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Biomedical subjects

B J Peter

Publications and source records attributed to B J Peter.

9 recordsLinked to original sources
PubMed2001

Topological challenges to DNA replication: conformations at the fork.

The unwinding of the parental DNA duplex during replication causes a positive linking number difference, or superhelical strain, to build up around the elongating replication fork. The branching at the fork and this strain bring about different conformations from that of (-) supercoiled DNA that is not being replicated. The replicating DNA can form (+) precatenanes, in which the daughter DNAs are intertwined, and (+) supercoils. Topoisomerases have the essential role of relieving the superhelical strain by removing these structures. Stalled replication forks of molecules with a (+) superhelical strain have the additional option of regressing, forming a four-way junction at the replication fork. This four-way junction can be acted on by recombination enzymes to restart replication. Replication and chromosome folding are made easier by topological domain barriers, which sequester the substrates for topoisomerases into defined and concentrated regions. Domain barriers also allow replicated DNA to be (-) supercoiled. We discuss the importance of replicating DNA conformations and the roles of topoisomerases, focusing on recent work from our laboratory.

DNA Replication↗
PubMed2000

Nitrogen regulatory protein C-controlled genes of Escherichia coli: scavenging as a defense against nitrogen limitation.

Nitrogen regulatory protein C (NtrC) of enteric bacteria activates transcription of genes/operons whose products minimize the slowing of growth under nitrogen-limiting conditions. To reveal the NtrC regulon of Escherichia coli we compared mRNA levels in a mutant strain that overexpresses NtrC-activated genes [glnL(Up)] to those in a strain with an ntrC (glnG) null allele by using DNA microarrays. Both strains could be grown under conditions of nitrogen excess. Thus, we could avoid differences in gene expression caused by slow growth or nitrogen limitation per se. Rearranging the spot images from microarrays in genome order allowed us to detect all of the operons known to be under NtrC control and facilitated detection of a number of new ones. Many of these operons encode transport systems for nitrogen-containing compounds, including compounds recycled during cell-wall synthesis, and hence scavenging appears to be a primary response to nitrogen limitation. In all, approximately 2% of the E. coli genome appears to be under NtrC control, although transcription of some operons depends on the nitrogen assimilation control protein, which serves as an adapter between NtrC and final sigma(70)-dependent promoters.

Artificial Gene Fusion↗
PubMed2000

DNA microarray analysis of gene expression in response to physiological and genetic changes that affect tryptophan metabolism in Escherichia coli.

We investigated the global changes in mRNA abundance in Escherichia coli elicited by various perturbations of tryptophan metabolism. To do so we printed DNA microarrays containing 95% of all annotated E. coli ORFs. We determined the expression profile that is predominantly dictated by the activity of the tryptophan repressor. Only three operons, trp, mtr, and aroH, exhibited appreciable expression changes consistent with this profile. The quantitative changes we observed in mRNA levels for the five genes of the trp operon were consistent within a factor of 2, with expectations based on established Trp protein levels. Several operons known to be regulated by the TyrR protein, aroF-tyrA, aroL, aroP, and aroG, were down-regulated on addition of tryptophan. TyrR can be activated by any one of the three aromatic amino acids. Only one operon, tnaAB, was significantly activated by the presence of tryptophan in the medium. We uncovered a plethora of likely indirect effects of changes in tryptophan metabolism on intracellular mRNA pools, most prominent of which was the sensitivity of arginine biosynthetic operons to tryptophan starvation.

Escherichia coli↗
PubMed2000

Analysis of topoisomerase function in bacterial replication fork movement: use of DNA microarrays.

We used DNA microarrays of the Escherichia coli genome to trace the progression of chromosomal replication forks in synchronized cells. We found that both DNA gyrase and topoisomerase IV (topo IV) promote replication fork progression. When both enzymes were inhibited, the replication fork stopped rapidly. The elongation rate with topo IV alone was 1/3 of normal. Genetic data confirmed and extended these results. Inactivation of gyrase alone caused a slow stop of replication. Topo IV activity was sufficient to prevent accumulation of (+) supercoils in plasmid DNA in vivo, suggesting that topo IV can promote replication by removing (+) supercoils in front of the chromosomal fork.

DNA Replication↗
PubMed1999

The topological mechanism of phage lambda integrase.

Bacteriophage lambda integrase (Int) is a versatile site-specific recombinase. In concert with other proteins, it mediates phage integration into and excision out of the bacterial chromosome. Int recombines intramolecular sites in inverse or direct orientation or sites on separate DNA molecules. This wide spectrum of Int-mediated reactions has, however, hindered our understanding of the topology of Int recombination. By systematically analyzing the topology of Int reaction products and using a mathematical method called tangles, we deduce a unified model for Int recombination. We find that, even in the absence of (-) supercoiling, all Int reactions are chiral, producing one of two possible enantiomers of each product. We propose that this chirality reflects a right-handed DNA crossing within or between recombination sites in the synaptic complex that favors formation of right-handed Holliday junction intermediates. We demonstrate that the change in linking number associated with excisive inversion with relaxed DNA is equally +2 and -2, reflecting two different substrates with different topology but the same chirality. Additionally, we deduce that integrative Int recombination differs from excisive recombination only by additional plectonemic (-) DNA crossings in the synaptic complex: two with supercoiled substrates and one with relaxed substrates. The generality of our results is indicated by our finding that two other members of the integrase superfamily of recombinases, Flp of yeast and Cre of phage P1, show the same intrinsic chirality as lambda Int.

Bacteriophage P1↗
PubMed1999

Knot what we thought before: the twisted story of replication.

DNA replication requires the unwinding of the parental duplex, which generates (+) supercoiling ahead of the replication fork. It has been thought that removal of these (+) supercoils was the only method of unlinking the parental strands. Recent evidence implies that supercoils can diffuse across the replication fork, resulting in interwound replicated strands called precatenanes. Topoisomerases can then act both in front of and behind the replication fork. A new study by Sogo et al. [J Mol Biol 1999;286:637-643 (Ref. 1)], using a topological analysis, provides the best evidence that precatenanes exist in negatively supercoiled, partially replicated molecules in vivo.

DNA Replication↗
PubMed1999

Mechanisms of separation of the complementary strands of DNA during replication.

This article is a perspective on the separation of the complementary strands of DNA during replication. Given the challenges of DNA strand separation and its vital importance, it is not surprising that cells have developed many strategies for promoting unlinking. We summarize seven different factors that contribute to strand separation and chromosome segregation. These are: (1) supercoiling promotes unlinking by condensation of DNA; (2) unlinking takes place throughout a replicating domain by the complementary action of topoisomerases on precatenanes and supercoils; (3) topological domains isolate the events near the replication fork and permit the supercoiling-dependent condensation of partially replicated DNA; (4) type-II topoisomerases use ATP to actively unlink DNA past the equilibrium position; (5) the effective DNA concentration in vivo is less than the global DNA concentration; (6) mechanical forces help unlink chromosomes; and (7) site-specific recombination promotes unlinking at the termination of replication by resolving circular dimeric chromosomes.

Chromosome Segregation↗
PubMed1998

The structure of supercoiled intermediates in DNA replication.

We studied the structure of replication intermediates accumulated by Tus-induced arrest of plasmid DNA replication at termination sites. For intermediates generated both in vitro with purified components and in vivo, superhelical stress is distributed throughout the entire partially replicated molecule; daughter DNA segments are wound around each other, and the unreplicated region is supercoiled. Thus, unlinking of parental DNA strands by topoisomerases can be carried out both behind and in front of the replication fork. We explain why previous studies with prokaryotic and eukaryotic replication intermediates discerned only supercoiling in the unreplicated portion.

DNA Replication↗