©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Conditional Lethality of Null Mutations in RTH1 That Encodes the Yeast Counterpart of a Mammalian 5`- to 3`-Exonuclease Required for Lagging Strand DNA Synthesis in Reconstituted Systems (*)

(Received for publication, December 14, 1994 )

Christopher H. Sommers (1)(§) Edward J. Miller (2)(¶) Bernard Dujon (3) Satya Prakash (1)(¶) Louise Prakash (2)(¶)(**)

From the  (1)Department of Biology and (2)Department of Biophysics, University of Rochester, Rochester, New York 14642 and (3)Unité de Génétique Moléculaire des Levures (URA 1149 du CNRS), Institut Pasteur, 25 rue du Dr. Roux, F-75724 Paris Cedex 15, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A 5`- to 3`-exonuclease of about 45 kDa has been purified from various mammalian sources and shown to be required for the completion of lagging strand synthesis in reconstituted DNA replication systems. RTH1 encodes the yeast Saccharomyces cerevisiae counterpart of the mammalian enzyme. To determine the in vivo biological role of RTH1-encoded 5`- to 3`-exonuclease, we have examined the effects of an rth1Delta mutation on various cellular processes. rth1Delta mutants grow poorly at 30 °C, and a cessation in growth occurs upon transfer of the mutant to 37 °C. At the restrictive temperature, the rth1Delta mutant exhibits a terminal cell cycle morphology similar to that of mutants defective in DNA replication, and levels of spontaneous mitotic recombination are elevated in the rth1Delta mutant even at the permissive temperature. The rth1Delta mutation does not affect UV or -ray sensitivity but enhances sensitivity to the alkylating agent methyl methanesulfonate. The role of RTH1 in DNA replication and in repair of alkylation damage is discussed.


INTRODUCTION

In Escherichia coli, the 5`- to 3`-exonuclease activity of DNA polymerase I is involved in the removal of RNA primers attached to the 5`-end of newly replicated DNA. The E. coli polA ex1 mutant is defective in 5`- to 3`-exonuclease activity and is temperature-sensitive for growth. The joining of Okazaki fragments is retarded in the polA ex1 mutant, and as a consequence, it exhibits elevated levels of genetic recombination (1, 2) .

In eukaryotes, a 5`- to 3`-exonuclease with a molecular size of 45 kDa has been purified from HeLa cells, mouse, and calf thymus(3, 4, 5) . Ishimi et al.(4) reconstituted the replication of simian virus 40 origin containing DNA using SV40 large T antigen and other purified components (single-stranded DNA binding protein RPA, DNA polymerase alpha-primase complex, topoisomerase II, ribonuclease H, 5`- to 3`-exonuclease, and DNA ligase) isolated from HeLa cells. The 5`- to 3`-exonuclease was essential for the production of replicating form I DNA. A 5`- to 3`-exonuclease from mouse cells has also been shown to be required in a polalpha-primase-dependent replication that used single-stranded circular DNA as a template(5) . Both of these studies indicated that the removal of RNA primers required the combined action of RNase H and 5`- to 3`-exonuclease. In another study(6) , the 5`- to 3`-exonuclease has been shown to functionally interact with DNA polymerase alpha, , or in the completion of lagging strand DNA synthesis. Using purified proteins from calf thymus, Turchi et al.(7) have shown that RNase H1 cleaves the primer RNA one nucleotide 5` of the RNA-DNA junction, and the remaining monoribonucleotide is removed by the 5`- to 3`-exonuclease activity.

The mouse and human 5`- to 3`-exonuclease genes have been cloned, and they encode highly homologous proteins of 378 and 380 amino acids, respectively(8, 9) . Both proteins share a high degree of homology with the Saccharomyces cerevisiae protein of 382 amino acids encoded by the YKL510 open reading frame(8, 9) , and the S. cerevisiae protein also has a 5`- to 3`-exonuclease activity(8) . All these exonucleases share homology with the S. cerevisiae and human nucleotide excision repair proteins RAD2 and XPG, respectively. Both the RAD2 and XPG proteins contain DNA endonuclease and 5`- to 3`-exonuclease activities(10, 11, 12) . RAD2 and XPG, however, are much larger proteins containing 1031 and 1186 residues, respectively, and the homology between RAD2/XPG and the above noted mammalian and yeast 5`- to 3`-exonucleases occurs in three regions(8, 13, 14) . Because of the homology of the S. cerevisiae YKL510 open reading frame encoded protein with RAD2, we have named the gene contained within the YKL510 DNA fragment RTH1 (RAD two homolog).

The strict dependence of reconstituted mammalian DNA replication systems on the 5`- to 3`-exonuclease suggested that RTH1 may be an essential gene. To define the biological role of RTH1, we have examined the effects of a null mutation in RTH1 on viability, cell cycle morphology, and spontaneous mitotic recombination. We find that the rth1Delta mutation is conditionally lethal, and at the restrictive temperature, mutant cells exhibit a cell cycle morphology characteristic of mutants defective in DNA replication.


MATERIALS AND METHODS

Construction of a Null Mutation of the RTH1 Gene

The plasmid pR2.10, a derivative of pUC19, was used to make a genomic deletion of the RTH1 gene. pR2.10 contains the RTH1 gene in which nucleotides +58 to +755 of the RTH1 1146 nucleotide open reading frame (15) have been replaced with the yeast URA3 gene flanked by Salmonella typhimurium HisG sequences(16) . An rth1Delta mutation was made by the gene replacement technique (17) by cutting pR2.10 with EcoRI and SalI and transforming yeast cells to Ura. Genomic deletion mutations of RTH1 and various RAD genes were constructed in S. cerevisiae strains EMY6 (MATalpha ade5 his7 leu2 lys1 met14 trp1Delta ura3) and LP3041-6D (MATaleu2-3 leu2-112 trp1Delta ura3-52).

Sensitivity to UV and Irradiation

Quantification of survival after UV irradiation of yeast strains was carried out as described(18) . Cultures were grown from single colonies to stationary phase in the appropriate selection media. Cells were sonicated for 1-3 min in a Branson water bath sonicator, diluted, and plated on the appropriate medium. Plates were exposed to UV irradiation, incubated at 25, 30, or 34 °C for 3-5 days in the dark to avoid photoreactivation, and the colonies were counted.

For -ray irradiation, RTH1 and rth1Delta strains were suspended in U-wells and transferred to yeast extract-peptone-dextrose (YPD) (^1)plates. Following irradiation with a cobalt-60 source at a dose rate of 9 kilorads/min, plates were incubated at either 25, 30, or 34 °C for 3-4 days and examined at regular intervals.

Sensitivity to the DNA Alkylating Agent Methyl Methanesulfonate (MMS)

RTH1 and rth1Delta strains were transferred to YPD plates containing either no MMS or MMS concentrations ranging from 0.02 to 0.035%. Plates were incubated at 25, 30, or 34 °C for 4-5 days and examined.

Rates of Mitotic Recombination

The his3Delta3`, his3Delta5` duplication was constructed in wild type strains LP3041-6D, EMY6, and their rth1Delta derivatives by introducing the plasmid pRS6, which contains an internal fragment of the HIS3 gene, the LEU2 gene, and pBR322 sequences. Integration of pRS6 at the genomic HIS3 site results in two copies of the his3 gene, one with a terminal deletion at the 3`-end and the other with a terminal deletion at the 5`-end. The two his3 alleles are separated by LEU2 and pBR322 sequences(19) .

Three 10-ml cultures were grown to a density of 10^7 cells/ml at either 25, 30, or 34 °C in synthetic liquid medium lacking leucine. As nearly all of the HIS3 recombinants show a simultaneous loss of the LEU2 gene and pBR322 sequences, HIS3 recombinants do not divide in medium lacking leucine. Therefore, the frequency of HIS3 recombinants measures the actual recombination rate. Log phase cultures were washed, resuspended in 1 ml of sterile water, diluted, and plated onto synthetic complete media to determine viability and onto synthetic complete media lacking histidine to determine the frequency of HIS3 recombinants. Plates were incubated for 3-4 days at 25, 30, or 34 °C, and the frequency of HIS3 recombinants was determined. Recombination rates were calculated by the method of median of Lea and Coulson (as described previously in (19) ).

Growth Rates and Cell Morphology

Doubling times were determined from growth curves obtained for the various yeast strains at 30 °C in liquid YPD medium. Cultures were diluted to A of about 0.05, and the densities followed to A of 4-5. For growth at 37 °C, cultures were divided with half of the culture left at 30 °C and the other half shifted to 37 °C and A determined at various times.

For examining terminal morphology at 37 °C, following ethanol fixation, cells were rehydrated in 40 mM KPO(4), pH 6.5 buffer containing 0.5 M MgCl(2) and 1.2 M sorbitol, and mounted on slides coated with 0.1% polylysine (M(r) 400,000). For visualization of nuclear morphology, a drop of mounting medium containing 4`,6`-diamidino-2-phenylindole was applied to the slide, and cells were photographed with a Leitz Laborlux D fluorescence microscope equipped with an Olympus PM-10AD photomicrographic system.


RESULTS

Sensitivity of the rth1Delta Mutation to DNA Damaging Agents

To test if RTH1 has a role in DNA repair, we examined the sensitivity of the rth1Delta mutant to ultraviolet irradiation, -irradiation, and the alkylating agent MMS. The rth1Delta mutation was constructed in two different yeast strains and their sensitivity to DNA damaging agents examined at three different temperatures, 25, 30, and 34 °C. As shown in Fig. 1, A and B, the rth1Delta mutation has no effect on UV sensitivity. The UV sensitivity of the rth1Delta mutation, in combination with the rad2Delta mutation defective in nucleotide excision repair, is the same as that of the rad2Delta single mutation, suggesting that RTH1 has no significant role in the RAD6 postreplicative bypass or the RAD52 recombinational repair pathways, which, in addition to nucleotide excision repair, constitute the three pathways for the repair of UV damage in yeast(20) . We also found no effect of the rth1Delta mutation on -ray sensitivity (data not shown). Sensitivity to MMS was, however, enhanced by the rth1Delta mutation (Fig. 1C).


Figure 1: UV survival of rth1Delta, rad2Delta, and rad2Delta rth1Delta strains. circle, wild type strain; bullet, rth1Delta; box, rad2Delta; , rad2Delta rth1Delta. A, wild type strain LP3041-6D and its rth1Delta, rad2Delta, and rth1Delta rad2Delta derivative mutant strains were grown at 30 °C, and cells were plated and UV-irradiated, followed by incubation of plates at 30 °C in the dark. Similar results were obtained at 25 or 34 °C. B, wild type strain EMY6 and its mutant derivatives were grown at 34 °C and following UV irradiation plates were incubated at 34 °C in the dark. Similar results were observed at 25 or 30 °C. C, MMS sensitivity of rth1Delta mutant. The wild type (WT) strain EMY6 and its rth1Delta derivative were grown on YPD + 0.03% MMS (+ MMS) or on YPD medium lacking MMS (-MMS) at 30 °C. Similar results were observed for LP3041-6D and its rth1Delta derivative.



Conditional Lethality of the rth1Delta Mutation

When grown at 30 °C, the rth1Delta mutation affects growth rate considerably, increasing the cell doubling time to twice to that in the wild type strain (Table 1). We also determined the growth rate of the rth1Delta mutation in combination with mutations in genes representing the three epistasis groups for the repair of UV damage in S. cerevisiae (Table 1). The growth rates of the rth1Delta rad2Delta and rth1Delta rad6Delta double mutants were about the same as that of the rth1Delta single mutant; the doubling time of the rth1Delta rad52Delta mutant was, however, about twice that of the rth1Delta or the rad52Delta single mutant, indicating that the RAD52 recombinational pathway plays an important role in repairing the DNA lesions that accumulate in the rth1Delta mutant.



The rth1Delta mutant stops growth upon transfer to 37 °C (Fig. 2A). Microscopic examination revealed that rth1Delta mutant cells stop division as two large cells consisting of the mother and the daughter cell, with a block in nuclear division. In some cases, the nucleus has migrated to the neck between the two cells, whereas in other cases, cells exhibit an elongated nucleus stretching between the mother and daughter cell (Fig. 2B). The cell cycle arrest phenotype of the rth1Delta mutant resembles that of the various S. cerevisiae mutants known to be defective in DNA replication. For example, cdc2 mutants with a defect in DNA polymerase stop at the restrictive temperature with the nucleus that has migrated to the neck of the mother cell but has not elongated, whereas the DNA ligase defective mutant cdc9 stops cell division with an elongated nucleus extending between the two cells(21) .


Figure 2: Lethality of rth1Delta mutant at 37 °C. A, rth1Delta mutation inhibits growth at 37 °C. Log phase cultures of LP3041-6D and its rth1Delta derivative were diluted to approximately A = 0.05, and the cultures were then split. One half of the culture was incubated at 30 °C and the other half at 37 °C. circle, RTH1 (30 °C); bullet, RTH1 (37 °C); box, rth1Delta (30 °C); , rth1Delta (37 °C). B, cell cycle morphology of rth1Delta strain. The terminal morphology of rth1Delta cells was examined by 4`,6`-diamidino-2-phenylindole staining.



Elevated Rates of Spontaneous Mitotic Recombination in the rth1Delta Mutant

In E. coli, mutations in the 5`- to 3`-exonuclease activity of DNA polymerase I and in DNA ligase confer a hyper recombination phenotype because of the retarded joining of Okazaki fragments(1, 2, 22) . In S. cerevisiae, mutations in DNA replication genes encoding DNA ligase(23) , DNA polymerases, and other enzymes (24) also result in elevated rates of spontaneous mitotic recombination. The requirement of 5`- to 3`-exonuclease encoded by the mammalian counterparts of RTH1 in the completion of lagging strand synthesis in reconstituted DNA replication systems suggested that retarded joining of Okazaki fragments in rth1Delta mutant cells should result in an increased level of spontaneous mitotic recombination. To verify this, we examined the effect of the rth1Delta mutation on spontaneous mitotic intrachromosomal recombination between two his3 genes (his3Delta3`, his3Delta5`) in two different RAD strains and in their rth1Delta derivatives. As shown in Table 2, the rate of mitotic recombination is elevated 15-30-fold in the rth1Delta mutant.




DISCUSSION

In vitro reconstitution of the DNA replication machinery from different mammalian sources has indicated the requirement of a 5`- to 3`-exonuclease activity in the completion of lagging strand DNA synthesis. RTH1 encodes the S. cerevisiae counterpart of the mammalian 5`- to 3`-exonuclease. In this study, we have determined the in vivo biological role of RTH1 by examining the effects of the rth1Delta mutation on viability, mitotic recombination, and DNA repair. We find that RTH1 is not an essential gene. However, growth rate is slowed very considerably in the rth1Delta mutant at the permissive temperature, and the rth1Delta mutation is inviable at the restrictive temperature of 37 °C. These results suggest the presence of an alternate 5`- to 3`-exonuclease activity that, at the permissive temperature, can substitute for the activity missing in the rth1Delta mutant; however, at the elevated temperature, the other 5`- to 3`-exonuclease activity is unable to support DNA replication. Because of the homology of RTH1-encoded protein with the S. cerevisiae RAD2 protein and the fact that RAD2 also possesses a 5`- to 3`-exonuclease activity(10) , we determined the effect of the rad2Delta mutation on viability in combination with the rth1Delta mutation. However, the rad2Delta mutation has no effect on viability or growth rate of the rth1Delta mutation, indicating that RAD2 does not fulfill the role of the alternate 5`- to 3`-exonuclease in DNA replication.

As expected for a mutant defective in DNA replication, the rth1Delta mutant stops division at the restrictive temperature as two large cells with a defect in nuclear division. At the permissive temperature, the rth1Delta mutation results in a reduction in growth rate, and a further decline in growth rate occurs in the rth1Delta rad52Delta double mutant. A slowdown in the removal of RNA primers in the rth1Delta mutant would retard the joining of nascent DNA fragments; subsequent channeling of these discontinuities into the RAD52 recombinational repair pathway would result in elevated levels of spontaneous mitotic recombination observed in the rth1Delta mutant. Elimination of recombinational repair by the rad52Delta mutation would leave the DNA lesions in the rth1Delta mutant unrepaired, resulting in a further reduction in growth rate of rth1Delta rad52Delta mutant strain over that of the rth1Delta and rad52Delta single mutants. We have shown previously that DNA ligase-deficient mutations are lethal in combination with mutations in the RAD52 gene(23) .

We find no evidence for the involvement of RTH1 in the repair of UV damage. The rth1Delta mutation, however, confers sensitivity to MMS, suggesting a role for the RTH1 5`- to 3`-exonuclease in the repair of alkylation damage. Following removal of the alkylated base by a DNA glycosylase and subsequent cleavage of the phosphodiester bond at the 5`-side of the apurinic/apyrimidinic (AP) residue by a class II AP endonuclease, the 5`- to 3`-exonuclease could effect the release of the AP residue, resulting in a 1-nucleotide gap, which could then be filled in by repair synthesis.


FOOTNOTES

*
This work was supported by Grant GM19261 from the National Institutes of Health and Grant CA41261 from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Xenometrix Inc., Boulder, CO 80301.

Present address: Sealy Center for Molecular Science, University of Texas Medical Branch, 6.104 Medical Research Bldg., Galveston, TX 77555-1061.

**
To whom correspondence should be addressed: Sealy Center for Molecular Science, University of Texas Medical Branch, 6.104 Medical Research Bldg., 11th & Mechanic St., Galveston, TX 77555-1061. Tel.: 409-747-8601; Fax: 409-747-8608.

(^1)
The abbreviations used are: YPD, yeast extract-peptone-dextrose; MMS, methyl methanesulfonate.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.