Oral lining mucosa development depends on mesenchymal microRNAs.

The oral mucosa plays critical roles in protection, sensation, and secretion and can be classified into masticatory, lining, and specialized mucosa that are known to be functionally, histologically, and clinically distinct. Each type of oral mucosa is believed to develop through discrete molecular mechanisms, which remain unclear. MicroRNAs (miRNAs) are 19 to 25nt non-coding small single-stranded RNAs that negatively regulate gene expression by binding target mRNAs. miRNAs are crucial for fine-tuning of molecular mechanisms. To investigate the role of miRNAs in oral mucosa development, we examined mice with mesenchymal (Wnt1Cre;Dicer(fl/fl)) conditional deletion of Dicer. Wnt1Cre;Dicer(fl/fl) mice showed trans-differentiation of lining mucosa into an epithelium with masticatory mucosa/ skin-specific characteristics. Up-regulation of Fgf signaling was found in mutant lining mucosal epithelium that was accompanied by an increase in Fgf7 expression in mutant mesenchyme. Mesenchyme miRNAs thus have an indirect effect on lining mucosal epithelial cell growth/differentiation.

Double-check probing of DNA bending and unwinding by XPA-RPA: an architectural function in DNA repair.

The multiprotein factor composed of XPA and replication protein A (RPA) is an essential subunit of the mammalian nucleotide excision repair system. Although XPA-RPA has been implicated in damage recognition, its activity in the DNA repair pathway remains controversial. By replacing DNA adducts with mispaired bases or non-hybridizing analogues, we found that the weak preference of XPA and RPA for damaged substrates is entirely mediated by indirect readout of DNA helix conformations. Further screening with artificially distorted substrates revealed that XPA binds most efficiently to rigidly bent duplexes but not to single-stranded DNA. Conversely, RPA recognizes single-stranded sites but not backbone bending. Thus, the association of XPA with RPA generates a double-check sensor that detects, simultaneously, backbone and base pair distortion of DNA. The affinity of XPA for sharply bent duplexes, characteristic of architectural proteins, is not compatible with a direct function during recognition of nucleotide lesions. Instead, XPA in conjunction with RPA may constitute a regulatory factor that monitors DNA bending and unwinding to verify the damage-specific localization of repair complexes or control their correct three-dimensional assembly.

DNA synthesis by CHO cell extracts on fork-like DNA templates containing the major cisplatin adduct requires a ligation step.

In this work we have examined the role of DNA ligation in the in vitro replication catalyzed by CHO crude extracts on fork-like oligonucleotide substrates containing a unique d(GpG) intrastrand cross-link produced by the antitumor drug cisplatin. We show here that this reaction involves a ligation step, which necessitates excision of the flap strand of the forked substrate. By constructing substrates in which the unannealed tail could not be degraded by a 5' exonuclease, we obtained evidence suggesting that this type of activity participates in the removal of the flap strand. Furthermore, we found that the ligation event played a predominant role in the synthesis of fully replicated products from both intact and platinated templates. Finally, we investigated whether translesion synthesis of the cisplatin lesion could occur concomitantly to ligation by monitoring the incorporation of labeled precursors downstream of the adduct. Our results are compatible with the possibility that some translesion syntheses of the Pt-d(GpG) adduct by the extracts also contributed to the generation of full length molecules.

The homeodomain protein vax1 is required for axon guidance and major tract formation in the developing forebrain.

The homeodomain protein Vax1 is expressed in a highly circumscribed set of cells at the ventral anterior midline of the embryonic CNS. These cells populate the choroid fissure of the optic disk, the body of the optic stalk and nerve, the optic chiasm and ventral diencephalon, and the anterior midline zones that abut developing commissural tracts. We have generated mutant mice that lack Vax1. In these mice (1) the optic disks fail to close, leading to coloboma and loss of the eye-nerve boundary; (2) optic nerve glia fail to associate with and appear to repulse ingrowing retinal axons, resulting in a fascicle of axons that are completely segregated from optic nerve astrocytes; (3) retinal axons fail to penetrate the brain in significant numbers and fail to form an optic chiasm; and (4) axons in multiple commissural tracts of the anterior CNS, including the corpus callosum and the hippocampal and anterior commissures, fail to cross the midline. These axon guidance defects do not result from the death of normally Vax1(+) midline cells but, instead, correlate with markedly diminished expression of attractive guidance cues in these cells. Vax1 therefore regulates the guidance properties of a set of anterior midline cells that orchestrate axon trajectories in the developing mammalian forebrain.

Regulation of DNA replication and repair proteins through interaction with the front side of proliferating cell nuclear antigen.

The DNA polymerase accessory factor proliferating cell nuclear antigen (PCNA) has been caught in interaction with an ever increasing number of proteins. To characterize the sites and functions of some of these interactions, we constructed four mutants of human PCNA and analysed them in a variety of assays. By targeting loops on the surface of the PCNA trimer and changing three or four residues at a time to alanine, we found that a region including part of the domain-connecting loop of PCNA and loops on one face of the trimer, close to the C-termini, is involved in binding to all of the following proteins: DNA polymerase delta, replication factor C, the flap endonuclease Fen1, the cyclin dependent kinase inhibitor p21 and DNA ligase I. An inhibition of DNA ligation caused by the interaction of PCNA with DNA ligase I was found, and we show that DNA ligase I and Fen1 can inhibit DNA synthesis by DNA polymerase delta/PCNA. We demonstrate that PCNA must be located below a 5' flap on a forked template to stimulate Fen1 activity, and considering the interacting region on PCNA for Fen1, this suggests an orientation for PCNA during DNA replication with the C-termini facing forwards, in the direction of DNA synthesis.

The solution structure of functionally active human proliferating cell nuclear antigen determined by small-angle neutron scattering.

The function of proliferating cell nuclear antigen (PCNA) in DNA replication and repair is to form a sliding clamp with replication factor C (RF-C) tethering DNA polymerase delta or epsilon to DNA. In addition, PCNA has been found to interact directly with various proteins involved in cell cycle regulation. The crystal structure of yeast PCNA shows that the protein forms a homotrimeric ring lining a hole through which double-stranded DNA can thread, thus forming a moving platform for DNA synthesis. Human and yeast PCNA are highly conserved at a structural and functional level. We determined the solution structure of functionally active human PCNA by small-angle neutron scattering. Our measurements strongly support a trimeric ring-like structure of functionally active PCNA in solution, and the data are in good agreement with model calculations based on the crystal structure from yeast PCNA. The human PCNA used in the small-angle neutron scattering experiments was active before and after the measurements in a RF-C independent and a RF-C dependent assay suggesting that the trimeric structure is the in vivo functional form.

Cloning, chromosomal localization, and interspecies interaction of mouse DNA polymerase delta small subunit (PolD2).

DNA polymerase delta core is a heterodimeric enzyme with a catalytic subunit of 125 kDa and a second subunit of 50 kDa with an as yet unknown function. It is an essential enzyme for DNA replication and DNA repair. We cloned the full-length cDNA encoding the DNA polymerase delta small subunit from mouse cells. The sequence of the predicted polypeptide of 51,336 Da is, like the catalytic subunit, highly conserved not only among mammals (93% identity and 96% similarity), but also between yeast and mammals (34% identity and 57% similarity). Fluorescence in situ hybridization experiments indicated that the gene for the small DNA polymerase delta of mouse is localized on chromosome 11, band A2. By using the yeast two-hybrid system we found that the mouse 125-kDa DNA polymerase catalytic subunit is able to interact with the 50-kDa subunit of the human enzyme, suggesting an in vivo interspecies interaction between the two subunits of DNA polymerase delta.

DNA polymerase delta, an essential enzyme for DNA transactions.

Many DNA transactions, such as replication, repair and recombination involve DNA synthesis and consequently require the action of DNA synthesizing enzymes called DNA polymerases (Pol). Eukaryotic cells contain at least six different Pols, named alpha, beta, gamma, delta, epsilon, and zeta. Among them Pol delta occupies important roles in DNA replication, nucleotide excision repair, base excision repair and VDJ recombination. Pol a has been extremely conserved in evolution from yeast to man. The function of Pol delta must be considered in the context of two other factors, called proliferating cell nuclear antigen and replication factor C, two protein complexes that build together the moving platform for Pol delta. This moving platform provides an important framework for dynamic properties of an accurate Pol delta such as its recruitment when its function is needed, the facilitation of Pol delta binding to the primer terminus, the increase in Pol delta processivity, the prevention of non-productive binding of the Pol delta to single-stranded DNA, the release of Pol delta after DNA synthesis and the bridging of Pol delta interactions to other replication proteins. In this review we summarize the current knowledge of Pol delta and will focus in particular to its structural conservation, its functional tasks in the cell and its interactions with other proteins.

Production of active mouse DNA polymerase delta in bacteria.

The entire cDNA encoding the large subunit of mouse DNA polymerase delta (mPol delta; EC 2.7.7.7) has been cloned and expressed in various bacterial expression systems. A soluble protein could only be obtained when mPol delta was produced as a glutathione S-transferase (GST) fusion protein and the incubation temperature of the expression strain was reduced to 30 degrees C. After purification over a glutathione-Sepharose column, the fractions containing the recombinant (re-) fusion protein showed both DNA Pol and 3'-->5' Exo activities. In situ activity gel analysis indicated that the Pol activity resides in the re-protein. This activity, however, was not stimulated by proliferating cell nuclear antigen (PCNA). Our data are discussed in the view of the findings of Goulian et al. [J. Biol. Chem., 265 (1990) 16402-16411] that the second mPol delta subunit, the 48-kDa protein, might play an important role in DNA Pol delta-PCNA interaction.

Cloning of a mouse cDNA encoding DNA polymerase delta: refinement of the homology boxes.

A mouse DNA polymerase delta (Pol delta)-encoding cDNA (pol delta) was isolated by PCR amplification and cDNA library screening. The sequenced cDNA has a length of 3386 bp and the open reading frame (ORF) encodes a protein of 1105 amino acids (aa) with an M(r) of 123,743. The aa identity to the proteins encoded by the corresponding cDNA from Bos taurus (93%) and Homo sapiens (92%) is very high. The identity to the Pol delta from Schizosaccharomyces pombe, Saccharomyces cerevisiae and Plasmodium falciparum is around 50%. An aa comparison between all available Pol delta sequences reveals several common structural motifs. Polyclonal antibodies raised against a 31-aa synthetic peptide deduced from the ORF specifically recognize Pol delta polymerases from human cells and calf thymus in an immunoblot.