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Biomedical subjects

S Tom

Publications and source records attributed to S Tom.

11 recordsLinked to original sources
PubMed2001

Regulatory roles of p21 and apurinic/apyrimidinic endonuclease 1 in base excision repair.

Many types of DNA damage induce a cellular response that inhibits replication but allows repair by up-regulating the p53 pathway and inducing p21(Cip1, Waf1, Sdi1). The p21 regulatory protein can bind proliferating cell nuclear antigen (PCNA) and prohibit DNA replication. We show here that p21 also inhibits PCNA stimulation of long patch base excision repair (BER) in vitro. p21 disrupts PCNA-directed stimulation of flap endonuclease 1 (FEN1), DNA ligase I, and DNA polymerase delta. The dilemma is to understand how p21 prevents DNA replication but allows BER in vivo. Differential regulation by p21 is likely to relate to the utilization of DNA polymerase beta, which is not sensitive to p21, in the repair pathway. We have also found that apurinic/apyrimidinic endonuclease 1 (APE1) stimulates long patch BER. Furthermore, neither APE1 activity nor its ability to stimulate long patch BER is significantly affected by p21 in vitro. We propose that APE1 serves as an assembly and coordination factor for long patch BER proteins. APE1 initially cleaves the DNA and then facilitates the sequential binding and catalysis by DNA polymerase beta, DNA polymerase delta, FEN1, and DNA ligase I. This model implies that BER can be regulated differentially, based upon the assembly of relevant proteins around APE1 in the presence or absence of PCNA.

Animals↗
PubMed2001

DNA ligase I and proliferating cell nuclear antigen form a functional complex.

DNA ligase I is responsible for joining Okazaki fragments during DNA replication. An additional proposed role for DNA ligase I is sealing nicks generated during excision repair. Previous studies have shown that there is a physical interaction between DNA ligase I and proliferating cell nuclear antigen (PCNA), another important component of DNA replication and repair. The results shown here indicate that human PCNA enhances the reaction rate of human DNA ligase I up to 5-fold. The stimulation is specific to DNA ligase I because T4 DNA ligase is not affected. Electrophoretic mobility shift assays indicate that PCNA improves the binding of DNA ligase I to the ligation site. Increasing the DNA ligase I concentration leads to a reduction in PCNA stimulation, consistent with PCNA-directed improvement of DNA ligase I binding to its DNA substrate. Two experiments show that PCNA is required to encircle duplex DNA to enhance DNA ligase I activity. Biotin-streptavidin conjugations at the ends of a linear substrate inhibit PCNA stimulation. PCNA cannot enhance ligation on a circular substrate without the addition of replication factor C, which is the protein responsible for loading PCNA onto duplex DNA. These results show that PCNA is responsible for the stable association of DNA ligase I to nicked duplex DNA.

Casein Kinase II↗
PubMed2000

Inhibition of flap endonuclease 1 by flap secondary structure and relevance to repeat sequence expansion.

Recent genetic evidence indicates that null mutants of the 5'-flap endonuclease (FEN1) result in an expansion of repetitive sequences. The substrate for FEN1 is a flap formed by natural 5'-end displacement of the short intermediates of lagging strand replication. FEN1 binds the 5'-end of the flap, tracks to the point of annealing at the base of the flap, and then cleaves. Here we examine mechanisms by which foldback structures within the flap could contribute to repeat expansions. Cleavage by FEN1 was reduced with increased length of the foldback. However, even the longest foldbacks were cleaved at a low rate. Substrates containing the repetitive sequence CTG also were cleaved at a reduced rate. Bubble substrates, likely intermediates in repeat expansions, were inhibitory. Neither replication protein A nor proliferating cell nuclear antigen were able to assist in the removal of secondary structure within a flap. We propose that FEN1 cleaves natural foldbacks at a reduced rate. However, although the cleavage delay is not likely to influence the overall process of chromosomal replication, specific foldbacks could inhibit cleavage sufficiently to result in duplication of the foldback sequence.

Base Sequence↗
PubMed2000

Mechanism whereby proliferating cell nuclear antigen stimulates flap endonuclease 1.

Human flap endonuclease 1 (FEN1), an essential DNA replication protein, cleaves substrates with unannealed 5'-tails. FEN1 apparently tracks along the flap from the 5'-end to the cleavage site. Proliferating cell nuclear antigen (PCNA) stimulates FEN1 cleavage 5-50-fold. To determine whether tracking, binding, or cleavage is enhanced by PCNA, we tested a variety of flap substrates. Similar levels of PCNA stimulation occur on both a cleavage-sensitive nicked substrate and a less sensitive gapped substrate. PCNA stimulates FEN1 irrespective of the flap length. Stimulation occurs on a pseudo-Y substrate that exhibits upstream primer-independent cleavage. A pseudo-Y substrate with a sequence requiring an upstream primer for cleavage was not activated by PCNA, suggesting that PCNA does not compensate for substrate features that inhibit cleavage. A biotin.streptavidin conjugation at the 5'-end of a flap structure prevents FEN1 loading. The addition of PCNA does not restore FEN1 activity. These results indicate that PCNA does not direct FEN1 to the cleavage site from solution. Kinetic analyses reveal that PCNA can lower the K(m) for FEN1 by 11-12-fold. Overall, our results indicate that after FEN1 tracks to the cleavage site, PCNA enhances FEN1 binding stability, allowing for greater cleavage efficiency.

Base Sequence↗
PubMed1999

Secretion of glycosylation site mutants can be rescued by the signal/pro sequence of tissue plasminogen activator.

Strategies that prevent the attachment of N-linked carbohydrates to nascent glycoproteins often impair intracellular transport and secretion. In the present study, we describe a method to rescue the intracellular transport and secretion of glycoproteins mutagenized to delete N-linked glycosylation sites. Site-directed mutagenesis was used to delete N-linked glycosylation sites from a chimeric protein, TNFR-IgG1. Deletion of any of the three glycosylation sites in the TNFR portion of the molecule, alone or in combination, resulted in a moderate or near total blockade of TNFR-IgG1 intracellular transport and secretion. Pulse chase experiments suggested that the glycosylation site mutants accumulated in the endoplasmic reticulum (ER) and were inefficiently exported to the Golgi apparatus (GA). Replacement of the TNFR signal sequence with the signal/pro sequence of human tissue plasminogen activator (tPA) overcame the blockade to intracellular transport, and restored secretion to levels comparable to those achieved with the fully glycosylated molecule. Ligand binding studies suggested that the secreted glycosylation variants possessed binding characteristics similar to the fully glycosylated protein. This study demonstrates that N-terminal sequences of tPA are unexpectedly efficient in facilitating transport from the ER to the GA and suggests that these sequences contain a previously unrecognized structural element that promotes intracellular transport.

Amino Acid Sequence↗
PubMed1999

Engineering Chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins.

We have engineered two Chinese hamster ovary cell lines secreting different recombinant glycoproteins to express high levels of human beta1,4-galactosyltransferase (GT, E.C. 2.4.1.38) and/or alpha2, 3-sialyltransferase (ST, E.C. 2.4.99.6). N-linked oligosaccharide structures synthesized by cells overexpressing the glycosyltransferases showed greater homogeneity compared with control cell lines. When GT was overexpressed, oligosaccharides terminating with GlcNAc were significantly reduced compared with controls, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. As expected, GT overexpression resulted in reduction of oligosaccharides terminating with GlcNAc, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. The more highly sialylated glycoproteins had a significantly longer mean residence time in a rabbit model of pharmacokinetics. These experiments demonstrate the feasibility of genetically engineering cell lines to produce therapeutics with desired glycosylation patterns.

Animals↗
PubMed1985

A new approach to the diagnosis of active Rh0(D) immunization in passively immunized pregnant women.

An assay has been developed to distinguish active from passive Rh0(D) immunization in a patient who had recently received hyperimmune anti-Rh0(D) immunoglobulin therapy. Isolated peripheral B lymphocytes from a pregnant woman at 32 weeks gestation were co-cultured with Epstein-Barr virus in a liquid growth medium. After 7 days, anti-Rh0(D) antibodies produced in vitro by the transformed lymphocytes were detected in culture supernatants, thereby proving active immunization and indicating the potential of hemolytic disease of the newborn in the current pregnancy. This assay was also performed with peripheral B lymphocytes from three groups of individuals: mothers known to be Rh0(D) immunized and who recently delivered Rh-positive infants, women with longstanding Rh0(D) immunization, and women who were treated with anti-Rh0(D) globulin. In the first group, anti-Rh0(D) antibodies were again detected after in vitro viral stimulation. In the latter two groups, essentially no anti-Rh0(D) activity was detected.

Adult↗