Ty1 RNA levels are not increased in rtt mutants. Northern blot analysis is shown for total RNA from congenic strains whose relevant genotypes are indicated above each lane. The spt3Δ strain is a negative control. The Northern blot was probed with ³²P-riboprobes that hybridize to Ty1his3AI RNA (top), total Ty1 RNA (middle), and PYK1 RNA (bottom), the last as a loading control. Autoradiograms of the blot hybridized to each probe are shown. The ratio of ³²P activity in the Ty1his3AI band to ³²P activity in the PYK1 band and the ratio of ³²P activity in the Ty1 band to ³²P activity in the PYK1 band were determined by phosphorimaging. Ty1his3AI/PYK1 and Ty1/PYK1 RNA ratios for each strain, normalized to that for wild-type strain JC3787, are provided below each lane. WT, wild type.

A rad24Δ mutation suppresses Ty1 hypermobility in most rtt/genome integrity mutants. Ty1his3AI mobility is shown for RAD24 (light gray, dark gray, or black bars) and rad24Δ (white bars) derivatives of a wild-type strain and isogenic rtt derivatives. Light gray bars, RTT strains; dark gray bars, rtt mutants without defects in genome maintenance; black bars, rtt/genome integrity mutants. Error bars; standard error.

Deletion of RTT101 results in increased levels of Ty1 IN and RT proteins and RT activity in VLPs derived from a pGTy1 element but not a pGTy1^PR-d1 element. (A) Western blot analysis of VLP-enriched sucrose gradient fractions. (Top) 0.7 μg of each VLP-enriched fraction was probed with anti-VLP (α-VLP) antibody, which recognizes p49-Gag and p45-Gag. (Middle) 2.1 μg of each VLP-enriched fraction was probed with anti-RT antibody, which recognizes the Gag-Pol fusion protein, processing intermediates (not labeled), and RT. (Bottom) 2.1 μg of each VLP-enriched fraction was probed with anti-IN antibody, which recognizes Gag-Pol, processing intermediates (not labeled), and IN. (B) RT activity of VLP fractions from each strain on an exogenously added template. Units are 10^{5 32}P counts μg⁻¹ h⁻¹.

The S-phase checkpoint pathways are activated when replication is impeded. Components of each pathway that are relevant to this study are shown. The replication stress and the DNA damage pathways are activated by replication pausing and replication blocks, respectively. The DNA is bound independently by two sensor complexes, represented by the individual components Mec1 and Rad24. Binding of sensor complexes activates the Chk kinases Rad53 and Chk1. The mediator proteins Mrc1 and Rad9 stimulate the activities of the Chk kinases in the replication stress and DNA damage pathways, respectively. Rad53 activates the Dun1 kinase, which has multiple downstream targets, including the ribonucleotide reductase inhibitor Sml1. The pathways are drawn separately but function in an integrated fashion.

High Ty1 cDNA levels are suppressed in the absence of S-phase checkpoint proteins in many rtt/genome integrity mutants. (A) Schematic of a genomic Ty1 element with flanking genomic DNA, denoted by solid black bars. LTRs are indicated by tripartite boxes. A gray rectangle indicates the location of the Ty1 riboprobe used in Southern analysis. The approximate locations of the furthest 3′ SphI site in Ty1 and the next SphI site in genomic DNA 3′ of the Ty1 element are indicated, as is the end of the unintegrated Ty1 cDNA. (B) A representative Southern blot containing SphI-digested genomic DNA hybridized to a ³²P-labeled Ty1 riboprobe. Each lane contains genomic DNA from an independent colony grown to a 10-ml saturated culture at 20°C. The ³²P activity in the 1.7-kb Ty1 cDNA band (denoted with a C), which extends from the SphI site in Ty1 to the end of the unintegrated cDNA molecule, was quantified by phosphorimaging. Bands from two genomic Ty1 elements (denoted by G1 and G2) were quantified to determine the level of Ty1 cDNA relative to genomic DNA in each sample. The rtt mutation present in each set of strains is indicated below the blot. In addition, the genotypes at the RAD9 and RAD24 loci in each strain are indicated (+, RAD9 or RAD24; Δ, rad9Δ or rad24Δ). (C) Table showing the average Ty1 cDNA level in one to five independent DNA samples of rttΔ, rttΔ rad9Δ, rttΔ rad24Δ, and rttΔ rad53Δ sml1Δ strains relative to the average Ty1 cDNA level in two independent DNA samples of wild-type strain JC3787 run on each Southern blot. The average level of Ty1 cDNA in wild-type strain JC3787 on each Southern blot was assigned a value of 1. Data from the Southern blot shown, and eight additional Southern blots are compiled in the table. The control strains med1Δ and fus3Δ are rtt mutants with no known effect on genome integrity. Asterisks indicate values determined from one DNA sample.

Ty1 is activated through S-phase checkpoint pathways in rtt/genome integrity mutants. (A) Ty1his3AI mobility in RAD53 SML1 (light gray bars, RTT strains; dark gray bars, rtt mutants without defects in genome integrity; black bars, rtt/genome integrity mutants) and rad53Δ sml1Δ (white bars) derivatives of a wild-type strain and congenic rtt mutants. WT, wild type. (B) Ty1his3AI mobility in wild-type and isogenic rad9Δ strains grown in the presence of increasing concentrations of HU. The level of Ty1 cDNA relative to that of genomic Ty1 DNA in two independent DNA samples was determined as described in the legend to Fig. 2. (C) Ty1his3AI mobility in a wild-type strain and three mutant rtt strains carrying the pGAL1 vector or one of three pGAL1:RTT gene expression constructs. Ty1his3AI mobility, which is the average of the number of His⁺ Ura⁺ prototrophs divided by the number of total Ura⁺ cells per culture, was determined for each strain. Error bars, standard errors.