A Requirement for Recombinational Repair in Saccharomyces cerevisiae Is Caused by DNA Replication Defects of mec1 Mutants

Genetics ◽  
1999 ◽  
Vol 153 (2) ◽  
pp. 595-605 ◽  
Author(s):  
Bradley J Merrill ◽  
Connie Holm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Synthetic Lethality ◽  
Velocity Sedimentation ◽  
Recombinational Repair ◽  
Repair Pathway ◽  
Dna Breaks ◽  
Single Stranded Dna ◽  
Double Stranded Dna

Abstract To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.

scholarly journals The RAD52 Recombinational Repair Pathway is Essential in pol30 (PCNA) Mutants That Accumulate Small Single-Stranded DNA Fragments During DNA Synthesis

Genetics ◽  
1998 ◽  
Vol 148 (2) ◽  
pp. 611-624 ◽  
Author(s):  
Bradley J Merrill ◽  
Connie Holm
Keyword(s):  
Dna Replication ◽  
Synthetic Lethality ◽  
Large Subunit ◽  
Group Analysis ◽  
Nuclear Antigen ◽  
Recombinational Repair ◽  
Dna Fragments ◽  
Single Stranded Dna ◽  
Factor C

Abstract To identify in vivo pathways that compensate for impaired proliferating cell nuclear antigen (PCNA or Pol30p in yeast) activity, we performed a synthetic lethal screen with the yeast pol30-104 mutation. We identified nine mutations that display synthetic lethality with pol30-104; three mutations affected the structural gene for the large subunit of replication factor C (rfc1), which loads PCNA onto DNA, and six mutations affected three members of the RAD52 epistasis group for DNA recombinational repair (rad50, rad52, and rad57). We also found that pol30-104 displayed synthetic lethality with mutations in other members of the RAD52 epistasis group (rad51 and rad54), but not with mutations in members of the RAD3 nor the RAD6 epistasis group. Analysis of nine different pol30 mutations shows that the requirement for the RAD52 pathway is correlated with a DNA replication defect but not with the relative DNA repair defect caused by pol30 mutations. In addition, mutants that require RAD52 for viability (pol30-100, pol30-104, rfc1-1 and rth1Δ) accumulate small single-stranded DNA fragments during DNA replication in vivo. Taken together, these data suggest that the RAD52 pathway is required when there are defects in the maturation of Okazaki fragments.

scholarly journals The Saccharomyces cerevisiae DNA Recombination and Repair Functions of the RAD52 Epistasis Group Inhibit Ty1 Transposition

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 543-556 ◽  
Author(s):  
Alison J Rattray ◽  
Brenda K Shafer ◽  
David J Garfinkel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Dna Recombination ◽  
Recombinational Repair ◽  
Repair Pathway ◽  
Low Frequencies ◽  
Double Stranded Dna ◽  
High Level ◽  
In Cells

Abstract RNA transcribed from the Saccharomyces cerevisiae retrotransposon Ty1 accumulates to a high level in mitotically growing haploid cells, yet transposition occurs at very low frequencies. The product of reverse transcription is a linear double-stranded DNA molecule that reenters the genome by either Ty1-integrase-mediated insertion or homologous recombination with one of the preexisting genomic Ty1 (or δ) elements. Here we examine the role of the cellular homologous recombination functions on Ty1 transposition. We find that transposition is elevated in cells mutated for genes in the RAD52 recombinational repair pathway, such as RAD50, RAD51, RAD52, RAD54, or RAD57, or in the DNA ligase I gene CDC9, but is not elevated in cells mutated in the DNA repair functions encoded by the RAD1, RAD2, or MSH2 genes. The increase in Ty1 transposition observed when genes in the RAD52 recombinational pathway are mutated is not associated with a significant increase in Ty1 RNA or proteins. However, unincorporated Ty1 cDNA levels are markedly elevated. These results suggest that members of the RAD52 recombinational repair pathway inhibit Ty1 post-translationally by influencing the fate of Ty1 cDNA.

scholarly journals The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway.

Genetics ◽  
1990 ◽  
Vol 124 (4) ◽  
pp. 817-831 ◽  
Author(s):  
R H Schiestl ◽  
S Prakash ◽  
L Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Gamma Ray ◽  
Dna Lesions ◽  
Recombinational Repair ◽  
Repair Pathway ◽  
Uv Mutagenesis ◽  
Uv Sensitivity ◽  
Suppressor Mutations ◽  
Induced Mutagenesis

Abstract rad6 mutants of Saccharomyces cerevisiae are defective in the repair of damaged DNA, DNA damage induced mutagenesis, and sporulation. In order to identify genes that can substitute for RAD6 function, we have isolated genomic suppressors of the UV sensitivity of rad6 deletion (rad6 delta) mutations and show that they also suppress the gamma-ray sensitivity but not the UV mutagenesis or sporulation defects of rad6. The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of gamma-ray sensitivity. The six suppressor mutations we isolated are all alleles of the same locus and are also allelic to a previously described suppressor of the rad6-1 nonsense mutation, SRS2. We show that suppression of rad6 delta is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6 delta SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes. Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into the RAD52 DNA repair pathway are discussed.

scholarly journals Archaeal Orc1 protein interacts with T-rich single-stranded DNA

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Katarzyna Wegrzyn ◽  
Igor Konieczny
Keyword(s):  
Dna Replication ◽  
Replication Initiation ◽  
Single Stranded Dna ◽  
Dna Replication Initiation ◽  
Double Stranded Dna ◽  
Nucleoprotein Complexes ◽  
Eukaryotic Organisms ◽  
Replication Initiation Proteins ◽  
Hydrolysis Of ◽  
Replication Activity

Abstract Objective The ability to form nucleoprotein complexes is a fundamental activity of DNA replication initiation proteins. They bind within or nearby the region of replication origin what results in melting of a double-stranded DNA (dsDNA) and formation of single-stranded DNA (ssDNA) region where the replication machinery can assemble. For prokaryotic initiators it was shown that they interact with the formed ssDNA and that this interaction is required for the replication activity. The ability to interact with ssDNA was also shown for Saccharomyces cerevisiae replication initiation protein complex ORC. For Archaea, which combine features of both prokaryotic and eukaryotic organisms, there was no evidence whether DNA replication initiators can interact with ssDNA. We address this issue in this study. Results Using purified Orc1 protein from Aeropyrum pernix (ApOrc1) we analyzed its ability to interact with ssDNA containing sequence of an AT-rich region of the A. pernix origin Ori1 as well as with homopolymers of thymidine (polyT) and adenosine (polyA). The Bio-layer interferometry, surface plasmon resonance and microscale thermophoresis showed that the ApOrc1 can interact with ssDNA and it binds preferentially to T-rich ssDNA. The hydrolysis of ATP is not required for this interaction.

scholarly journals Yeast Meiotic Mutants Proficient for the Induction of Ectopic Recombination

Genetics ◽  
1998 ◽  
Vol 148 (2) ◽  
pp. 581-598
Author(s):  
JoAnne Engebrecht ◽  
Sherie Masse ◽  
Luther Davis ◽  
Kristine Rose ◽  
Therese Kessel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Meiotic Division ◽  
Ectopic Recombination ◽  
Meiotic Mutants ◽  
Chromosome Synapsis

Abstract A screen was designed to identify Saccharomyces cerevisiae mutants that were defective in meiosis yet proficient for meiotic ectopic recombination in the return-to-growth protocol. Seven mutants alleles were isolated; two are important for chromosome synapsis (RED1, MEK1) and five function independently of recombination (SPO14, GSG1, SPOT8/MUM2, 3, 4). Similar to the spoT8-1 mutant, mum2 deletion strains do not undergo premeiotic DNA synthesis, arrest prior to the first meiotic division and fail to sporulate. Surprisingly, although DNA replication does not occur, mum2 mutants are induced for high levels of ectopic recombination. gsg1 diploids are reduced in their ability to complete premeiotic DNA synthesis and the meiotic divisions, and a small percentage of cells produce spores. mum3 mutants sporulate poorly and the spores produced are inviable. Finally, mum4-1 mutants produce inviable spores. The meiotic/sporulation defects of gsg1, mum2, and mum3 are not relieved by spo11 or spo13 mutations, indicating that the mutant defects are not dependent on the initiation of recombination or completion of both meiotic divisions. In contrast, the spore inviability of the mum4-1 mutant is rescued by the spo13 mutation. The mum4-1 spo13 mutant undergoes a single, predominantly equational division, suggesting that MUM4 functions at or prior to the first meiotic division. Although recombination is variably affected in the gsg1 and mum mutants, we hypothesize that these mutants define genes important for aspects of meiosis not directly related to recombination.

scholarly journals Genome-wide analysis of DNA turnover and gene expression in stationary-phase Saccharomyces cerevisiae

Microbiology ◽  
2010 ◽  
Vol 156 (6) ◽  
pp. 1758-1771 ◽  
Author(s):  
A. de Morgan ◽  
L. Brodsky ◽  
Y. Ronin ◽  
E. Nevo ◽  
A. Korol ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Stationary Phase ◽  
Dna Synthesis ◽  
Exponential Phase ◽  
Yeast Cells ◽  
Genome Wide ◽  
Dna Turnover ◽  
Genomic Scale ◽  
Dna Maintenance

Exponential-phase yeast cells readily enter stationary phase when transferred to fresh, carbon-deficient medium, and can remain fully viable for up to several months. It is known that stationary-phase prokaryotic cells may still synthesize substantial amounts of DNA. Although the basis of this phenomenon remains unclear, this DNA synthesis may be the result of DNA maintenance and repair, recombination, and stress-induced transposition of mobile elements, which may occur in the absence of DNA replication. To the best of our knowledge, the existence of DNA turnover in stationary-phase unicellular eukaryotes remains largely unstudied. By performing cDNA-spotted (i.e. ORF) microarray analysis of stationary cultures of a haploid Saccharomyces cerevisiae strain, we demonstrated on a genomic scale the localization of a DNA-turnover marker [5-bromo-2′-deoxyuridine (BrdU); an analogue of thymidine], indicative of DNA synthesis in discrete, multiple sites across the genome. Exponential-phase cells on the other hand, exhibited a uniform, total genomic DNA synthesis pattern, possibly the result of DNA replication. Interestingly, BrdU-labelled sites exhibited a significant overlap with highly expressed features. We also found that the distribution among chromosomes of BrdU-labelled and expressed features deviates from random distribution; this was also observed for the overlapping set. Ty1 retrotransposon genes were also found to be labelled with BrdU, evidence for transposition during stationary phase; however, they were not significantly expressed. We discuss the relevance and possible connection of these results to DNA repair, mutation and related phenomena in higher eukaryotes.

scholarly journals TEN1 Is Essential for CDC13-Mediated Telomere Capping

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 793-810 ◽  
Author(s):  
Ling Xu ◽  
Ruben C. Petreaca ◽  
Hovik J. Gasparyan ◽  
Stephanie Vu ◽  
Constance I. Nugent
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
High Temperatures ◽  
De Novo ◽  
Temperature Sensitive ◽  
Single Stranded Dna ◽  
Growth Defects ◽  
Telomere Capping ◽  
Telomere Replication ◽  
Essential Function

Telomere binding proteins protect chromosome ends from degradation and mask chromosome termini from checkpoint surveillance. In Saccharomyces cerevisiae, Cdc13 binds single-stranded G-rich telomere repeats, maintaining telomere integrity and length. Two additional proteins, Ten1 and Stn1, interact with Cdc13 but their contributions to telomere integrity are not well defined. Ten1 is known to prevent accumulation of aberrant single-stranded telomere DNA; whether this results from defective end protection or defective telomere replication is unclear. Here we report our analysis of a new group of ten1 temperature-sensitive (ts) mutants. At permissive temperatures, ten1-ts strains display greatly elongated telomeres. After shift to nonpermissive conditions, however, ten1-ts mutants accumulate extensive telomeric single-stranded DNA. Cdk1 activity is required to generate these single-stranded regions, and deleting the EXO1 nuclease partially suppresses ten1-ts growth defects. This is similar to cdc13-1 mutants, suggesting ten1-ts strains are defective for end protection. Moreover, like Cdc13, our analysis reveals Ten1 promotes de novo telomere addition. Interestingly, in ten1-ts strains at high temperatures, telomeric single-stranded DNA and Rad52-YFP repair foci are strongly induced despite Cdc13 remaining associated with telomeres, revealing Cdc13 telomere binding is not sufficient for end protection. Finally, unlike cdc13-1 mutants, ten1-ts strains display strong synthetic interactions with mutations in the POLα complex. These results emphasize that Cdc13 relies on Ten1 to execute its essential function, but leave open the possibility that Ten1 has a Cdc13-independent role in DNA replication.

scholarly journals Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (7) ◽  
pp. 2329-2334 ◽  
Author(s):  
J R Simon ◽  
P D Moore
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Gene Conversion ◽  
Mutant Gene ◽  
Transformation Process ◽  
Single Stranded Dna ◽  
Autonomous Replication ◽  
Double Stranded Dna ◽  
Chromosomal Genes ◽  
Chromosomal Sequences

Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA.

scholarly journals Evidence that single-stranded DNA breaks are a normal feature of koala sperm chromatin, while double-stranded DNA breaks are indicative of DNA damage

Reproduction ◽  
2009 ◽  
Vol 138 (2) ◽  
pp. 267-278 ◽  
Author(s):  
Yeng Peng Zee ◽  
Carmen López-Fernández ◽  
F Arroyo ◽  
Stephen D Johnston ◽  
William V Holt ◽  
...  
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Dna Fragmentation ◽  
Sperm Nucleus ◽  
Severe Form ◽  
Sperm Chromatin ◽  
Dna Breaks ◽  
Single Stranded Dna ◽  
Double Stranded Dna ◽  
Dispersion Test

In this study, we have used single and double comet assays to differentiate between single- and double-stranded DNA damage in an effort to refine the interpretation of DNA damage in mature koala spermatozoa. We have also investigated the likelihood that single-stranded DNA breakage is part of the natural spermiogenic process in koalas, where its function would be the generation of structural bends in the DNA molecule so that appropriate packaging and compaction can occur. Koala spermatozoa were examined using the sperm chromatin dispersion test (SCDt) and comet assays to investigate non-orthodox double-stranded DNA. Comet assays were conducted under 1) neutral conditions; and 2) neutral followed by alkaline conditions (double comet assay); the latter technique enabled simultaneous visualisation of both single-stranded and double-stranded DNA breaks. Following the SCDt, there was a continuum of nuclear morphotypes, ranging from no apparent DNA fragmentation to those with highly dispersed and degraded chromatin. Dispersion morphotypes were mirrored by a similar diversity of comet morphologies that could be further differentiated using the double comet assay. The majority of koala spermatozoa had nuclei with DNA abasic-like residues that produced single-tailed comets following the double comet assay. The ubiquity of these residues suggests that constitutive alkali-labile sites are part of the structural configuration of the koala sperm nucleus. Spermatozoa with ‘true’ DNA fragmentation exhibited a continuum of comet morphologies, ranging from a more severe form of alkaline-susceptible DNA with a diffuse single tail to nuclei that exhibited both single- and double-stranded breaks with two comet tails.

scholarly journals Interaction of replication factor Sld3 and histone acetyl transferase Esa1 alleviates gene silencing and promotes the activation of late and dormant replication origins

2020 ◽  
Author(s):  
Seiji Tanaka
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Gene Silencing ◽  
Dna Synthesis ◽  
Histone Acetyltransferase ◽  
Replication Origins ◽  
Histone Acetyl Transferase ◽  
Step Process ◽  
Helicase Loading ◽  
Rate Limiting

SUMMARYDNA replication in eukaryotes is a multi-step process that consists of three main reactions: helicase loading (licensing), helicase activation (firing), and nascent DNA synthesis (elongation). Although the contributions of some chromatin regulatory factors in the licensing and elongation reaction have been determined, their functions in the firing reaction remain elusive. In the budding yeast Saccharomyces cerevisiae, Sld3, Sld7, and Cdc45 (3-7-45) are rate-limiting in the firing reaction and simultaneous overexpression of 3-7-45 causes untimely activation of late and dormant replication origins. Here we found that 3-7-45 overexpression not only activated dormant origins in the silenced locus, HMLα, but also exerted an anti-silencing effect at this locus. For these, interaction between Sld3 and Esa1, a conserved histone acetyltransferase, was responsible. Moreover, the Sld3–Esa1 interaction was required for untimely activation of late origins. These results reveal the Sld3–Esa1 interaction as a novel level of regulation in the firing reaction.

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