Flap endonuclease 1 (FEN1) proteins, which are present in all kingdoms of life, catalyze the sequence-independent hydrolysis of the bifurcated nucleic acid intermediates formed during DNA replication and repair. How FEN1s have evolved to preferentially cleave flap structures is of great interest especially in light of studies wherein mice carrying a catalytically deficient FEN1 were predisposed to cancer. Structural studies of FEN1s from phage to human have shown that, although they share similar folds, the FEN1s of higher organisms contain a 3-extrahelical nucleotide (3-flap) binding pocket. When presented with 5-flap substrates having a 3-flap, archaeal and eukaryotic FEN1s display enhanced reaction rates and cleavage site specificity. To investigate the role of this interaction, a kinetic study of human FEN1 (hFEN1) employing well defined DNA substrates was conducted. The presence of a 3-flap on substrates reduced K m and increased multiple-and single turnover rates of endonucleolytic hydrolysis at near physiological salt concentrations. Exonucleolytic and fork-gap-endonucleolytic reactions were also stimulated by the presence of a 3-flap, and the absence of a 3-flap from a 5-flap substrate was more detrimental to hFEN1 activity than removal of the 5-flap or introduction of a hairpin into the 5-flap structure. hFEN1 reactions were predominantly rate-limited by product release regardless of the presence or absence of a 3-flap. Furthermore, the identity of the stable enzyme product species was deduced from inhibition studies to be the 5-phosphorylated product. Together the results indicate that the presence of a 3-flap is the critical feature for efficient hFEN1 substrate recognition and catalysis.In eukaryotic DNA replication and repair, various bifurcated nucleic acid structure intermediates are formed and must be processed by the appropriate nuclease. Two examples of biological processes that create bifurcated DNA intermediates are Okazaki fragment maturation (1, 2) and long patch excision repair (3). In both models, a polymerase executes strand-displacement synthesis to create a double-stranded DNA (dsDNA) 6 two-way junction from which a 5Ј-flap structure protrudes. The penultimate step of both pathways is the cleavage of this flap structure to create a nicked DNA that is then ligated. Because the bifurcated DNA structures that are formed in the aforementioned processes can theoretically occur anywhere in the genome, the nuclease associated with the cleavage of 5Ј-flap structures in eukaryotic cells, which is called flap endonuclease 1 (FEN1), must be capable of cleavage regardless of sequence. Therefore, FEN1 nucleases, which are found in all kingdoms of life (4), have evolved to recognize substrates based upon nucleic acid structure and strand polarity (5, 6).The Okazaki fragment maturation pathway of yeast has become a paradigm of eukaryotic lagging strand DNA synthesis. In the yeast model, bifurcated intermediates with large single-stranded DNA (ssDNA) 5Ј-flap structures are imprecisely cleaved by DNA2 ...

TL1A is a member of the TNF superfamily and its expression is increased in the mucosa of inflammatory bowel disease patients. Neutralizing anti-mouse TL1A Ab attenuates chronic colitis in two T-cell driven murine models, suggesting that TL1A is a central modulator of gut mucosal inflammation in inflammatory bowel disease. We showed previously that TL1A is induced by immune complexes via the FccR signaling pathway. In this study, we report that multiple bacteria, including gram negative organisms (E. coli, E. coli Nissle 1917, Salmonella typhimurium), gram positive organisms (Listeria monocytogenes, Staphylococcus epidermidis), partial anaerobes (Campylobacter jejuni), and obligate anaerobes (Bacteroides thetaiotaomicron, Bifidobacterium breve, Clostridium A4) activate TL1A expression in human APC, including monocytes and monocyte-derived DC. Bacterially induced TL1A mRNA expression correlates with the detection of TL1A protein levels. TL1A induced by bacteria is mediated in part by the TLR signaling pathway and inhibited by downstream blockade of p38 MAPK and NF-jB activation. Microbial induction of TL1A production by human APC potentiated CD4 1 T-cell effector function by augmenting IFN-c production. Our findings suggest a role for TL1A in pro-inflammatory APC-T cell interactions and implicate TL1A in host responses to enteric microorganisms.

There is much evidence to indicate that FEN-1 efficiently cleaves single-stranded DNA flaps but is unable to process double-stranded flaps or flaps adopting secondary structures. However, the absence of Fen1 in yeast results in a significant increase in trinucleotide repeat (TNR) expansion. There are then two possibilities. One is that TNRs do not always form stable secondary structures or that FEN-1 has an alternative approach to resolve the secondary structures. In the present study, we test the hypothesis that concerted action of exonuclease and gap-dependent endonuclease activities of FEN-1 play a role in the resolution of secondary structures formed by (CTG) n and (GAA) n repeats. Employing a yeast FEN-1 mutant, E176A, which is deficient in exonuclease (EXO) and gap endonuclease (GEN) activities but retains almost all of its flap endonuclease (FEN) activity, we show severe defects in the cleavage of various TNR intermediate substrates. Precise knock-in of this point mutation causes an increase in both the expansion and fragility of a (CTG) n tract in vivo. Taken together, our biochemical and genetic analyses suggest that although FEN activity is important for singlestranded flap processing, EXO and GEN activities may contribute to the resolution of structured flaps. A model is presented to explain how the concerted action of EXO and GEN activities may contribute to resolving structured flaps, thereby preventing their expansion in the genome.

Coactivator-associated arginine methyltransferase-1 (CARM1) is known to enhance transcriptional activation by nuclear receptors through interactions with the coactivators p160 and cAMP response element binding protein-binding protein (CBP) and methylation of histone H3 at arginine 17 (H3-R17). Here, we show that CARM1 can act as a coactivator for the transcription factor nuclear factor-kappaB (NF-kappaB) and enhance NF-kappaB activity in a CBP (p300)-dependent manner. This enhancement in 293T cells was abolished by cotransfection with a specific short hairpin RNA targeted to knockdown CARM1. Chromatin immunoprecipitation demonstrated CARM1 recruitment in vivo to the promoters of NF-kappaB p65-regulated genes along with CBP and steroid receptor coactivator-1. This was accompanied by an increase in histone H3-R17 methylation as well as H3-K9 and H3-K14 acetylation, and a decrease in H3-citrulline. Immunoprecipitation with anti-p65 antibody revealed that CARM1 physically interacts with NF-kappaB p65. Furthermore, we demonstrated the physiological significance by observing that similar events occurred when THP-1 monocytic cells were stimulated with TNF-alpha or with S100b, a ligand for the receptor of advanced glycation end products, both of which are associated with diabetic complications and also known inducers of NF-kappaB and inflammatory genes in monocytes. These results demonstrate that CARM1 participates in NF-kappaB-mediated transcription through H3-R17 methylation and support a nonnuclear receptor-associated function for CARM1. They also demonstrate for the first time that CARM1 occupancy, histone H3-R17 methylation, and citrullination are regulated at the promoters of inflammatory genes in monocytes, thereby suggesting a novel role for histone arginine modifications in inflammatory diseases.

Flap endonuclease 1 (FEN-1) is a multifunctional and structure-specific nuclease that plays a critical role in maintaining human genome stability through RNA primer removal, long-patch base excision repair, resolution of DNA secondary structures and stalled DNA replication forks, and apoptotic DNA fragmentation. How FEN-1 is involved in multiple pathways, some of which are seemingly contradictory, is of considerable interest. To date, at least twenty proteins are known to interact with FEN-1; some form distinct complexes that affect one or more FEN-1 activities presumably to direct FEN-1 to a particular DNA metabolic pathway. FEN-1 consists of a nuclease core domain and a Cterminal extension. While the core domain harbors the nuclease activity, the C-terminal extension may be important for protein-protein interactions. Here, we have truncated or mutated the C-terminus of FEN-1 to identify amino acid residues that are critical for interaction with five proteins representing roles in different DNA replication and repair pathways. We found with all five proteins that the Cterminus is important for binding and that each protein uses a subset of amino acid residues. Replacement of one or more residues with an alanine in many cases leads to the complete loss of interaction, which may consequently lead to severe biological defects in mammals.