Graduation date: 2001

DNA is the central storage molecule for genetic information in the cell. Therefore, the DNA must be protected from damage that will otherwise be passed on to future generations as deleterious mutations. Although many different pathways have evolved for repairing different classes of damage there are certain features that are common to all repair pathways. Generically, for DNA damage to be repaired it must first be recognized, then excised and replaced with undamaged DNA. DNA damage recognition is highly varied since specific interactions are required between the protein and the damaged DNA. DNA damage repair, paradoxically, requires the action of highly processive nucleases. The nucleases may digest hundreds if not thousands of nucleotides, sometimes for the repair of a single mutant nucleotide. We have chosen to focus on Exonuclease VII (ExoVII), one of the processive nucleases that have been implicated in the process of Mismatch Repair (MMR). ExoVII is a hetero-pentameric enzyme composed of one large subunit (XseA) and four small subunits (XseB). It has been previously characterized as a processive, single-strand specific nuclease able to digest DNA in either the 5'->3' or 3'->5' direction by a metalindependent mechanism. Early studies have shown that although ExoVII is a hydrolytic nuclease it was completely active in the presence of large amounts of EDTA and was strongly stimulated by phosphate. This feature is unusual because hydrolytic DNA nucleases typically function by a mechanism that requires coordination of a divalent cation. To further our understanding of the mechanism ExoVII we have identified and characterized the ExoVII homolog from Thermotoga maritima (T. maritima, Tm), a hyperthermophilic bacterium. The genes responsible for Tm ExoVII (TM1768 and TM1769) were cloned into an overexpression construct and the resulting proteins were overexpressed, co-purified and characterized. Consistent with previous studies, we found that Tm ExoVII is a processive, single-strand specific nuclease. Surprisingly, unlike Ec ExoVII, the T. maritima homolog was found to have an absolute requirement for the divalent cation magnesium and was strongly inhibited by the presence of either phosphate or sulfate in the reaction buffer. Using multiple sequence alignments of the large subunit we have identified a conserved core present within the C-terminal ExoVII_Large domain. This conserved core, RGGGx27GHx2Dx4Dx9P, although unique among nucleases, is reminiscent of a metal-coordinating hydrolytic active site. We have tested this putative active site using site-directed mutagenesis to create the TmD235A/TmD240A double mutant. This mutant protein was purified and the resulting protein was found to be inactive. We propose that this conserved core represents the metal-coordinating active site of all ExoVII homologs and that the group of E. coli-like homologs are unique in their EDTA resistance and anion (phosphate and sulfate) stimulation. Since ExoVII is a bi-directional nuclease (both 5'->3' and 3'->5' activity), and MMR is a bi-directional process, our model was that ExoVII was the primary nuclease associated with MMR. To test this model and determine if, in fact, a minimal conserved MMR pathway can be defined, we performed an analysis of the genomic occurrence profiles for the genes involved in MMR. To do this we have developed a bioinformatic application, Magma, which assists in the creation of a searchable relational database. Using Magma we have found that MutH, the enzyme responsible for generating a nick that directs MMR to excise the newly synthesized DNA strand including a DNA mispair, is only present in E. coli and a subset of gamma-proteobacteria, suggesting that MutH is not a core component of MMR. Instead, most organisms employ a nicking activity found in the MutL subunit. We also show that, although four nucleases have been implicated as having "redundant" roles in bacterial mismatch repair, RecJ is the primary nuclease responsible for degrading the mutated DNA strand and that 5'->3' single-strand exonuclease activity is a core MMR component. From this analysis, it appears that prokaryotic mismatch repair is more similar to eukaryotic mismatch repair than was previously thought, from the genetic and biochemical work done in E. coli. We offer a model for a universal minimal MMR system.

DNA crosslinking agents exhibit a variety of DNA lesions, such as monoadducts, DNA-DNA interstrand or intrastrand crosslinks or DNA-protein crosslinks. Agents that produce interstrand crosslinks (ICLs) exist naturally and are widely used in chemotherapy. Therefore, it is important to understand how the lesions induced by these agents are repaired. In bacteria, the repair is mainly dependent on nucleotide excision repair (NER) together with homologous recombination (HR) or translesion synthesis (TLS). In human cells, it is not clear how these lesions are repaired, and it is believed to be a more complicated process in which NER does not play as important a role as in prokaryotes. Here, we investigated the repair mechanisms mainly after treatment with psoralen but also with acetaldehyde, cisplatin and mitomycin C in some studies. As expected from studies on plasmids and in bacteria, we used new techniques to confirm that various ICL-inducing agents block replication fork elongation in mammalian cells. We also found that the replication fork was unable to bypass these lesions. We confirmed that ERCC1/XPF and the HR proteins BRCA2 and XRCC2/3 are vital for protection against ICL treatments. These proteins were also found to be equally important for the repair of monoadducts. To better understand ICL repair in mammalian cells, we developed a method to study the induction and unhooking of ICL in human fibroblasts. We found that ICLs were repaired and that 50% of the induced ICLs were unhooked within 3 hours following exposure. Additionally, we determined that XPA, but not XPE, is involved in ICL unhooking, although not affecting lethality. A step in ICL repair is the formation of double-strand breaks (DSBs), and we identified a replication-dependent formation of DSBs following ICL treatment. Furthermore, ERCC1/XPF was not necessary for DSB formation. The repair of these DSBs was performed by HR and involved ERCC1/XPF. Additionally, we were able to quantify the ICL unhooking in human fibroblasts and found that they can unhook ~2500 ICL/h. We also determined that a dose of approximately 400 ICL/cell is lethal to 50% of the cells, indicating that ICL unhooking is not the most critical step during the repair process. / DNA-skadande ämnen är vanligt i cancerbehandling, då snabbt växande celler, såsom cancerceller är betydligt känsligare än normala celler för DNA skador. En grupp av ämnen som vanligen används i cancerbehandling är korsbindare av DNA. Dessa ämnen kommer reagera två gånger med DNA och skapa två bindningar mitt emot varandra. DNA strängen, som består av två delar, måste kunna separeras och kopieras (replikation) på ett tillförlitligt sätt för att cellerna ska kunna dela sig och bli flera. DNA strängen måste också kunna dela sig och bli avläst rätt för att nya proteiner ska kunna bildas (transkription). När korsbindarna har bundit till DNA strängarna, hindrar detta deras separation och därigenom förhindras även avläsningen och kopieringen.  För att göra undersökningarna av DNA korsbindande ämnen ännu lite svårare, så ger korsbindare flera olika typer av skador. Dels kan det bli flera olika typer av korsbindningar, både mellan två DNA-strängar (ICL) vilket är den farligaste och mest svårreparerade typen, men det kan också ske inom samma DNA-sträng (intrastrand crosslink) eller mellan en DNA-sträng och ett protein (DNA-protein crosslink). Korsbindare kan även bilda enbindningsskador (monoaddukt), vilket innebär den bara binder en gång till DNA. För att cellen ska kunna överleva, så måste den reparera skadorna och ta bort korsbindningen eller monoaddukten. Hur detta sker i människor är inte helt klarlagt men det verkar som det sker i flera steg. Till att börja med klipps DNA sönder i ena strängen på båda sidorna om korsbindningen, detta gör att den kvarvarande delen av korsbindningen kan böjas bort. Därefter kommer cellen att skapa nytt DNA för att fylla mellanrummet som bildats. Cellen använder sig av den andra DNA strängen som mall för att sätta in rätt DNA baser, men i fallet med korsbindande ämnen så är även den strängen skadad och därför finns det en stor risk för att fel DNA baser sätts in och då uppstår mutationer. Nästa steg är att klippa den kvarvarande delen av korsbindningen, även denna gång skapas ett mellanrum som måste fyllas med nya baser. Den första artikeln i avhandlingen handlar om att försöka reda ut om det är ICLen eller monoaddukten som är orsak till olika effekter som påträffas efter behandling med korsbindande ämnen. Det vi fann var att även om det bara var från ICLs som vi kunde mäta en effekt på replikationen, så fick vi nästan lika stark effekt från monoaddukterna, som från ICL, för en av de vanligast använda markörerna (kännetecknen) för båda DNA strängarna var brutna på samma ställe (dubbelstränsbrott). Detta berodde dock inte på att även monoaddukterna skapade dubbelsträngsbrott, utan på att markören vi använde var ospecifik. Vi fann även att även om ICLs har mycket större effekt än monoaddukten på cellens överlevnad m.m., så kan man inte bortse ifrån effekten av monoaddukten och att den troligen har en betydande roll för de korsbindande ämnen som endast ger en liten del ICLs. I artikel två har vi utvecklat en ny metod, som gör det möjligt att mäta hur många ICLs som bildas vid en viss dos av de korsbindande ämnen vi undersöker. Vi kan även mäta hur fort ICLerna kan repareras i mänskliga celler med hjälp av metoden. Tack vare en kombination av våra mätningar och med hjälp av datorsimuleringar, kunde vi räkna ut hur många ICLs som bildades per dos för tre vanliga korsbindare. Vi kunde även visa att 50 % av ICLen har påbörjat reparationen och kommit så långt att de var bortklippta från ena stängen inom 3 timmar efter behandlingen. I artikel tre undersöker vi vilka proteiner som är inblandade i den tidiga delen av ICL reparationen, alltså fram till och med att celler klipper ut korsbindningen på båda sidorna om skadan i ena strängen. Här visar vi att celler som är defekta i reparationsprotein kallat XPA, har en betydligt långsammare borttagning av ICLer än vad båda normala celler och celler defekta i reparationsprotein XPE har. Vi visar även att detta inte påverkar cellens replikationshastighet, eller har någon effekt på cellens överlevnad. Den fjärde artikeln handlar om acetaldehyd, som bildas när alkohol förbränns i kroppen. Acetaldehyd har föreslagits bilda ICL och därför undersökte vi vilka effekter den har på cellerna. Vi visar i den här artikeln att det krävs nysyntes av DNA för att acetaldehyd ska leda till dubbelsträngsbrott. Celler kan reparera dessa dubbelsträngsbrott med hjälp av reparationssystem, som kallas homolog rekombination, men att reparationen ibland blir felaktig. I den femte och sista artikeln i avhandligen undersöker vi ett av de vanligast föreslagna proteinen för att sköta klippningen av DNA (ERCC1/XPF) och hur den är inblandad i reparationen av korsbindningar. Vi kan här visa att även det krosbindande ämnet mitomycin C bromsar replikationshastigheter och att ERCC1/XPF är nödvändigt för att kunna fullfölja homolog rekombination av ICLs. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Submitted. Paper 2: Manuscript. Paper 3: Manuscript. Paper 4: Submitted.</p>

<p>Both prokaryotic and eukaryotic cells are equipped with DNA repair mechanisms to protect the integrity of their genome in case of DNA damage. In the eukaryotic organism <i>Saccharomyces cerevisiae</i>, MMS2 encodes a ubiquitin-conjugating enzyme variant protein belonging to the RAD6 repair pathway; MMS2 functions in error-free postreplication repair (PRR), a subpathway parallel to REV3 mutagenesis. A mutation in MMS2 does not result in extreme sensitivity to DNA damaging agents; however, deletion of both subpathways of PRR results in a synergistic phenotype. By taking advantage of the synergism between error-free PRR and mutagenesis pathway mutations, a conditional synthetic lethal screen was used to identify novel genes genetically involved in PRR. A synthetic lethal screen was modified to use extremely low doses of MMS that would not affect the growth of single mutants, but would effectively kill the double mutants. Fifteen potential mutants were characterized, of which twelve were identified as known error-prone PRR genes. Characterization of mutations in strains SLM-9 and SLM-11, that are conditionally synthetically lethal with mms2Ä, revealed functions for both checkpoints and mating-type heterozygosity in regulating PRR. Cell cycle checkpoints monitor the integrity of the genome and ensure that cell cycle progression is deferred until chromosome damage is repaired. The checkpoint genes genetically interact with both the error-free and error-prone branches of PRR, potentially for delaying cell cycle progression to allow time for DNA repair, and for signaling the stage of the cell cycle and thus DNA content. Other potential monitors for DNA content are the a1 and á2 proteins encoded by the mating type genes MATa and MATá, respectively. Diploid cells heterozygous for mating type (a/á) show an increased resistance to UV damage and are more recombination-proficient than haploid cells. Haploid PRR mutants expressing both mating type genes show an increased resistance to DNA-damaging agents. This phenomenon is specific to PRR: it was not seen in excision repair-deficient and recombination-deficient mutants tested. The rescuing effect seen in PRR mutants heterozygous for mating type is likely the result of channeling lesions into a recombination repair pathway and away from the non-operational PRR pathway. Both checkpoint and mating type genes play a role in regulating PRR. Almost certainly these genes are required to monitor the cell cycle stage and DNA content to determine the best mechanism to repair the damaged DNA thus preventing genomic instability.</p>

<p>Both prokaryotic and eukaryotic cells are equipped with DNA repair mechanisms to protect the integrity of their genome in case of DNA damage. In the eukaryotic organism <i>Saccharomyces cerevisiae</i>, MMS2 encodes a ubiquitin-conjugating enzyme variant protein belonging to the RAD6 repair pathway; MMS2 functions in error-free postreplication repair (PRR), a subpathway parallel to REV3 mutagenesis. A mutation in MMS2 does not result in extreme sensitivity to DNA damaging agents; however, deletion of both subpathways of PRR results in a synergistic phenotype. By taking advantage of the synergism between error-free PRR and mutagenesis pathway mutations, a conditional synthetic lethal screen was used to identify novel genes genetically involved in PRR. A synthetic lethal screen was modified to use extremely low doses of MMS that would not affect the growth of single mutants, but would effectively kill the double mutants. Fifteen potential mutants were characterized, of which twelve were identified as known error-prone PRR genes. Characterization of mutations in strains SLM-9 and SLM-11, that are conditionally synthetically lethal with mms2Ä, revealed functions for both checkpoints and mating-type heterozygosity in regulating PRR. Cell cycle checkpoints monitor the integrity of the genome and ensure that cell cycle progression is deferred until chromosome damage is repaired. The checkpoint genes genetically interact with both the error-free and error-prone branches of PRR, potentially for delaying cell cycle progression to allow time for DNA repair, and for signaling the stage of the cell cycle and thus DNA content. Other potential monitors for DNA content are the a1 and á2 proteins encoded by the mating type genes MATa and MATá, respectively. Diploid cells heterozygous for mating type (a/á) show an increased resistance to UV damage and are more recombination-proficient than haploid cells. Haploid PRR mutants expressing both mating type genes show an increased resistance to DNA-damaging agents. This phenomenon is specific to PRR: it was not seen in excision repair-deficient and recombination-deficient mutants tested. The rescuing effect seen in PRR mutants heterozygous for mating type is likely the result of channeling lesions into a recombination repair pathway and away from the non-operational PRR pathway. Both checkpoint and mating type genes play a role in regulating PRR. Almost certainly these genes are required to monitor the cell cycle stage and DNA content to determine the best mechanism to repair the damaged DNA thus preventing genomic instability.</p>

Abstract Deinococcus radiodurans is highly resistant to radiation when it grown in tryptone-glucose-yeast extraxt (TGY) medium. It oxidized glucose slowly mainly by the pentose phosphate pathway (PPP) and showed little glycolytic Embden-Meyerhof pathway (EMP) activity. The addition of 10 &#x00B5;M Mn(II) into the stationary phase cultures, could induced new round of cell division (Mn-CD effect) and the EMP activity. Glucose metabolized by Mn-CD cells at a EMP:PPP=6:1 ratio. In analyzing the metabolites for DNA repair, we found that after the addition of Mn(II) , the concentrations of PPP metabolites such as insione monophosphate (IMP)¡Buridine monophosphate (UMP) and NAD (nicotine adenine dinucleotide) were greatly reduced. This event is also occurred when replacing the glucose by fructose, sodium acetate, or removing glucose from the TGY culture medium. Besides, we also found that the TGY and TFY grown cells contained more PPP metabolites than those of TAY and TY cells. This finding suggested that glucose and fructose were metabolized by the PPP pathway in D. radiodurans. Finally, the concentrations of IMP¡BUMP and NAD in the cells were greatly decreased after UV irradiation. This indicated that these metabolites were probably employed to repair the DNA damage causing by UV irradiation.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 158-166). Available also in a digital version from Dissertation Abstracts.

published_or_final_version / Biological Sciences / Master / Master of Philosophy

Friedreich ataxia (FRDA) is the most common inherited ataxia disorder, caused by a GAA repeat expansion mutation within the first intron of the FXN gene. The subsequent deficiency of frataxin protein leads to neurological disability, increased risk of diabetes mellitus, cardiomyopathy and premature death. The exact FRDA disease mechanism is not yet clear, despite some understanding of epigenetic, transcriptional and DNA repair system effects that lead to frataxin reduction. Previous studies have shown that mismatch repair (MMR) genes can affect other trinucleotide repeat disorders by destabilisation of the repeats. Furthermore, it has been proposed that frataxin deficiency might lead to cell malignancy by an as yet undefined mode of action. Therefore, the principle aim of this thesis was to use human and genetically altered mouse cells and tissues to understand the effects of MMR proteins on GAA repeat instability and FXN transcription, and also to identify potential changes in MMR transcription that might cause malignancy in FXN-defective human cells. Firstly, by using FXN and MMR genetically altered mice, MMR proteins were shown to be involved in both intergenerational and somatic GAA repeat instability, although their effects in the two systems were different. Thus, Msh2 or Msh3 were both found to protect against intergenerational transmission of GAA contractions, while loss of Msh2 or Msh3 reduced somatic GAA repeat expansions and increased levels of FXN transcription in brain and cerebellum tissues. Loss of Msh6 induced both intergenerational GAA repeat expansions and contractions, while the frequency of somatic GAA repeat expansions was reduced. Curiously, the level of FXN transcription was also reduced in Msh6-deficient brain and cerebellum tissues. On the other hand, Pms2 was found to protect against both intergenerational and somatic GAA repeat expansions, with loss of Pms2 causing increased GAA repeat expansions and decreased levels of FXN transcription in brain and cerebellum tissues. Finally, loss of Mlh1 led to a reduced frequency of both intergenerational and somatic GAA repeat expansions, but the level of FXN transcription was also reduced in brain and cerebellum tissues. Furthermore, upregulation of MMR mRNA expression was detected in human FRDA fibroblast cells, but downregulation was seen in FRDA cerebellum tissues, suggesting tissue-dependent control of FXN and MMR expression. In summary, these studies indicate that the MMR system can affect GAA repeat expansion instability and FXN transcription through different mechanisms of action. Furthermore, frataxin deficiency can also affect the levels of MMR mRNA expression in a tissue-dependent manner. These findings will assist future investigations aimed at identifying novel FRDA therapies.

When the DNA of Escherichia coli is damaged a set of events termed "SOS functions" occur to aid cellular survival. The recA and lexA proteins are involved in the regulation of these functions. To determine the role of the lexA protein, amber mutations, designated spr-55(amber), were isolated in the lexA-3 gene. The lack of the lexA-3 gene product abolished sensitivity to ultraviolet light and resulted in the constitutive synthesis of recA protein. Introduction of amber suppressor mutations restored the original lexA-3 phenotype. It was concluded that spr mutations inactivate lexA protein resulting in the constitutive expression of the SOS functions. These data provide evidence that the lexA protein is the repressor for the recA gene. The repair of phage lambda (λ⁺) by ultraviolet light was determined in the strains carrying alleles of the spr, uvrA, and recA genes. The survival of the phage was more in the spr-51 uvrA⁺ strain as compared to wild type. These results were not dependent on the recA genotype. Introduction of the uvrA-6 mutation into the spr-51 uvrA⁺ recA⁺ strains resulted in the same relative decrease of phage survival. These results suggest that lexA protein is involved in the regulation of uvrA-dependent excision repair and that inactivation of lexA leads to the constitutive expression of excision repair. New mutant forms of lexaA protein were isolated. The lexA⁺, lexA-3, lexA-10, and lexA-27 proteins displayed identical mobilities in the Weber and Osborn gel system. The lexA-10 and lexA-27 genes showed different phenotypes and encoded proteins of different mobilities in the Laemmli gel system. It was concluded that the differences in mobilities observed in the Laemmli gel system are due to alterations in charge or amino acid, not in size; furthermore; the molecular weight of lexA⁺ protein was determined to be 24 kilodaltons.