Yeast DNA Repair Gene Research Articles

RNA polymerase II transcription attenuation at the yeast DNA repair gene DEF1 is biologically significant and dependent on the Hrp1 RNA-recognition motif.

Premature transcription termination (i.e. attenuation) is a potent gene regulatory mechanism that represses mRNA synthesis. Attenuation of RNA polymerase II is more prevalent than once appreciated, targeting 10-15% of mRNA genes in yeast through higher eukaryotes, but its significance and mechanism remain obscure. In the yeast Saccharomyces cerevisiae, polymerase II attenuation was initially shown to rely on Nrd1-Nab3-Sen1 termination, but more recently our laboratory characterized a hybrid termination pathway involving Hrp1, an RNA-binding protein in the 3'-end cleavage factor. One of the hybrid attenuation gene targets is DEF1, which encodes a repair protein that promotes degradation of polymerase II stalled at DNA lesions. In this study, we characterized the chromosomal DEF1 attenuator and the functional role of Hrp1. DEF1 attenuator mutants overexpressed Def1 mRNA and protein, exacerbated polymerase II degradation, and hindered cell growth, supporting a biologically significant DEF1 attenuator function. Using an auxin-induced Hrp1 depletion system, we identified new Hrp1-dependent attenuators in MNR2, SNG1, and RAD3 genes. An hrp1-5 mutant (L205S) known to impair binding to cleavage factor protein Rna14 also disrupted attenuation, but surprisingly no widespread defect was observed for an hrp1-1 mutant (K160E) located in the RNA-recognition motif. We designed a new RNA recognition motif mutant (hrp1-F162W) that altered a highly conserved residue and was lethal in single copy. In a heterozygous strain, hrp1-F162W exhibited dominant-negative readthrough defects at several gene attenuators. Overall, our results expand the hybrid RNA polymerase II termination pathway, confirming that Hrp1-dependent attenuation controls multiple yeast genes and may function through binding cleavage factor proteins and/or RNA.

RNA Polymerase II Transcription Attenuation at the Yeast DNA Repair Gene, DEF1, Involves Sen1-Dependent and Polyadenylation Site-Dependent Termination.

Termination of RNA Polymerase II (Pol II) activity serves a vital cellular role by separating ubiquitous transcription units and influencing RNA fate and function. In the yeast Saccharomyces cerevisiae, Pol II termination is carried out by cleavage and polyadenylation factor (CPF-CF) and Nrd1-Nab3-Sen1 (NNS) complexes, which operate primarily at mRNA and non-coding RNA genes, respectively. Premature Pol II termination (attenuation) contributes to gene regulation, but there is limited knowledge of its prevalence and biological significance. In particular, it is unclear how much crosstalk occurs between CPF-CF and NNS complexes and how Pol II attenuation is modulated during stress adaptation. In this study, we have identified an attenuator in the DEF1 DNA repair gene, which includes a portion of the 5′-untranslated region (UTR) and upstream open reading frame (ORF). Using a plasmid-based reporter gene system, we conducted a genetic screen of 14 termination mutants and their ability to confer Pol II read-through defects. The DEF1 attenuator behaved as a hybrid terminator, relying heavily on CPF-CF and Sen1 but without Nrd1 and Nab3 involvement. Our genetic selection identified 22 cis-acting point mutations that clustered into four regions, including a polyadenylation site efficiency element that genetically interacts with its cognate binding-protein Hrp1. Outside of the reporter gene context, a DEF1 attenuator mutant increased mRNA and protein expression, exacerbating the toxicity of a constitutively active Def1 protein. Overall, our data support a biologically significant role for transcription attenuation in regulating DEF1 expression, which can be modulated during the DNA damage response.

Regulation of Translesion Synthesis DNA Polymerase η by Monoubiquitination

DNA polymerase eta is a Y family polymerase involved in translesion synthesis (TLS). Its action is initiated by simultaneous interaction between the PIP box in pol eta and PCNA and between the UBZ in pol eta and monoubiquitin attached to PCNA. Whereas monoubiquitination of PCNA is required for its interaction with pol eta during TLS, we now show that monoubiquitination of pol eta inhibits this interaction, preventing its functions in undamaged cells. Identification of monoubiquitination sites within pol eta nuclear localization signal (NLS) led to the discovery that pol eta NLS directly contacts PCNA, forming an extended pol eta-PCNA interaction surface. We name this the PCNA-interacting region (PIR) and show that its monoubiquitination is downregulated by various DNA-damaging agents. We propose that this mechanism ensures optimal availability of nonubiquitinated, TLS-competent pol eta after DNA damage. Our work shows how monoubiquitination can either positively or negatively regulate the assembly of a protein complex, depending on which substrates are targeted by ubiquitin.

Structure and expression of AtPMS1, the Arabidopsis ortholog of the yeast DNA repair gene PMS1

The DNA mismatch repair (MMR) machinery of eukaryotes involves several genes whose products interact to repair mispaired bases resulting from DNA replication errors or homoeologous recombination. We report here the cloning and characterization of AtPMS1, a gene that codes for a predicted polypeptide sequence of 924 amino acids containing the typical MutL motifs. It is one of three homologs of the Escherichia coli MutL gene in Arabidopsis thaliana and constitutes the first ortholog of the yeast PMS1 gene to be characterized in plants. The gene is present in a single copy in the Arabidopsis genome and is located in the top portion of chromosome IV. Northern and RT-PCR analyses indicate that the AtPMS1 gene is expressed at very low levels in mature leaves but is much more highly expressed in cultured cells. Furthermore, an alternate form of the messenger, apparently coding for an incomplete protein lacking a key portion of the C-terminus, appears to co-exist in some plant tissues, albeit at a lower abundance. Primer extension and 5′-RACE experiments suggest that this gene has multiple transcription start sites, observations that are in agreement with the lack of a detectable TATA box. These results underline the importance of experimentally-derived results to confirm and, occasionally, to correct and extend the information provided by genome projects.

Structure and expression of AtPMS1, the Arabidopsis ortholog of the yeast DNA repair gene PMS1

The DNA mismatch repair (MMR) machinery of eukaryotes involves several genes whose products interact to repair mispaired bases resulting from DNA replication errors or homoeologous recombination. We report here the cloning and characterization of AtPMS1 , a gene that codes for a predicted polypeptide sequence of 924 amino acids containing the typical MutL motifs. It is one of three homologs of the Escherichia coli MutL gene in Arabidopsis thaliana and constitutes the first ortholog of the yeast PMS1 gene to be characterized in plants. The gene is present in a single copy in the Arabidopsis genome and is located in the top portion of chromosome IV. Northern and RT-PCR analyses indicate that the AtPMS1 gene is expressed at very low levels in mature leaves but is much more highly expressed in cultured cells. Furthermore, an alternate form of the messenger, apparently coding for an incomplete protein lacking a key portion of the C-terminus, appears to co-exist in some plant tissues, albeit at a lower abundance. Primer extension and 5′-RACE experiments suggest that this gene has multiple transcription start sites, observations that are in agreement with the lack of a detectable TATA box. These results underline the importance of experimentally-derived results to confirm and, occasionally, to correct and extend the information provided by genome projects.

Expression of genes involved in mammalian meiosis during the transition from egg to embryo.

The ooplasm of higher eukaryotes provides substances necessary for completing the last stages of meiosis and initiating the first mitotic division. These processes are firmly attuned to other events in the egg and newly formed embryo, such as switching from the use of maternal transcripts to the onset of zygotic transcription. In mammals little is known about the molecular mechanisms guiding this transition, largely due to the lack of information about genes expressed in the egg and early embryos. Studies of yeast mitosis have contributed much of what is known about the vertebrate cell cycle, and recent reports indicate that homologs of yeast DNA repair genes also function during mammalian gametogenesis. To examine whether this conservation can be expanded to include genes operative in oocyte meiosis, we performed a computer-based search for homologs of yeast genes that are induced during sporulation in C. elegans, Drosophila, and mammals. Results from this study suggest that yeast and higher eukaryotes share genes that coordinate the overall process of meiosis. However intriguing differences exist, reflecting the distinctive mechanisms governing the progression of meiosis in each organism. ESTs representing more than half of the mammalian homologs are present in mouse cDNA libraries that contains genes controlling the meiosis/mitosis transition. About 50% of these genes contain potential cis-elements for cytoplasmic polyadenylation in their 3'-UTR, suggesting the importance of controlled translation in the egg and zygote.

Isolation of a Schizosaccharomyces pombe rad21ts mutant that is aberrant in chromosome segregation, microtubule function, DNA repair and sensitive to hydroxyurea: possible involvement of Rad21 in ubiquitin-mediated proteolysis.

The fission yeast DNA repair gene rad21+ is essential for cell growth. To investigate the function essential for cell proliferation, we have isolated a temperature-sensitive mutant of the rad21+ gene. The mutant, rad21-K1, showed abnormal mitosis at the nonpermissive temperature. Some cells contained abnormal nuclear structures, such as condensed chromosomes with short spindles, or chromosomes stretched or unequally separated by elongating spindles. Other cells exhibited the displaced nucleus or a cut-like phenotype. Similar abnormalities were observed when the Rad21 protein was depleted from cells. We therefore concluded that Rad21 is essential for proper segregation of chromosomes. Moreover, the rad21-K1 mutant is sensitive not only to UV and gamma-ray irradiation but to thiabendazole and hydroxyurea, indicating that Rad21 plays important roles in microtubule function, DNA repair, and S phase function. The relation to the microtubule function was further confirmed by the fact that rad21+ genetically interacts with tubulin genes, nda2+ and nda3+. Finally, the growth of the rad21-K1 mutant was inhibited at the permissive temperature by introduction of another mutation in the cut9+ gene, coding for a component of the 20S cyclosome/anaphase promoting complex, which is involved in ubiquitin-mediated proteolysis. The results suggest that these diverse functions of Rad21 may be facilitated through ubiquitin-mediated proteolysis.

Expression of yeast but not human apurinic/apyrimidinic endonuclease renders Chinese hamster cells more resistant to DNA damaging agents

Abasic sites represent ubiquitous DNA lesions that arise spontaneously or are induced by DNA-damaging agents. They block DNA replication and are considered to be cytotoxic and mutagenic. The key enzymes involved in the repair of abasic sites are apurinic/apyrimidinic (AP) endonucleases which process these lesions in an error-free mechanism. To analyze the role of AP endonuclease in the protection of mammalian cells against DNA damaging agents, we have transfected both the human (APE) and the yeast (APN1) AP endonuclease in Chinese hamster cells and compared the effects of expression of these genes in stable transfectants as to survival of cells and formation of chromosomal aberrations. Although APE was markedly expressed on RNA and protein level, nuclear extracts of human APE transfectants did not show a higher AP endonuclease activity than the parental line and became not more resistant to the cell killing and clastogenic effect of methyl methanesulfonate (MMS) and hydrogen peroxide (H2O2). In contrast, cells transfected with the yeast APN1 gene expressed higher AP endonuclease activity and became clearly more resistant to the cytotoxic and chromosome breakage inducing activity of the agents. The results indicate that the excision repair capacity and correspondingly the mutagen resistance can be elevated by introducing, in mammalian cells, a yeast DNA repair gene and verify that AP sites are both cytotoxic and clastogenic lesions.

Allelism of PSO4 and PRP19 links pre-mRNA processing with recombination and error-prone DNA repair in Saccharomyces cerevisiae.

The radiation-sensitive mutant pso4-1 of Saccharomyces cerevisiae shows a pleiotropic phenotype, including sensitivity to DNA cross-linking agents, nearly blocked sporulation and reduced mutability. We have cloned the putative yeast DNA repair gene PSO4 from a genomic library by complementation of the blocked UV-induced mutagenesis and of sporulation in diploids homozygous for pso4-1. Sequence analysis revealed that gene PSO4 consists of 1512 bp located upstream of UBI4 on chromosome XII and encodes a putative protein of 56.7 kDa. PSO4 is allelic to PRP19, a gene encoding a spliceosome-associated protein, but shares no significant homology with other yeast genes. Gene disruption with a destroyed reading frame of our PSO4 clone resulted in death of haploid cells, confirming the finding that PSO4/PRP19 is an essential gene. Thus, PSO4 is the third essential DNA repair gene found in the yeast S.cerevisiae.

(CGG) trinucleotide repeat polymorphism in the 5' region of the HHR6B gene: the human homolog of the yeast DNA repair gene RAD6.

The polymerase chain reaction was used to detect polymorphisms in a (CGG) trinucleotide repeat sequence in the 5′ region of the human HHR6B DNA repair gene on chromosome 5q23-31

Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes.

Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes.

Synergistic Interactions between RAD5, RAD16 and RAD54, Three Partially Homologous Yeast DNA Repair Genes Each in a Different Repair Pathway

Considerable homology has recently been noted between the proteins encoded by the RAD5, RAD16 and RAD54 genes of Saccharomyces cerevisiae. These genes are members of the RAD6, RAD3 and RAD50 epistasis groups, respectively, which correspond to the three major DNA repair pathways in yeast. These proteins also share homology with other eucaryotic proteins, including those encoded by SNF2 and MOT1 of yeast, brahma and lodestar of Drosophila and the human ERCC6 gene. The homology shares features with known helicases, suggesting a newly identified helicase subfamily. We have constructed a series of congenic single-, double- and triple-deletion mutants involving RAD5, RAD16 and RAD54 to examine the interactions between these genes. Each deletion mutation alone has only a moderate effect on survival after exposure to UV radiation. Each pairwise-double mutant exhibits marked synergism. The triple-deletion mutant displays further synergism. These results confirm the assignment of the RAD54 gene to the RAD50 epistasis group and suggest that the RAD16 gene play a larger role in DNA repair after exposure to UV radiation than has been suggested previously. Additionally, the proteins encoded by RAD5, RAD16 and RAD54 may compete for the same substrate after damage induced by UV radiation, possibly at an early step in their respective pathways.

An ERCC5 gene with homology to yeast RAD2 is involved in group G xeroderma pigmentosum

We have isolated a human excision repair gene ERCC5 which complements the defect of the mouse UV-sensitive mutant XL216 (rodent complementation group 5). Here we report cDNA cloning of human and mouse ERCC5 genes using an exon containing an ERCC5 fragment as a probe. The ERCC5 cDNA encodes a predicted 133-kDa nuclear protein that shares some homology with the product of the yeast DNA repair gene RAD2. Transfection with mouse ERCC5 cDNA restored normal levels of UV resistance to XL216 cells. Microinjection of ERCC5 cDNA specifically restored the defect of xeroderma pigmentosum group G cells (XP-G) as measured by unscheduled DNA synthesis, and XP-G cells stably transformed with ERCC5 cDNA showed nearly normal UV resistance.

Yeast DNA-repair gene RAD14 encodes a zinc metalloprotein with affinity for ultraviolet-damaged DNA.

Xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers due to a defect in excision repair of UV light-damaged DNA. Of the seven XP complementation groups, A-G, group A represents a severe and frequent form of the disease. The Saccharomyces cerevisiae RAD14 gene is a homolog of the XP-A correcting (XPAC) gene. Like XP-A cells, rad14-null mutants are defective in the incision step of excision repair of UV-damaged DNA. We have purified RAD14 protein to homogeneity from extract of a yeast strain genetically tailored to overexpress RAD14. As determined by atomic emission spectroscopy, RAD14 contains one zinc atom. We also show in vitro that RAD14 binds zinc but does not bind other divalent metal ions. In DNA mobility-shift assays, RAD14 binds specifically to UV-damaged DNA. Removal of cyclobutane pyrimidine dimers from damaged DNA by enzymatic photoreactivation has no effect on binding, strongly suggesting that RAD14 recognizes pyrimidine(6-4)pyrimidone photoproduct sites. These findings indicate that RAD14 functions in damage recognition during excision repair.

Expression cloning of a human DNA repair gene involved in xeroderma pigmentosum group C.

Xeroderma pigmentosum (XP) is a rare human autosomal recessive disease characterized by solar sensitivity, high predisposition for developing cancers on areas exposed to sunlight, and, in some cases, neurological abnormalities. XP cells are defective in DNA repair, and complementation of this defect has been used to identify eight genetic groups (A-G and variant). We have developed a simple, highly efficient complementary DNA expression system for use in human cells. Here we use this system to isolate a cDNA clone that restores the ultraviolet sensitivity and unscheduled DNA synthesis of XP-C cells to normal levels. The XP-C complementing clone XPCC encodes a highly hydrophilic protein which is composed of a predicted 823 amino acids and shares limited homology with the product of the yeast DNA repair gene RAD4. The XPCC transcript is undetectable by northern blotting in most XP-C cell lines examined.

Specific complex formation between proteins encoded by the yeast DNA repair and recombination genes RAD1 and RAD10.

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for excision repair of ultraviolet light-damaged DNA, and they also function in a mitotic recombination pathway that is distinct from the double-strand-break recombination pathway controlled by RAD52. Here, we show that the RAD1 and RAD10 proteins are complexed with each other in vivo. Immunoprecipitation of yeast cell extracts with either anti-RAD1 antibody or anti-RAD10 antibody coprecipitated quantitative amounts of both RAD1 and RAD10 proteins. The level of coprecipitable RAD1 and RAD10 increased when both proteins were overproduced together, but not if only one of the proteins was overproduced. The RAD1/RAD10 complex is highly stable, being refractory to 1 M NaCl and to low concentrations of SDS. By hydroxylamine mutagenesis, we have identified a rad1 mutant allele whose encoded protein fails to complex with RAD10. The interaction-defective rad1 mutant resembles the rad1 or rad10 null mutant in defective DNA repair and recombination, implying that complex formation is essential for the expression of biological activities controlled by RAD1 and RAD10.

A rabbit reticulocyte ubiquitin carrier protein that supports ubiquitin-dependent proteolysis (E214k) is homologous to the yeast DNA repair gene RAD6.

The two isoforms of the 14-kDa ubiquitin carrier protein (E2(14k)) are unique among rabbit E2s in efficiently supporting ubiquitin-protein ligase (E3)-mediated ubiquitination of proteins destined for degradation. To begin determining the structural basis for this property, we have isolated a cDNA encoding the predominant reticulocyte isoform of the E2 from a rabbit skeletal muscle library. The sequence predicts a protein of 152 amino acids with a molecular weight of 17,293. Expression of the cDNA in Escherichia coli and purification of the recombinant protein revealed an E2 with high affinity for E3 and ubiquitin activating enzyme (E1). The latter high affinity interaction appears to be between the ubiquitin charged form of E1 and the uncharged form of E2 and does not result in a stable complex between these two enzymes. The predicted sequence shows regions of strong homology with other sequenced E2s, suggesting that these regions may be involved in binding to E1 and/or in ubiquitin transfer from E1, functions common to all E2s. Surprisingly, the E2(14k)) sequence is markedly more similar to Saccharomyces cerevisiae RAD6 (69% identity) than to its proposed homologs UBC4/UBC5 (38% identity). The sequence is identical to that recently reported for a human 17-kDa E2 which can complement rad6 mutants thereby identifying rabbit E2(14k) as a RAD6 homologue. The biochemical properties of this previously uncharacterized human 17-kDa E2 are now defined and its misassignment as a homologue of rabbit E2(17k) is corrected. Our findings resolve current confusion regarding relationships among E2s and define yeast RAD6, rabbit E2(14k), and the human 17-kDa E2 as a subclass of E2s which biochemically support E3-mediated conjugation and ubiquitin-dependent proteolysis and physiologically play a role in DNA repair.

Localization of two human homologs, HHR6A and HHR6B, of the yeast DNA repair gene RAD6 to chromosomes Xq24–q25 and 5q23–q31

The chromosomal localizations of two closely related human DNA repair genes, HHR6A and HHR6B, were determined by in situ hybridization with biotinylated probes. HHR6A and HHR6B (human homolog of yeast RAD6) encode ubiquitin-conjugating enzymes (E2 enzymes), likely to be involved in postreplication repair and induced mutagenesis. The HHR6B gene was assigned to human chromosome 5q23–q31, whereas the HHR6A gene was localized on the human X chromosome (Xq24–q25). This latter assignment was confirmed with an X-specific human-mouse/hamster somatic cell hybrid panel. Southern blot analysis points to an X and an autosomal localization of HHR6A and HHR6B, respectively, in the mouse. The potential involvement of these genes in human genetic disorders is discussed.

Induced accumulation of polyubiquitin gene transcripts in HeLa cells after UV-irradiation and TPA-treatment.

There are three species of ubiquitin gene transcripts in HeLa cells, termed UbA (approximately 0.7 kb), UbB (approximately 1.1 kb) and UbC (approximately 2.5 kb). In the present report, the UbC transcript was shown to accumulate up to 2.5-fold after irradiation with UV light or treatment with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The kinetic analysis indicated that the induced accumulation of UbC was rapid and transient; maximal accumulation of UbC was induced at 2.5 h after UV irradiation or 3 h after TPA treatment. Inhibition of a de novo protein synthesis by cycloheximide did not repress the induction of UbC after treatment with UV light and TPA. On the other hand, induction of UbA and UbB, in most cases, was not observed. UV-inducibility of human ubiquitin conjugating enzyme, E2(17k), was also tested. E2(17k) is a protein with high sequence similarity to the product of yeast DNA repair gene, RAD6. While the RAD6 gene has been reported to be inducible by UV light, no change in E2(17k) gene transcript was observed after UV irradiation.

A damage-responsive DNA binding protein regulates transcription of the yeast DNA repair gene PHR1.

The PHR1 gene of Saccharomyces cerevisiae encodes the DNA repair enzyme photolyase. Transcription of PHR1 increases in response to treatment of cells with 254-nm radiation and chemical agents that damage DNA. We report here the identification of a damage-responsive DNA binding protein, termed photolyase regulatory protein (PRP), and its cognate binding site, termed the PHR1 upstream repression sequence, that together regulate induction of PHR1 transcription after DNA damage. PRP activity, monitored by electrophoretic-mobility-shift assay, was detected in cells during normal growth but disappeared within 30 min after irradiation. Copper-phenanthroline footprinting of PRP-DNA complexes revealed that PRP protects a 39-base-pair region of PHR1 5' flanking sequence beginning 40 base pairs upstream from the coding sequence. A prominent feature of the foot-printed region is a 22-base-pair palindrome. Deletion of the PHR1 upstream repression sequence increased the basal level expression of PHR1 in vivo and decreased induction after exposure of cells to UV radiation or methyl methanesulfonate, whereas insertion of the PRP binding site between the CYC1 upstream activation sequence and "TATA" sequence reduced basal level expression and conferred damage responsiveness upon a reporter gene. Thus these observations establish that PRP is a damage-responsive repressor of PHR1 transcription.