as a processivity factor necessary to maintain ongoing synthesis of DNA. PCNA has critical functions in DNA replication and DNA damage tolerance, and these functions depend on its interaction with other proteins. In addition, post-translational modification of PCNA has emerged as a critical step in regulating PCNA functions (1, 2).In response to DNA damage or replication fork stalling, PCNA is modified at a conserved site, Lys-164, by either a single ubiquitin moiet...

Background: Rare plants that contain corrinoid compounds mostly comprise cobalamin analogues, which may compete with cobalamin (vitamin B12 (B12)) metabolism. We examined the presence of B12 in a cultivated strain of an aquatic plant: Wolffia globosa (Mankai), and predicted functional pathways using gut-bioreactor, and the effects of long-term Mankai consumption as a partial meat substitute, on serum B12 concentrations. Methods: We used microbiological assay, liquid-chromatography/electrospray-ionization-tandem-mass-spectrometry (LC-MS/MS), and anoxic bioreactors for the B12 experiments. We explored the effect of a green Mediterranean/low-meat diet, containing 100 g of frozen Mankai shake/day, on serum B12 levels during the 18-month DIRECT-PLUS (ID:NCT03020186) weight-loss trial, compared with control and Mediterranean diet groups. Results: The B12 content of Mankai was consistent at different seasons (p = 0.76). Several cobalamin congeners (Hydroxocobalamin(OH-B12); 5-deoxyadenosylcobalamin(Ado-B12); methylcobalamin(Me-B12); cyanocobalamin(CN-B12)) were identified in Mankai extracts, whereas no pseudo B12 was detected. A higher abundance of 16S-rRNA gene amplicon sequences associated with a genome containing a KEGG ortholog involved in microbial B12 metabolism were observed, compared with control bioreactors that lacked Mankai. Following the DIRECT-PLUS intervention (n = 294 participants; retention-rate = 89%; baseline B12 = 420.5 ± 187.8 pg/mL), serum B12 increased by 5.2% in control, 9.9% in Mediterranean, and 15.4% in Mankai-containing green Mediterranean/low-meat diets (p = 0.025 between extreme groups). Conclusions: Mankai plant contains bioactive B12 compounds and could serve as a B12 plant-based food source.

Background: Polyphenols are secondary metabolites produced by plants to defend themselves from environmental stressors. We explored the effect of Wolffia globosa ‘Mankai’, a novel cultivated strain of a polyphenol-rich aquatic plant, on the metabolomic-gut clinical axis in vitro, in-vivo and in a clinical trial. Methods: We used mass-spectrometry-based metabolomics methods from three laboratories to detect Mankai phenolic metabolites and examined predicted functional pathways in a Mankai artificial-gut bioreactor. Plasma and urine polyphenols were assessed among the 294 DIRECT-PLUS 18-month trial participants, comparing the effect of a polyphenol-rich green-Mediterranean diet (+1240 mg/polyphenols/day, provided by Mankai, green tea and walnuts) to a walnuts-enriched (+440 mg/polyphenols/day) Mediterranean diet and a healthy controlled diet. Results: Approximately 200 different phenolic compounds were specifically detected in the Mankai plant. The Mankai-supplemented bioreactor artificial gut displayed a significantly higher relative-abundance of 16S-rRNA bacterial gene sequences encoding for enzymes involved in phenolic compound degradation. In humans, several Mankai-related plasma and urine polyphenols were differentially elevated in the green Mediterranean group compared with the other groups (p < 0.05) after six and 18 months of intervention (e.g., urine hydroxy-phenyl-acetic-acid and urolithin-A; plasma Naringenin and 2,5-diOH-benzoic-acid). Specific polyphenols, such as urolithin-A and 4-ethylphenol, were directly involved with clinical weight-related changes. Conclusions: The Mankai new plant is rich in various unique potent polyphenols, potentially affecting the metabolomic-gut-clinical axis.