DNA polymerases required for repair of UV-induced damage in Saccharomyces cerevisiae

The ability of yeast DNA polymerase mutant strains to carry out repair synthesis after UV irradiation was studied by analysis of postirradiation molecular weight changes in cellular DNA. Neither DNA polymerase alpha, delta, epsilon, nor Rev3 single mutants evidenced a defect in repair. A mutant defective in all four of these DNA polymerases, however, showed accumulation of single-strand breaks, indicating defective repair. Pairwise combination of polymerase mutations revealed a repair defect only in DNA polymerase delta and epsilon double mutants. The extent of repair in the double mutant was no greater than that in the quadruple mutant, suggesting that DNA polymerases alpha and Rev3p play very minor, if any, roles. Taken together, the data suggest that DNA polymerases delta and epsilon are both potentially able to perform repair synthesis and that in the absence of one, the other can efficiently substitute. Thus, two of the DNA polymerases involved in DNA replication are also involved in DNA repair, adding to the accumulating evidence that the two processes are coupled

Dna2 Helicase/Nuclease Causes Replicative Fork Stalling and Double-strand Breaks in the Ribosomal DNA of Saccharomyces cerevisiae

We have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae and contributes to the shortened lifespan of dna2 mutants. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We show directly that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the early aging, hypomorphic dna2-2 helicase mutant. Deletion of FOB1, encoding the fork barrier protein, suppresses the elevated pausing and DSB formation, and represses initiation at rDNA ARSs. The dna2-2 mutation is synthetically lethal with {Delta}rrm3, encoding another DNA helicase involved in rDNA replication. It does not appear to be the case that the rDNA is the only determinant of genome stability during the yeast lifespan however since strains carrying deletion of all chromosomal rDNA but with all rDNA supplied on a plasmid, have decreased rather than increased lifespan. We conclude that the replication-associated defects that we can measure in the rDNA are symbolic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones

The Nuclease Activity of the Yeast Dna2 Protein, Which Is Related to the RecB-like Nucleases, Is Essential in Vivo

Saccharomyces cerevisiae Dna2 protein is required for DNA replication and repair and is associated with multiple biochemical activities: DNA-dependent ATPase, DNA helicase, and DNA nuclease. To investigate which of these activities is important for the cellular functions of Dna2, we have identified separation of function mutations that selectively inactivate the helicase or nuclease. We describe the effect of six such mutations on ATPase, helicase, and nuclease after purification of the mutant proteins from yeast or baculovirus-infected insect cells. A mutation in the Walker A box in the C-terminal third of the protein affects helicase and ATPase but not nuclease; a mutation in the N-terminal domain (amino acid 504) affects ATPase, helicase, and nuclease. Two mutations in the N-terminal domain abolish nuclease but do not reduce helicase activity (amino acids 657 and 675) and identify the putative nuclease active site. Two mutations immediately adjacent to the proposed nuclease active site (amino acids 640 and 693) impair nuclease activity in the absence of ATP but completely abolish nuclease activity in the presence of ATP. These results suggest that, although the Dna2 helicase and nuclease activities can be independently affected by some mutations, the two activities appear to interact, and the nuclease activity is regulated in a complex manner by ATP. Physiological analysis shows that both ATPase and nuclease are important for the essential function of DNA2 in DNA replication and for its role in double-strand break repair. Four of the nuclease mutants are not only loss of function mutations but also exhibit a dominant negative phenotype

Significance of the dissociation of Dna2 by flap endonuclease 1 to Okazaki fragment processing in Saccharomyces cerevisiae

Okazaki fragments are initiated by short RNA/DNA primers, which are displaced into flap intermediates for processing. Flap endonuclease 1 (FEN1) and Dna2 are responsible for flap cleavage. Replication protein A (RPA)-bound flaps inhibit cleavage by FEN1 but stimulate Dna2, requiring that Dna2 cleaves prior to FEN1. Upon cleavage, Dna2 leaves a short flap, which is then cut by FEN1 forming a nick for ligation. Both enzymes require a flap with a free 5'-end for tracking to the cleavage sites. Previously, we demonstrated that FEN1 disengages the tracking mechanism of Dna2 to remove it from the flap. To determine why the disengagement mechanism evolved, we measured FEN1 dissociation of Dna2 on short RNA and DNA flaps, which occur during flap processing. Dna2 tracked onto these flaps but could not cleave, presenting a block to FEN1 entry. However, FEN1 disengaged these nonproductively bound Dna2 molecules, proceeding on to conduct proper cleavage. These results clarify the importance of disengagement. Additional results showed that flap substrate recognition and tracking by FEN1, as occur during fragment processing, are required for effective displacement of the flap-bound Dna2. Dna2 was recently shown to dissociate flap-bound RPA, independent of cleavage. Using a nuclease-defective Dna2 mutant, we reconstituted the sequential dissociation reactions in the proposed RPA/Dna2/FEN1 pathway showing that, even without cutting, Dna2 enables FEN1 to cleave RPA-coated flaps. In summary, RPA, Dna2, and FEN1 have evolved highly coordinated binding properties enabling one protein to succeed the next for proper and efficient Okazaki flap processing

Operational and research aspects of a radio-controlled model flight test program

The operational and research aspects of a subscale, radio-controlled model flight test program are presented. By using low-cost free-flying models, an approach was developed for obtaining research-quality vehicle performance and aerodynamic information. The advantages and limitations learned by applying this approach to a specific flight test program are described. The research quality of the data acquired shows that model flight testing is practical for obtaining consistent and repeatable flight data

Promising new cherries

In the summer of 1882 the writer had a fine opportunity for studying the character of tree and fruit of the European cherries from the valley of the Moselle in East France, eastward to the Volga in Russia. In the spring of 1883 we imported one year old trees o f the varieties which we decided to be most promising for trial on the prairies of the Northwest. These trees were set in orchards and have had hard U 6 a g e , as they have been exposed to the recent test summers and winters, that have killed out the trees young and old of the grade o f hardiness of the Early Richmond and English Morello, and in addition they have been cut mercilessly for scions in autumn - and buds in summer. A better opportunity for determining the relative hardiness of trees and perfection of foliage has not been given in the history of prairie settlement. With this severe trial we are now pleased to report that many of the varieties have endured the tests as perfectly as our native plums such as DeSoto asmf Wolf, and have proven quite as hardy in fruit buds

Promising new pears

Prior to 1882 the writer—in common with all experienced orchardists—believed that pear growing would never prove profitable on the great prairies west of Lake Michigan. The varieties from southwest Europe, and their American grown seedlings, had failed with us as completely as had the strawberries, raspberries, and grapes from that source, and our available periodicals and books made no mention of successful pear growing on the great steppes of east Europe and central Asia. Hence we were utterly surprised to find healthy pear orchards, loaded with fine pears, in the parts of south central Russia where our native Black Locust winter kills as the common peach does with us, and we were still more surprised to find the pear used as a street tree at points on the Upper Volga where the Duchess apple will not endure the winters, and where with scanty snow falls the thermometer often goes down to fifty or more degrees below zero

Stocks for the cherry, plum, prune and apricot

Mahaleb Stock.— Investigation will determine the fact that the use of the Mahaleb as a stock on which to bud the cultivated varieties of the cherry is becoming general in the nurseries o f the Eastern, Middle and Western States. Millions of the plants are annually imported and grown in the Eastern States,and a visit to any one of our larger nurseries in August will show busy gangs inserting the buds of about every variety catalogued, under the bark of the cheap and easily worked Prunus mahaleb. Yet at every horticultural meeting, where the subject comes up for disscussion, the most diverse views are expressed by practical fruit growers as to the value o f the stock for all varieties and all soils. Turning to any one of the standard works on propagation of the Eastern or Western Continent, we discover some o f the reasons for the well known opposing views of orchardists. As to its adaptation to varied soils Loudon says: “The most effectual dwarfing stock for the cherry is the Mahaleb, which however will not succeed on the generality of soils in England. Dubreuil recommends the wild red cherries for clayey and light soils, and the Mahaleb for soils of a light, sandy or chalky nature.

The college vineyard

The two vacant pages of the Bulletin give limited space for notes on our twelve years experience with the one acre on the college grounds known as the vineyard. Previous to my taking charge of the Department, this plot had been twice planted with vines which were lost almost wholly by root killing in winter. Believing the loss to result from too shallow planting the rows, were reset with strong, one year old plants in the bottom of holes eighteen inches deep, filling the holes gradually as the canes made growth. With this deep planting no vines have been lost of hardy varieties in twelve years, except a few on a gravelly point where we shall re-set at a still greater depth