Polymerases ε and ∂ repair dysfunctional telomeres facilitated by salt.

Damaged DNA can be repaired by removal and re-synthesis of up to 30 nucleotides during base or nucleotide excision repair. An important question is what happens when many more nucleotides are removed, resulting in long single-stranded DNA (ssDNA) lesions. Such lesions appear on chromosomes during telomere damage, double strand break repair or after the UV damage of stationary phase cells. Here, we show that long single-stranded lesions, formed at dysfunctional telomeres in budding yeast, are re-synthesized when cells are removed from the telomere-damaging environment. This process requires Pol32, an accessory factor of Polymerase δ. However, re-synthesis takes place even when the telomere-damaging conditions persist, in which case the accessory factors of both polymerases δ and ε are required, and surprisingly, salt. Salt added to the medium facilitates the DNA synthesis, independently of the osmotic stress responses. These results provide unexpected insights into the DNA metabolism and challenge the current view on cellular responses to telomere dysfunction.

Mechanism of Lagging-Strand DNA Replication in Eukaryotes.

This chapter focuses on the enzymes and mechanisms involved in lagging-strand DNA replication in eukaryotic cells. Recent structural and biochemical progress with DNA polymerase α-primase (Pol α) provides insights how each of the millions of Okazaki fragments in a mammalian cell is primed by the primase subunit and further extended by its polymerase subunit. Rapid kinetic studies of Okazaki fragment elongation by Pol δ illuminate events when the polymerase encounters the double-stranded RNA-DNA block of the preceding Okazaki fragment. This block acts as a progressive molecular break that provides both time and opportunity for the flap endonuclease 1 (FEN1) to access the nascent flap and cut it. The iterative action of Pol δ and FEN1 is coordinated by the replication clamp PCNA and produces a regulated degradation of the RNA primer, thereby preventing the formation of long-strand displacement flaps. Occasional long flaps are further processed by backup nucleases including Dna2.

The catalytic subunit of Arabidopsis DNA polymerase α ensures stable maintenance of histone modification.

Mitotic inheritance of identical cellular memory is crucial for development in multicellular organisms. The cell type-specific epigenetic state should be correctly duplicated upon DNA replication to maintain cellular memory during tissue and organ development. Although a role of DNA replication machinery in maintenance of epigenetic memory has been proposed, technical limitations have prevented characterization of the process in detail. Here, we show that INCURVATA2 (ICU2), the catalytic subunit of DNA polymerase α in Arabidopsis, ensures the stable maintenance of repressive histone modifications. The missense mutant allele icu2-1 caused a defect in the mitotic maintenance of vernalization memory. Although neither the recruitment of CURLY LEAF (CLF), a SET-domain component of Polycomb Repressive Complex 2 (PRC2), nor the resultant deposition of the histone mark H3K27me3 required for vernalization-induced FLOWERING LOCUS C (FLC) repression were affected, icu2-1 mutants exhibited unstable maintenance of the H3K27me3 level at the FLC region, which resulted in mosaic FLC de-repression after vernalization. ICU2 maintains the repressive chromatin state at additional PRC2 targets as well as at heterochromatic retroelements. In icu2-1 mutants, the subsequent binding of LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1), a functional homolog of PRC1, at PRC2 targets was also reduced. We demonstrated that ICU2 facilitates histone assembly in dividing cells, suggesting a possible mechanism for ICU2-mediated epigenetic maintenance.

Significance of DNA polymerase I in in vivo processing of clustered DNA damage.

We examined the biological consequences of bi-stranded clustered damage sites, consisting of a combination of DNA lesions, such as a 1-nucleotide gap (GAP), an apurinic/apyrimidinic (AP) site, and an 8-oxo-7,8-dihydroguanine (8-oxoG), using a bacterial plasmid-based assay. Following transformation with the plasmid containing bi-stranded clustered damage sites into the wild type strain of Escherichia coli, transformation frequencies were significantly lower for the bi-stranded clustered GAP/AP lesions (separated by 1bp) than for either a single GAP or a single AP site. When the two lesions were separated by 10-20bp, the transformation efficiencies were comparable with those of the single lesions. This recovery of transformation efficiency for separated lesions requires DNA polymerase I (Pol I) activity. Analogously, the mutation frequency was found to depend on the distance separating lesions in a bi-stranded cluster containing a GAP and an 8-oxoG, and Pol I was found to play an important role in minimising mutations induced as a result of clustered lesions. The mutagenic potential of 8-oxoG within the bi-stranded lesions does not depend on whether it is situated on the leading or lagging strand. These results indicate that the biological consequences of clustered DNA damage strongly depend on the extent of repair of the strand breaks as well as the DNA polymerase in lesion-avoidance pathways during replication.

Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication.

DNA polymerase I (pol I) processes RNA primers during lagging-strand synthesis and fills small gaps during DNA repair reactions. However, it is unclear how pol I and pol III work together during replication and repair or how extensive pol I processing of Okazaki fragments is in vivo. Here, we address these questions by analyzing pol I mutations generated through error-prone replication of ColE1 plasmids. The data were obtained by direct sequencing, allowing an accurate determination of the mutation spectrum and distribution. Pol I's mutational footprint suggests: (i) during leading-strand replication pol I is gradually replaced by pol III over at least 1.3 kb; (ii) pol I processing of Okazaki fragments is limited to ∼20 nt and (iii) the size of Okazaki fragments is short (∼250 nt). While based on ColE1 plasmid replication, our findings are likely relevant to other pol I replicative processes such as chromosomal replication and DNA repair, which differ from ColE1 replication mostly at the recruitment steps. This mutation footprinting approach should help establish the role of other prokaryotic or eukaryotic polymerases in vivo, and provides a tool to investigate how sequence topology, DNA damage, or interactions with protein partners may affect the function of individual DNA polymerases.

Roles of the four DNA polymerases of the crenarchaeon Sulfolobus solfataricus and accessory proteins in DNA replication.

The hyperthermophilic crenarchaeon Sulfolobus solfataricus P2 encodes three B-family DNA polymerase genes, B1 (Dpo1), B2 (Dpo2), and B3 (Dpo3), and one Y-family DNA polymerase gene, Dpo4, which are related to eukaryotic counterparts. Both mRNAs and proteins of all four DNA polymerases were constitutively expressed in all growth phases. Dpo2 and Dpo3 possessed very low DNA polymerase and 3' to 5' exonuclease activities in vitro. Steady-state kinetic efficiencies (k(cat)/K(m)) for correct nucleotide insertion by Dpo2 and Dpo3 were several orders of magnitude less than Dpo1 and Dpo4. Both the accessory proteins proliferating cell nuclear antigen and the clamp loader replication factor C facilitated DNA synthesis with Dpo3, as with Dpo1 and Dpo4, but very weakly with Dpo2. DNA synthesis by Dpo2 and Dpo3 was remarkably decreased by single-stranded binding protein, in contrast to Dpo1 and Dpo4. DNA synthesis in the presence of proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein was most processive with Dpo1, whereas DNA lesion bypass was most effective with Dpo4. Both Dpo2 and Dpo3, but not Dpo1, bypassed hypoxanthine and 8-oxoguanine. Dpo2 and Dpo3 bypassed uracil and cis-syn cyclobutane thymine dimer, respectively. High concentrations of Dpo2 or Dpo3 did not attenuate DNA synthesis by Dpo1 or Dpo4. We conclude that Dpo2 and Dpo3 are much less functional and more thermolabile than Dpo1 and Dpo4 in vitro but have bypass activities across hypoxanthine, 8-oxoguanine, and either uracil or cis-syn cyclobutane thymine dimer, suggesting their catalytically limited roles in translesion DNA synthesis past deaminated, oxidized base lesions and/or UV-induced damage.

Mutation in the catalytic subunit of DNA polymerase alpha influences transcriptional gene silencing and homologous recombination in Arabidopsis.

REPRESSOR OF SILENCING 1 (ROS1) encodes a DNA demethylase that actively removes DNA methylation. Mutation in ROS1 leads to transcriptional gene silencing of a T-DNA locus that contains two genes, RD29A-LUC and 35S-NPTII, originally expressed in the C24 wild type. These units have different silencing regulation mechanisms: the former mechanism is dependent on small interfering RNA (siRNA)-directed DNA methylation, but the latter is not. We studied the latter gene silencing mechanism by screening the suppressors of the ros1 mutant using the silenced 35S-NPTII as a selection marker gene. The polalpha/incurvata2 (icu2) gene was isolated as one ros1 suppressor because its mutation leads to the reactivation of the silenced 35S-NPTII gene. POLalpha/ICU2 encodes a catalytic subunit of DNA polymerase alpha. Mutation of POLalpha/ICU2 did not affect DNA methylation, but reduced histone H3 Lys9 dimethylation (H3K9me2) modification in the 35S promoter. The polalpha mutation also influences the development of the shoot apical meristem, and delays the G2/M phase with high expression of a G2/M marker gene CycB1;1:GUS. Furthermore, the frequency of homologous recombination is greater in the polalpha/icu2 mutant than in the C24 wild type. Our results suggest that DNA polymerase alpha is involved in mediating epigenetic states and in DNA homologous recombination in Arabidopsis.

Evidence that errors made by DNA polymerase alpha are corrected by DNA polymerase delta.

Eukaryotic replication begins at origins and on the lagging strand with RNA-primed DNA synthesis of a few nucleotides by polymerase alpha, which lacks proofreading activity. A polymerase switch then allows chain elongation by proofreading-proficient pol delta and pol epsilon. Pol delta and pol epsilon are essential, but their roles in replication are not yet completely defined . Here, we investigate their roles by using yeast pol alpha with a Leu868Met substitution . L868M pol alpha copies DNA in vitro with normal activity and processivity but with reduced fidelity. In vivo, the pol1-L868M allele confers a mutator phenotype. This mutator phenotype is strongly increased upon inactivation of the 3' exonuclease of pol delta but not that of pol epsilon. Several nonexclusive explanations are considered, including the hypothesis that the 3' exonuclease of pol delta proofreads errors generated by pol alpha during initiation of Okazaki fragments. Given that eukaryotes encode specialized, proofreading-deficient polymerases with even lower fidelity than pol alpha, such intermolecular proofreading could be relevant to several DNA transactions that control genome stability.

Reducing DNA polymerase alpha in the absence of Drosophila ATR leads to P53-dependent apoptosis and developmental defects.

The ability to respond to DNA damage and incomplete replication ensures proper duplication and stability of the genome. Two checkpoint kinases, ATM and ATR, are required for DNA damage and replication checkpoint responses. In Drosophila, the ATR ortholog (MEI-41) is essential for preventing entry into mitosis in the presence of DNA damage. In the absence of MEI-41, heterozygosity for the E(mus304) mutation causes rough eyes. We found that E(mus304) is a mutation in DNApol-alpha180, which encodes the catalytic subunit of DNA polymerase alpha. We did not find any defects resulting from reducing Polalpha by itself. However, reducing Polalpha in the absence of MEI-41 resulted in elevated P53-dependent apoptosis, rough eyes, and increased genomic instability. Reducing Polalpha in mutants that lack downstream components of the DNA damage checkpoint (DmChk1 and DmChk2) results in the same defects. Furthermore, reducing levels of mitotic cyclins rescues both phenotypes. We suggest that reducing Polalpha slows replication, imposing an essential requirement for the MEI-41-dependent checkpoint for maintenance of genome stability, cell survival, and proper development. This work demonstrates a critical contribution of the checkpoint function of MEI-41 in responding to endogenous damage.

Role of the second-largest subunit of DNA polymerase alpha in the interaction between the catalytic subunit and hyperphosphorylated retinoblastoma protein in late S phase.

DNA polymerase alpha (pol-alpha) is a heterotetrameric enzyme (p180-p68-p58-p48 in mouse) that is essential for the initiation of chain elongation during DNA replication. The catalytic (p180) and p68 subunits of pol-alpha are phosphorylated by Cdk-cyclin complexes, with p68 being hyperphosphorylated by cyclin-dependent kinases in G(2) phase of the cell cycle. The activity of Cdk2-cyclin A increases during late S phase and peaks in G(2) phase. We have now examined the role of p68 in the interaction between the catalytic subunit of pol-alpha and hyperphosphorylated retinoblastoma protein (ppRb) and in the stimulation of the polymerase activity of pol-alpha by ppRb. With the use of recombinant proteins, we found that nonphosphorylated p68 inhibited the stimulation of pol-alpha activity by ppRb, suggesting that p68 might impede the association of ppRb with p180. Phosphorylation of p68 by Cdk2-cyclin A greatly reduced its inhibitory effect. Immunofluorescence analysis also revealed that ppRb localized at sites of DNA replication specifically in late S phase. These results suggest that Cdk-cyclin A can phosphorylate pol-alpha which may result in a conformational change in pol-alpha facilitating its interaction with and activation by ppRb.

The mechanism of base excision repair in Chlamydiophila pneumoniae.

Repair of damaged DNA is of great importance in maintaining genome integrity, and there are several pathways for repair of damaged DNA in almost all organisms. Base excision repair (BER) is a main process for repairing DNA carrying slightly damaged bases. Several proteins are required for BER; these include DNA glycosylases, AP endonuclease, DNA polymerase, and DNA ligase. In some bacteria the single-stranded specific exonuclease, RecJ, is also involved in BER. In this research, six Chlamydiophila pneumoniae (C. pneumoniae) genes, encoding uracil DNA glycosylase (CpUDG), endonuclease IV (CpEndoIV), DNA polymerase I (CpDNApolI), endonuclease III (CpEndoIII), single-stranded specific exonuclease RecJ (CpRecJ), and DNA ligase (CpDNALig), were inserted into the expression vector pET28a. All proteins, except for CpDNALig, were successfully expressed in E. coli, and purified proteins were characterized in vitro. C. pneumoniae BER was reconstituted in vitro with CpUDG, CpEndoIV, CpDNApolI and E. coli DNA ligase (EcDNALig). After uracil removal by CpUDG, the AP site could be repaired by two BER pathways that involved in the replacement of either one (short patch BER) or multiple nucleotides (long patch BER) at the lesion site. CpEndoIII promoted short patch BER via its 5'-deoxyribophosphodiesterase (5'-dRPase) activity, while CpRecJ had little effect on short patch BER. The flap structure generated during DNA extension could be removed by the 5'-exonuclease activity of CpDNApolI. Based on these observations, we propose a probable mechanism for BER in C. pneumoniae.

NTP concentration effects on initial transcription by T7 RNAP indicate that translocation occurs through passive sliding and reveal that divergent promoters have distinct NTP concentration requirements for productive initiation.

The hypothesis that active site translocation during initial transcription occurs by a passive sliding mechanism which allows the pre- and post-translocated states to equilibrate on the time scale of bond formation was tested by evaluating the effects of NTP concentration on individual transcript extension steps in the presence of translocation roadblocks created by proteins bound immediately downstream of a T7 promoter, as well as by evaluating the effects of NTP concentration on competing transcript extension pathways (iterative synthesis and "normal" extension). Results are consistent with a passive sliding mechanism for translocation which is driven by NTP binding, and are inconsistent with mechanisms in which the pre- and post-translocated states fail to equilibrate with each other on the time scale of bond formation or in which translocation is driven by NTP hydrolysis. We also find, in agreement with many previous studies, that divergence from consensus in the ITS (initially transcribed sequence) of the T7 promoter decreases productive initiation. However, this appears to be largely due to increases in the NTP concentration requirements for efficient transcription on the divergent ITSs.

A global profile of replicative polymerase usage.

Three eukaryotic DNA polymerases are essential for genome replication. Polymerase (Pol) α-primase initiates each synthesis event and is rapidly replaced by processive DNA polymerases: Polɛ replicates the leading strand, whereas Polδ performs lagging-strand synthesis. However, it is not known whether this division of labor is maintained across the whole genome or how uniform it is within single replicons. Using Schizosaccharomyces pombe, we have developed a polymerase usage sequencing (Pu-seq) strategy to map polymerase usage genome wide. Pu-seq provides direct replication-origin location and efficiency data and indirect estimates of replication timing. We confirm that the division of labor is broadly maintained across an entire genome. However, our data suggest a subtle variability in the usage of the two polymerases within individual replicons. We propose that this results from occasional leading-strand initiation by Polδ followed by exchange for Polɛ.

Replicative enzymes and ageing: importance of DNA polymerase alpha function to the events of cellular ageing.

A hallmark of cellular ageing is the failure of senescing cells to initiate DNA synthesis and transition from G1 into S phase of the cell cycle. This transition is normally dependent on or concomitant with expression of a set of genes specifying cellular proteins, some of which directly participate in DNA replication. Deregulation of this gene expression may play a pivotal role in the ageing process. The number of known enzymes and co-factors required to maintain integrity of the genome during eukaryotic DNA replication has increased significantly in the past few years, and includes proteins essential for DNA replication and repair, as well as for cell cycle regulation. In eukaryotic cells, ranging from yeast to man, a replicative enzyme essential for initiation of transcription is DNA polymerase alpha (pol alpha), the activity of which is coordinately regulated with the initiation of DNA synthesis. DNA pol alpha, by means of its primase subunit, has the unique ability to initiate de novo DNA synthesis, and as a consequence, is required for the initiation of continuous (leading-strand) DNA synthesis at an origin of replication, as well as for initiation of discontinuous (lagging-strand) DNA synthesis. The dual role of the pol alpha-primase complex makes it a potential interactant with the regulatory mechanisms controlling entry into S phase. The purpose of this review is to address the regulation and/or modulation of DNA pol alpha during ageing that may play a key role in the cascade of events which ultimately leads to the failure of old cells to enter or complete S phase of the cell cycle.

Correlation between molecular clock ticking, codon usage fidelity of DNA repair, chromosome banding and chromatin compactness in germline cells.

The vertebrate genome is built of long DNA regions, relatively homogeneous in GC content, which likely correspond to bands on stained chromosomes. Large differences in composition have been found among DNA regions belonging to the same genome. They are paralleled by differences in codon usage in genes differently localized. The hypothesis presented here asserts that these differences in composition are caused by different mutational bias of alpha and beta DNA polymerases, these polymerases being involved to different extents in the repair of DNA lesions in compact and relaxed chromatin, respectively, in germline cells.

Replicative enzymes, DNA polymerase alpha (pol alpha), and in vitro ageing.

Normal cells in culture are used to investigate the underlying mechanisms of DNA synthesis because they retain regulatory characteristics of the in vivo replication machinery. During the last few years new studies have identified a number of genetic changes that occur during in vitro ageing, providing insight into the progressive decline in biological function that occurs during ageing. Maintaining genomic integrity in eukaryotic organisms requires precisely coordinated replication of the genome during mitosis, which is the most fundamental aspect of living cells. To achieve this coordinated replication, eukaryotic cells employ an ordered series of steps to form several key protein assemblies at origins of replication. Major progress has recently been made in identifying the enzymes, and other proteins, of DNA replication that are recruited to origin sites and the order in which they are recruited during the process of replication. More than 20 proteins, including DNA polymerases, have been identified as essential components that must be preassembled at replication origins for the initiation of DNA synthesis. Of the polymerases, DNA polymerase alpha-primase (pol alpha) is of particular importance since its function is fundamental to understanding the initiation mechanism of eukaryotic DNA replication. DNA must be replicated with high fidelity to ensure the accurate transfer of genetic information to progeny cells, and decreases in DNA pol alpha activity and fidelity, which are coordinated with cell cycle progression, have been shown to be important facets of a probable intrinsic cause of genetic alterations during in vitro ageing. This has led to the proposal that pol alpha activity and function is one of the crucial determinants in ageing. In this review we summarize the current state of knowledge of DNA pol alpha function in the regulation of DNA replication and focus in particular on its interactive tasks with other proteins during in vitro ageing.

Evidence for reduced copying fidelity of DNA polymerases alpha, delta, and epsilon from Novikoff hepatoma cells.

To investigate whether or not DNA polymerases alpha, delta, and epsilon from tumor cells have acquired properties that might be responsible for mutations found in tumor development, we investigated copying fidelities of DNA polymerases alpha, delta, and epsilon from the highly malignant Novikoff hepatoma cells and compared them to the corresponding enzymes from normal rat liver. DNA polymerases were purified more than 300-fold by three chromatographic steps. Copying fidelity was studied using steady-state kinetics and an 18-mer oligonucleotide primed with a 12-mer (13-mer for extension experiments) as DNA primer-template. Three experimental approaches were chosen: i) extension of DNA primers with mismatched 3'-OH ends opposite dGMP, ii) DNA insertion of nucleotides opposite m6G in the template and iii) extension of DNA primers with mismatched 3'-OH ends opposite m6G. i) Extension of DNA primers with mismatched 3'-OH ends opposite dGMP. DNA primer templates containing G:T and G:A mispairs at the 3'-OH position of the primer were easily extended by DNA polymerases alpha, delta and epsilon from both normal rat liver and Novikoff hepatoma cells. The G:G mismatch was elongated with low efficiency. Notably, DNA polymerase alpha from Novikoff hepatoma cells extended G:A and G:G mismatches significantly faster than the enzyme from normal cells. ii) Insertion of nucleotides opposite m6G. DNA polymerases alpha, delta, and epsilon from normal rat liver preferably catalyzed incorporation of dAMP opposite m6G at dNTP concentrations < 100 microM. When dNTP concentrations were raised to > or = 100 microM, dCMP (DNA polymerases delta and epsilon) and dTMP (DNA polymerase alpha) were also incorporated. The same insertion characteristics were found for the enzymes from Novikoff cells, however, insertion efficiencies of dAMP and dCMP were significantly higher for polymerases delta and epsilon. iii) Extension of primers with mismatched 3'-OH ends opposite m6G. Only m6G:dAMP and m6G:dCMP mismatches were extended by DNA polymerases alpha, delta and epsilon from both sources. No differences in extension efficiency were observed between the enzymes from normal and hepatoma cells. Taken together, our results suggest that DNA polymerases alpha, delta, and epsilon from Novikoff cells catalyzed incorporation of the wrong nucleotides more readily and extended mismatches more easily. These results may provide a rationale why numerous mutations accumulate during tumor development.

DNA polymerase alpha and beta activities during the cell cycle and their role in heat radiosensitization in Chinese hamster ovary cells.

The levels of DNA polymerase alpha and beta activities were measured during the cell cycle using a total cell homogenate technique. The results indicate a decrease in the levels of both enzyme activities during the G1 phase and a gradual increase as cells enter the S phase. The recovery of DNA polymerase activities was measured after heating for 10 min at 45.5 degrees C during the G1 phase (1.5 hr after mitotic release) or S phase (8-9 hr after mitotic release). This treatment reduced the levels of DNA polymerase beta activity to 20-30% and DNA polymerase alpha activity to 40-50% of their control activity for both G1 and S phase. The activity of DNA polymerase beta recovered fully during 20-25 hr after heating for both G1 phase or S phase cells. There was no recovery of the activity of DNA polymerase alpha during this time. Survival was measured when cells were irradiated (4 Gy) at various times after hyperthermia (10 min at 45.5 degrees C), and for both G1 and S phase the interaction between heat and X rays disappeared by 20-25 hr after heating and the same increase was observed for the recovery of DNA polymerase beta activity. Furthermore, treatment with cycloheximide inhibited protein synthesis and prevented recovery from heat damage assayed in terms of both cell survival and beta polymerase activity. These results, in addition to experiments with heat protection by glycerol or thermotolerance induced with sodium arsenite or fractionated heat doses, suggest that beta polymerase may be an important enzyme involved in repairing X-ray-induced damage that can result in cell lethality.

A comparison of the enhancement of radiation sensitivity and DNA polymerase inactivation by hyperthermia in human glioma cells.

Two human glioma cell lines (U87MG and U373MG) were evaluated for their thermal enhancement of radiation sensitivity and its correlation to the degree of inactivation of DNA polymerase alpha and beta. The data showed that hyperthermia increased radiation sensitivity in a time- and temperature-dependent manner. The differential heat sensitivity of the two cell lines was reflected in the degree of polymerase inactivation. Polymerase inactivation was also dependent on time and temperature and was greater for polymerase beta than alpha. The degree of polymerase inactivation correlated well with the thermal enhancement ratio (TER) calculated at the 1.0% survival level. This correlation was poor for the TER at the 50% survival level. The correlations were better for polymerase beta than alpha. The small differences in thermal sensitivity between the two cell lines primarily at 41 and 42 degrees C could not be explained by correlation between polymerase inactivation and TER. Incubation between hyperthermia and irradiation resulted in recovery of polymerase activity and loss of radiosensitization. Levels of polymerase beta after hyperthermia may be used to predict thermal enhancement of radiosensitivity for low survival levels, but possibly not in the shoulder region of the radiation survival curve. Small cell line-dependent differences in thermal sensitivity may not be resolved in these comparisons.

The involvement of topoisomerases and DNA polymerase I in the mechanism of induced thermal and radiation resistance in yeast.

Either an ionizing radiation exposure or a heat shock is capable of inducing both thermal tolerance and radiation resistance in yeast. Yeast mutants, deficient in topoisomerase I, in topoisomerase II, or in DNA polymerase I, were used to investigate the mechanism of these inducible resistances. The absence of either or both topoisomerase activities did not prevent induction of either heat or radiation resistance. However, if both topoisomerase I and II activities were absent, the sensitivity of yeast to become thermally tolerant (in response to a heat stress) was markedly increased. The absence of only topoisomerase I activity (top1) resulted in the constitutive expression of increased radiation resistance equivalent to that induced by a heat shock in wild-type cells, and the topoisomerase I-deficient cells were not further inducible by heat. This heat-inducible component of radiation resistance (or its equivalent constitutive expression in top1 cells) was, in turn, only a portion of the full response inducible by radiation. The absence of polymerase I activity had no detectable effect on either response. Our results indicate that the actual systems that confer resistance to heat or radiation are independent of either topoisomerase activity or DNA polymerase function, but suggest that topoisomerases may have a regulatory role during the signaling of these mechanisms. The results of our experiments imply that maintenance of correct DNA topology prevents induction of the heat-shock response, and that heat-shock induction of a component of the full radiation resistance in yeast may be the consequence of topoisomerase I inactivation.