Proliferating cell nuclear antigen-dependent abasic site repair in Xenopus laevis oocytes: an alternative pathway of base excision DNA repair.

DNA damage frequently leads to the production of apurinic/apyrimidinic (AP) sites, which are presumed to be repaired through the base excision pathway. For detailed analyses of this repair mechanism, a synthetic analog of an AP site, 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran), has been employed in a model system. Tetrahydrofuran residues are efficiently repaired in a Xenopus laevis oocyte extract in which most repair events involve ATP-dependent incorporation of no more than four nucleotides (Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 9:3750-3757, 1989; Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 11:4441-4447, 1991). Using a series of column chromatography procedures to fractionate X. laevis ovarian extracts, we developed a reconstituted system of tetrahydrofuran repair with five fractions, three of which were purified to near homogeneity: proliferating cell nuclear antigen (PCNA), AP endonuclease, and DNA polymerase delta. This PCNA-dependent system repaired natural AP sites as well as tetrahydrofuran residues. DNA polymerase beta was able to replace DNA polymerase delta only for repair of natural AP sites in a reaction that did not require PCNA. DNA polymerase alpha did not support repair of either type of AP site. This result indicates that AP sites can be repaired by two distinct pathways, the PCNA-dependent pathway and the DNA polymerase beta-dependent pathway.     

New pUC-derived expression vectors for rapid construction of cDNA libraries

We have constructed a new series of the pUC-derived plasmids with an extended polylinker obtained from M13tg131 [Kieny et al., Gene 26 (1983) 91-99]. These vectors allowed us to design a simplified version of the method of Okayama and Berg [Mol. Cell. Biol. 2 (1982) 161-170] for establishing complete cDNA libraries. Improvements included easy recovery of the inserted cDNA due to the extended polylinkers; use of these vectors for gene expression in Escherichia coli, and amenability to supercoil sequencing with the universal primers of the M13 system [Chen and Seeburg, DNA 4 (1985) 165-170], which speeds up the identification of positive clones. Moreover, there is no need for an additional linker fragment, as was required by the Okayama and Berg [Mol. Cell. Biol. 2 (1982) 161-170] method. We have successfully used this system to obtain cDNA clones coding for the different chains of the large basement membrane proteins type IV collagen and laminin.     

Sequence similarities among monkey ori-enriched (ors) fragments

Nucleotide sequences have been determined for eight ors (ori-enriched sequence) fragments isolated from monkey DNA by a method that was designed to enrich for origins of DNA replication [Kaufmann et al., Mol. Cell. Biol. 5 (1985) 721-727]. Evidence has been presented that some or possibly all of these sequences can serve, albeit inefficiently, as oris in vivo [Frappier and Zannis-Hadjopoulos, Proc. Natl. Acad. Sci. USA 84 (1987) 6668-6672]. Two of the fragments were found to contain the long terminal repeat-like elements of the 'O-family' of moderately repetitive sequences that are present in human DNA as a transposon-like element [Paulson et al., Nature 315 (1985) 359-361]. Extensive pair-wise comparisons of the sequences failed to detect any statistically significant common sequences, except for long asymmetrically distributed A + T-rich stretches. Nonetheless, when the ors fragments were examined for the presence of published consensus sequences, seven of eight were found to contain the control sequence described by Dierks et al. [Cell 32 (1983) 695-706], and the same seven of eight were found to contain both the scaffold attachment region T consensus [Gasser and Laemmli, Cell 46 (1986) 521-530] and the minimal Saccharomyces cerevisiae autonomously replicating sequence consensus [e.g., Palzkill and Newlon, Cell 53 (1988) 441-450].     

Actin's propensity for dynamic filament patterning

Actin, through its various forms of assembly, provides the basic framework for cell motility, cell shape and intracellular organization in all eukaryotic cells. Many other cellular processes, for example endocytosis and cytokinesis, are also associated with dynamic changes of the actin cytoskeleton. Important prerequisites for actin's functional diversity are its intrinsic ability to rapidly assemble and disassemble filaments and its spatially and temporally well-controlled supramolecular organization. A large number of proteins that interact with actin, collectively referred to as actin-binding proteins (ABPs), carefully orchestrate different scenarios. Since its isolation in 1994 [Machesky, L.M. et al. (1994) J. Cell Biol. 127, 107-115], the Arp2/3 complex containing the actin-related proteins Arp2 and Arp3 has evolved to be one of the main players in the assembly and maintenance of many actin-based structures in the cell (for review see [Borths, E.L. and Welch, M.D. (2002) Structure 10, 131-135; May, R.C. (2001) Cell Mol. Life Sci. 58, 1607-1626; Pollard, T.D. et al. (2000) Rev. Biophys. Biomol. Struct. 29, 545-576; Welch, M.D. (1999) Trends Cell Biol. 11, 423-427]). In particular, when it comes to the assembly of the intricate branched actin network at the leading edge of lamellipodia, the Arp2/3 complex seems to have received all the attention in recent years. In parallel, but not so much in the spotlight, several reports showed that actin on its own can assume different conformations [Bubb, M.R. et al. (2002) J. Biol. Chem. 277, 20999-21006; Schoenenberger, C.-A. et al. (1999) Microsc. Res. Tech. 47, 38-50; Steinmetz, M.O. et al. (1998) J. Mol. Biol. 278, 793-811; Steinmetz, M.O. et al. (1997) J. Cell Biol. 138, 559-574; Millonig, R., Salvo, H. and Aebi, U. (1988) J. Cell Biol. 106, 785-796] through which it drives its supramolecular patterning, and which ultimately generate its functional diversity.     

Evidence for the extent of insertion of the active site loop of intact alpha 1 proteinase inhibitor in beta-sheet A.

The extent of insertion of beta-strand s4A into sheet A in intact serpin alpha 1-proteinase inhibitor (alpha 1PI has been probed by peptide annealing experiments [Schulze et al. (1990) Eur. J. Biochem. 194, 51-56]. Twelve synthetic peptides of systematically varied length corresponding in sequence to the unprimed (N-terminal) side of the active site loop were complexed with alpha 1PI. The complexes were then characterized by circular dichroism spectroscopy and tested for inhibitory activity. Four peptides formed complexes which retained inhibitory activity, one of which was nearly as effective as the native protein. Comparison with the three dimensional structures of cleaved alpha 1PI [L?bermann et al. (1984) J. Mol. Biol. 177, 531-556] and plakalbumin [Wright et al. (1990) J. Mol. Biol. 213, 513-528] supports a model in which alpha 1PI requires the insertion of a single residue, Thr345, into sheet A for activity.     

Striatin, a calmodulin-dependent scaffolding protein, directly binds caveolin-1.

Caveolins are scaffolding proteins able to collect on caveolae a large number of signalling proteins bearing a caveolin-binding motif. The proteins of the striatin family, striatin, SG2NA, and zinedin, are composed of several conserved, collinearly aligned, protein-protein association domains, among which a putative caveolin-binding domain [Castets et al. (2000) J. Biol. Chem. 275, 19970-19977]. They are associated in part with membranes. These proteins are mainly expressed within neurons and thought to act both as scaffolds and as Ca(2+)-dependent signalling proteins [Bartoli et al. (1999) J. Neurobiol. 40, 234-243]. Here, we show that (1) rat brain striatin, SG2NA and zinedin co-immunoprecipitate with caveolin-1; (2) all are pulled down by glutathione-S-transferase (GST)-caveolin-1; (3) a fragment of recombinant striatin containing the putative caveolin-binding domain binds GST-caveolin-1. Hence, it is likely that the proteins of the striatin family are addressed to membrane microdomains by their binding to caveolin, in accordance with their putative role in membrane trafficking [Baillat et al. (2001) Mol. Biol. Cell 12, 663-673].     

Interaction of MAR-sequences with nuclear matrix proteins.

The recent discovery of DNA sequences responsible for the specific attachment of chromosomal DNA to the nuclear skeleton (MARs/SARs) was an important step towards our understanding of the functional and structural organization of eukaryotic chromatin [Mirkovitch et al.: Cell 44:273-282, 1984; Cockerill and Garrard: Cell 44:273-282, 1986]. A most important question, however, remains the nature of the matrix proteins involved in the specific binding of the MARs. It has been shown that topoisomerase II and histone H1 were capable of a specific interaction with SARs by the formation of precipitable complexes [Adachi et al.: EMBO J8:3997-4006, 1989; Izaurralde et al.: J Mol Biol 210:573-585, 1989]. Here, applying a different approach, we were able to "visualize" some of the skeletal proteins recognizing and specifically binding MAR-sequences. It is shown that the major matrix proteins are practically the same in both salt- and LIS-extracted matrices. However, the relative MAR-binding activity of the individual protein components may be different, depending on the method of matrix preparation. The immunological approach applied here allowed us to identify some of the individual MAR-binding matrix proteins. Histone H1 and nuclear actin are shown to be not only important components of the matrix, but to be involved in a highly efficient interaction with MAR-sequences as well. Evidence is presented that proteins recognized by the anti-HMG antibodies also participate in MAR-interactions.     

Functional interactions between DNA damage signaling and mutagenic translesion synthesis at post-replicative gaps

Comment on: Jansen JG, Tsaalbi-Shtylik A, Hendriks G, Gali H, Hendel A, Johansson F, et al. Separate domains of Rev1 mediate two modes of DNA damage bypass in mammalian cells. Mol Cell Biol 2009; 29:3113-23.     

Tubulin folding cofactor D is a microtubule destabilizing protein

A rapid switch between growth and shrinkage at microtubule ends is fundamental for many cellular processes. The main structural components of microtubules, the alphabeta-tubulin heterodimers, are generated through a complex folding process where GTP hydrolysis [Fontalba et al. (1993) J. Cell Sci. 106, 627-632] and a series of molecular chaperones are required [Sternlicht et al. (1993) Proc. Natl. Acad. Sci. USA 90, 9422-9426; Campo et al. (1994) FEBS Lett. 353, 162-166; Lewis et al. (1996) J. Cell Biol. 132, 1-4; Lewis et al. (1997) Trends Cell Biol. 7, 479-484; Tian et al. (1997) J. Cell Biol. 138, 821-823]. Although the participation of the cofactor proteins along the tubulin folding route has been well established in vitro, there is also evidence that these protein cofactors might contribute to diverse microtubule processes in vivo [Schwahn et al. (1998) Nature Genet. 19, 327-332; Hirata et al. (1998) EMBO J. 17, 658-666; Fanarraga et al. (1999) Cell Motil. Cytoskel. 43, 243-254]. Microtubule dynamics, crucial during mitosis, cellular motility and intracellular transport processes, are known to be regulated by at least four known microtubule-destabilizing proteins. OP18/Stathmin and XKCM1 are microtubule catastrophe-inducing factors operating through different mechanisms [Waters and Salmon (1996) Curr. Biol. 6, 361-363; McNally (1999) Curr. Biol. 9, R274-R276]. Here we show that the tubulin folding cofactor D, although it does not co-polymerize with microtubules either in vivo or in vitro, modulates microtubule dynamics by sequestering beta-tubulin from GTP-bound alphabeta-heterodimers.     

Primary structure of human salivary alpha-amylase gene

A recombinant clone which covers the human salivary alpha-amylase gene in a single insert has been isolated from a human genomic DNA library using a human salivary alpha-amylase cDNA as a probe. Restriction mapping and nucleotide (nt) sequence analysis revealed that this gene is approx. 10 kb long and is separated into eleven exons by ten introns. Its 5'-flanking region has some sequence homology with that of mouse salivary alpha-amylase gene [Schibler et al., J. Mol. Biol. 155 (1982) 247-266].     

Rheb: Enrichment beyond the brain

Comment on: Goorden SM, et al. Mol Cell Biol 2011; 31:1672-8.     

ALTered telomeres in response to telomere dysfunction

Comment on: Brault ME, et al. Mol Biol Cell 2011; 22:179-88.     

TIS11/TTP gene family: It's never too late for tumor suppressors

Comment on: Vignudelli T, et al. Mol Biol Cell 2010; 21:3340-51.     

Generation of cells that ignore the effects of PIP3 on cytoskeleton

Comment on: Tang M, et al. Mol Biol Cell 2011; 22:437-47.     

p21Cip1 regulation by ERK2: A post-translational mechanism that relays a proliferation signal

Comment on: Hwang CY, et al. Mol Cell Biol 2009; 29:3379-89.     

Busy traveling Ras

Comment on: Cheng CM, et al. Mol Cell Biol 2011; 31:983-97.     

Genome reduplication: Redeployment of cyclin-CDK complexes and spontaneous oscillation of APC/C

Comment on: Ma HT, et al. Mol Cell Biol 2009; 29:6500-14.     

Developmental regulation of replication-coupled protein destruction

Comment on: Lee HO, et al. Mol Biol Cell 2010; 21:3639-53.