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Biomedical subjects

K A Heichman

Publications and source records attributed to K A Heichman.

6 recordsLinked to original sources
PubMed1996

The yeast CDC16 and CDC27 genes restrict DNA replication to once per cell cycle.

CDC16 and CDC27 were identified as genes in S. cerevisiae necessary to limit DNA replication to once per cell cycle. A screen for mutants that overreplicated their DNA uncovered new conditional alleles that cause accumulation of up to 8C DNA. DNA overreplication involves all chromosomes and does not require passage through mitosis or another START. It occurs within a single cell cycle and can cause arrest at the MEC1 checkpoint. Remarkably, Clb2-Cdc28 activity remains elevated in the overreplicating cells. These observations distinguish CDC16 and CDC27 from other mutants that accumulate extra DNA after completing an aberrent mitosis, or skipping mitosis altogether, and entering a second, inappropriate G1 and S phase. CDC16 and CDC27 may contribute to replication control by targeted proteolysis of an S phase initiator.

Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome↗
PubMed1991

Configuration of DNA strands and mechanism of strand exchange in the Hin invertasome as revealed by analysis of recombinant knots.

The Hin recombinase of Salmonella normally catalyzes a site-specific DNA inversion reaction that is very efficient when the Fis protein and a recombinational enhancer sequence are present. The mechanism of this recombination reaction has been investigated by analyzing the formation and structure of knots generated in different plasmid substrates in vitro. Hin seldom knots the wild-type substrate under standard recombination conditions. However, we show that increasing the length of DNA between the recombination sites and the enhancer and changing the sequence of the core nucleotides where strand exchange occurs increases the efficiency of the knotting reaction. The structure of the knots generated by different mutant substrates strongly supports a model involving a unique configuration of DNA strands at synapsis and DNA strand exchange mediated by rotation of one set of Hin subunits after DNA cleavage. Analysis of the stereostructure of the knots by electron microscopy of RecA-coated DNA molecules demonstrates that the direction of subunit rotation is exclusively clockwise. Because multiple subunit rotations generating knotted molecules do not occur efficiently when the enhancer is located in its native position, we suggest that the enhancer normally remains associated with the two recombination sites in the invertasome structure during strand exchange to limit strand rotation once it has been initiated. Under certain conditions, however, complex knots are formed that are probably the result of the premature release of the enhancer and multiple, unrestrained subunit exchanges.

Base Sequence↗
PubMed1991

Alignment of recombination sites in Hin-mediated site-specific DNA recombination.

The Hin site-specific recombination system normally promotes inversion of DNA between two recombination sites in inverted orientation. We show that the rate of deletion of DNA between two directly repeated recombination sites is 10-300 times slower than inversion between sites in their native configuration as measured in vivo and in vitro, respectively. In vitro studies have shown that the deletion reaction has the same requirement for Fis, a recombinational enhancer, and DNA supercoiling as the inversion reaction. These requirements, together with the finding that the deletion products are interlinked once suggest that the deletion synaptic complex is similar to the invertasome intermediate that generates inversion. The inefficiency of the deletion reaction is not a function of a reduced ability to recognize or synapse recombination sites in direct orientation. Not only do these substrates support an efficient knotting reaction, but directly repeated recombination sites with symmetric core sequences also invert efficiently. These findings demonstrate that the recombination sites are preferentially assembled into the invertasome structure with the sites aligned in the configuration for inversion regardless of their starting orientation. We propose that the dynamics of a supercoiled DNA molecule biases the geometric assembly of specific intermediates. In the case of Hin-mediated recombination, inversion is overwhelmingly preferred over deletion because DNA supercoiling favors a specific alignment of DNA strands in the synaptic complex.

Bacterial Proteins↗
PubMed1990

The Hin invertasome: protein-mediated joining of distant recombination sites at the enhancer.

The Hin protein binds to two cis-acting recombination sites and catalyzes a site-specific DNA inversion reaction that regulates the expression of flagellin genes in Salmonella. In addition to the Hin protein and the two recombination sites that flank the invertible segment, a third cis-acting recombinational enhancer sequence and the Fis protein, which binds to two sites within the enhancer, are required for efficient recombination. Intermediates of this reaction were trapped during DNA strand cleavage and analyzed by gel electrophoresis and electron microscopy in order to determine their structure and composition. The analyses demonstrate that the recombination sites are assembled at the enhancer into a complex nucleo-protein structure (termed the invertasome) with the looping of the three segments of intervening DNA. Antibody studies indicated that Fis physically interacts with Hin and that both proteins are intimately associated with the invertasome. In order to achieve this protein-protein interaction and assemble the invertasome, the substrate DNA must be supercoiled.

Bacterial Proteins↗
PubMed1988

Expression of the yeast UB14 gene increases in response to DNA-damaging agents and in meiosis.

The polyubiquitin gene, UB14, of Saccharomyces cerevisiae is regulated by a variety of environmental stresses and physiological conditions. After exposure of rapidly growing yeast cells to DNA-damaging agents (4-nitroquinoline-1-oxide and N-methyl-N'-nitro-N-nitrosoguanidine), intracellular levels of UB14 transcript increased rapidly. Induction of UB14 transcripts occurred within 30 to 60 min of exposure to 4-nitroquinoline-1-oxide in RAD+, rad52, and rad6 repair-deficient yeast strains. In high-density RAD+ cultures, the effect of alkylating agents on UB14 transcript levels is attenuated, in part because of significant increases in the basal level of this message in untreated cells. We also observed that the levels of UB14 transcripts increased significantly when diploid cells were exposed to sporulation conditions. Maximal levels of UB14 transcripts were reached after 6 to 8 h in sporulation medium. Accumulation of UB14 transcripts occurred in a/alpha diploids that undergo meiosis but not in asporogenous alpha/alpha diploids exposed to the same nutritional conditions.

4-Nitroquinoline-1-oxide↗