Jack W. Szostak

The double-strand-break repair model for recombination

Gene conversion is the nonreciprocal transfer of information from one DNA duplex to another; in meiosis, it is frequently associated with crossing-over. We review the genetic properties of meiotic recombination and previous models of conversion and crossing-over. In these models, recombination is initiated by single-strand nicks, and heteroduplex DNA is generated. Gene conversion is explained by the repair of mismatches present in heteroduplex DNA. We propose a new mechanism for meiotic recombination, in which events are initiated by double-strand breaks that are enlarged to double-strand gaps. Gene conversion can then occur by the repair of a double-strand gap, and postmeiotic segregation can result from heteroduplex DNA formed at the boundaries of the gap-repair region. The repair of double-strand gaps is an efficient process in yeast, and is known to be associated with crossing-over. The genetic implications of the double-strand-break repair model are explored.

Thermostability of model protocell membranes

The earliest cells may have consisted of a self-replicating genetic polymer encapsulated within a self-replicating membrane vesicle. Here, we show that vesicles composed of simple single-chain amphiphiles such as fatty acids, fatty alcohols, and fatty-acid glycerol esters are extremely thermostable and retain internal RNA and DNA oligonucleotides at temperatures ranging from 0°C to 100°C. The strands of encapsulated double-stranded DNA can be separated by denaturation at high temperature while being retained within vesicles, implying that strand separation in primitive protocells could have been mediated by thermal fluctuations without the loss of genetic material from the protocell. At elevated temperatures, complex charged molecules such as nucleotides cross fatty-acid-based membranes very rapidly, suggesting that high temperature excursions may have facilitated nutrient uptake before the evolution of advanced membrane transporters. The thermostability of these membranes is consistent with the spontaneous replication of encapsulated nucleic acids by the alternation of template-copying chemistry at low temperature with strand-separation and nutrient uptake at high temperature.

A mutant with a defect in telomere elongation leads to senescence in yeast

We describe a general assay designed to detect mutants of yeast that are defective for any of several aspects of telomere function. Using this assay, we have isolated a mutant that displays a progressive decrease in telomere length as well as an increased frequency of chromosome loss. This mutation defines a new gene, designated EST1 (for ever shorter telomeres). Null alleles of EST1 are not immediately inviable; instead, they have a senescence phenotype, due to the gradual loss of sequences essential for telomere function, leading to a progressive decrease in chromosomal stability and subsequent cell death.

Telomeres and telomerase: the path from maize, Tetrahymena and yeast to human cancer and aging

Telomeres and telomerase: the path from maize, Tetrahymena and yeast to human cancer and aging

Template-directed Synthesis of a Genetic Polymer in a Model Protocell

Contemporary phospholipid-based cell membranes are formidable barriers to the uptake of polar and charged molecules ranging from metal ions to complex nutrients. Modern cells therefore require sophisticated protein channels and pumps to mediate the exchange of molecules with their environment. The strong barrier function of membranes has made it difficult to understand the origin of cellular life and has been thought to preclude a heterotrophic lifestyle for primitive cells. Although nucleotides can cross dimyristoyl phosphatidylcholine membranes through defects formed at the gel-to-liquid transition temperature, phospholipid membranes lack the dynamic properties required for membrane growth. Fatty acids and their corresponding alcohols and glycerol monoesters are attractive candidates for the components of protocell membranes because they are simple amphiphiles that form bilayer membrane vesicles that retain encapsulated oligonucleotides and are capable of growth and division. Here we show that such membranes allow the passage of charged molecules such as nucleotides, so that activated nucleotides added to the outside of a model protocell spontaneously cross the membrane and take part in efficient template copying in the protocell interior. The permeability properties of prebiotically plausible membranes suggest that primitive protocells could have acquired complex nutrients from their environment in the absence of any macromolecular transport machinery; that is, they could have been obligate heterotrophs.

Functional proteins from a random-sequence library

Functional primordial proteins presumably originated from random sequences, but it is not known how frequently functional, or even folded, proteins occur in collections of random sequences. Here we have used in vitro selection of messenger RNA displayed proteins, in which each protein is covalently linked through its carboxy terminus to the 3′ end of its encoding mRNA, to sample a large number of distinct random sequences. Starting from a library of 6 × 1012 proteins each containing 80 contiguous random amino acids, we selected functional proteins by enriching for those that bind to ATP. This selection yielded four new ATP-binding proteins that appear to be unrelated to each other or to anything found in the current databases of biological proteins. The frequency of occurrence of functional proteins in random-sequence libraries appears to be similar to that observed for equivalent RNA libraries.

Cloning yeast telomeres on linear plasmid vectors

We have constructed a linear yeast plasmid by joining fragments from the termini of Tetrahymena ribosomal DNA to a yeast vector. Structural features of the terminus region of the Tetrahymena rDNA plasmid maintained in the yeast linear plasmid include a set of specifically placed single-strand interruptions within the cluster of hexanucleotide (C4A2) repeat units. An artificially constructed hairpin terminus was unable to stabilize a linear plasmid in yeast. The fact that yeast can recognize and use DNA ends from the distantly related organism Tetrahymena suggests that the structural features required for telomere replication and resolution have been highly conserved in evolution. The linear plasmid was used as a vector to clone chromosomal telomeres from yeast. One Tetrahymena end was removed by restriction digestion, and yeast fragments that could function as an end on a linear plasmid were selected. Restriction mapping and hybridization analysis demonstrated that these fragments were yeast telomeres, and suggested that all yeast chromosomes might have a common telomere sequence. Yeast telomeres appear to be similar in structure to the rDNA of Tetrahymena, in which specific nicks or gaps are present within a simple repeated sequence near the terminus of the DNA.

[14] Genetic applications of yeast transformation with linear and gapped plasmids

Techniques for high frequency yeast transformation have been described. A double-strand break introduced by restriction enzyme cleavage can be used to direct a plasmid to integrate into a particular chromosomal locus. Plasmids containing a double-strand gap can be used in a straightforward method for the isolation and mapping of chromosomal alleles. These techniques extend the genetic applications of yeast transformation.

The use of mRNA display to select high-affinity protein-binding peptides

We report the use of “mRNA display,” an in vitro selection technique, to identify peptide aptamers to a protein target. mRNA display allows for the preparation of polypeptide libraries with far greater complexity than is possible with phage display. Starting with a library of ≈1013 random peptides, 20 different aptamers to streptavidin were obtained, with dissociation constants as low as 5 nM. These aptamers function without the aid of disulfide bridges or engineered scaffolds, yet possess affinities comparable to those for monoclonal antibody–antigen complexes. The aptamers bind streptavidin with three to four orders of magnitude higher affinity than those isolated previously by phage display from lower complexity libraries of shorter random peptides. Like previously isolated peptides, they contain an HPQ consensus motif. This study shows that, given sufficient length and diversity, high-affinity aptamers can be obtained even from random nonconstrained peptide libraries. By engineering structural constraints into these ultrahigh complexity peptide libraries, it may be possible to produce binding agents with subnanomolar binding constants.

HIV-1 rev regulation involves recognition of non-Watson-Crick base pairs in viral RNA

We have used an iterative in vitro genetic selection to identify the important structural features of the viral RNA element bound by the Rev protein of human immunodeficiency virus type 1 (HIV-1). Functional Rev-binding RNAs were selected from a pool of 10(13) variants of the wild-type Rev-binding domain. Bases conserved among the binding species define a 20 nucleotide core binding element. Covariation of some of these conserved bases indicates that the Rev-binding element is a stem-bulge-stem with a G:G base pair in the bulge. Mutational studies show that this non-Watson-Crick base pair is required for Rev binding in vitro and Rev responsiveness in vivo. We propose that the G:G base pair distorts the sugar-phosphate backbone of viral RNA and that this distortion is a critical determinant of recognition by Rev.