Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Thesis (Ph.D. in Biochemistry) -- University of Colorado, 2006. / Typescript. Includes bibliographical references (leaves 153-171). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

DNA repair proteins in mycobacteria and their physiological importance Mycobacterium tuberculosis, the causative organism of tuberculosis, resides in the host macrophages where it is subjected to a plethora of stresses like reactive oxygen species (ROS) and reactive nitrogen intermediate(RNI) which are generated as a part of the host’s primary immune response. These stresses can damage the cellular components of the pathogen including DNA and its precursors. Two common damages to DNA and its precursors caused by ROS and RNI are oxidation of guanine to 8-oxo-guanine and deamination of cytosine to uracil. Mycobacteria, which are known to have high G+C content, must be more susceptible to such damages, and are thus equipped with the mechanisms to counteract these damages. One such mechanism is to hydrolyse the 8-oxo-dGTP into 8-oxo-dGMP to avoid its incorporation in the DNA during its synthesis. This job is done by a protein called MutT.In mycobacteria four homologs of MutT, namely MutT1, MutT2, MutT3 and MutT4 have been annotated. The second mechanism deals with the repair of uracil residues present in DNA which are generated by deamination of cytosines or incorporation of dUTP during DNA synthesis. This is taken care of by a protein called uracil DNA glycosylase (UDG) which excises uracil by cleaving the N-C1’ glycosidic bond between the uracil and the deoxyribose sugar in a DNA repair pathway called the base excision repair (BER). In this study, the biochemical properties and physiological role of mycobacterial MutT2 and, MSMEG_0265 (MsmUdgX), a novel uracil DNA glycosylase superfamily protein, have been investigated. I.Biochemical characterization of MutT2 from mycobacteria and its antimutator role. Nucleotide pool, the substrate for DNA synthesis is one of the targets of ROS which is generated in the macrophage upon Mycobacterium tuberculosis infection. Thus, the pathogen is at increased risk of accumulating oxidised guanine nucleotides such as 8-oxo-dGTP and 8-oxo-GTP. By hydrolysing the damaged guanine nucleotides before their incorporation into nucleic acids, MutT proteins play a critical role inallowing organisms to avoid their deleterious effects. Mycobacteria possess several MutT proteins. Here, we have purified recombinantM. tuberculosisMutT2 (MtuMutT2) andM. smegmatisMutT2 (MsmMutT2) proteins as representative of slow and fast growing mycobacteria, for the purpose of biochemical characterization. UnlikeEscherichia coliMutT, which hydrolyzes 8-oxo-dGTP and 8-oxo-GTP, the mycobacterial proteins hydrolyze not only 8-oxo-dGTP and 8-oxo-GTP but also dCTP and 5-methyl-dCTP. Determination of kinetic parameters (KmandVmax) revealed thatwhileMtuMutT2 hydrolyzes dCTP nearly four times better than it does 8-oxo-dGTP,MsmMutT2 hydrolyzes them almost equally well. Also,MsmMutT2 is about 14 times more efficient thanMtuMutT2 in its catalytic activity of hydrolyzing 8-oxo-dGTP.Consistent with these observations,MsmMutT2 but notMtuMutT2 rescuesE. colifor MutT deficiency by decreasing both themutation frequency and A to C mutations (a hallmark of MutT deficiency). We discuss these findings in the context of the physiological significance of MutT proteins. II.Understanding the biochemical properties of MSMEG_0265 (MsmUdgX), a novel uracil DNA glycosylase superfamily protein Uracil DNA glycosylases (UDGs) are base excision repair enzymes which excise uracil from DNA by cleaving the N-glycosidic bond. UDGs are classified into 6 different families based on their two functional motifs, i. e.,motif A and motif B. In mycobacteria, there are two uracil DNA glycosylases, Ung and UdgB which belong to Family 1 and Family 5, respectively. In this study, based on the presence of the two functional motifs, we have discovered yet another uracil DNA glycosylase in M. smegmatis, which we have called MsmUdgX.The motif A and motif B of this protein indicate that it does not belong to any of the UDG families already classified but has highest similarity with Family 4 UDGs. Homologs of this protein are also present in several other organisms like M. avium, Streptomyces ceolicolor, Rhodococcus etc., but absent in M. tuberculosis, archaea and eukaryotes. Activity assays of this protein show that unlike other UDGs, MsmUdgX does not excise uracil, but forms a tight complex with uracil containing single stranded (ss) and double stranded (ds) DNAs, as observed by a shifted band in 8M urea-PAGE as well as SDS-PAGE. It also does not recognize other modified nucleotides that we investigated, in DNA. The protein binds to uracil-DNA in a wide range of pH and the minimum substrate required for its binding is pNUNN. Like Family 4 UDG, the protein has Fe-S cluster but it is not as thermostable as the Family 4 UDGs. Addition of different metal ions does not affect its binding property, and even the presence of M. smegmatis cell free extract does not diminish its binding activity. Since this protein binds specifically to uracil in DNA, an application of the protein for detection of uracil in the genomic DNA is proposed. III. Elucidation of the role of KRRIH loop in MsmUdgX by mutational analysis MsmUdgX is a novel uracil DNA glycosylase superfamily protein which has the highest homology to Family 4 UDGs. However, alignment of MsmUdgX amino acid sequence with that of Family 4 UDGs shows that there is an extra stretch of amino acids which is unique to this group of proteins. This stretch, defined by AGGKRRIH is absent in all Family 4 UDGs and the region KRRIH of the strtch is quite conserved amongst all UdgX proteins. Homology modelling of MsmUdgX, using a Family 4 UDG (TthUdgA) shows that this extra stretch of amino acids forms an outloop near the enzyme active site. Another unique difference between MsmUdgX and Family 4 UDGs is in the motif A where MsmUdgX has GEQPG and the Family 4 UDGs haveGE(A/G)PG. Our work on MsmUdgX has shown that, unlike other UDGs, this protein does not excise uracils, but forms a tight complex with the uracil containing DNA. This unique tight uracil binding property as well as KRRIH amino acid stretch has not been observed for any uracil DNA glycosylase superfamily proteins. So, to gain insight into the role of KRRIH and glutamine (Q) of motif A in MsmUdgX family of proteins, site directed mutagenesis was done in this region and we observed that mutation of His109 of the KRRIH loop to serine (S) leads to a gain of uracil excision activity, whereas changing the R107 to S, ‘RRIH’ to ‘SSAS’ or deleting the loop altogether leads to loss of its complex formation activity. Further, mutation of H109 to other amino acids like G, Q and A also shows uracil excision activity. Mutation of the glutamine in the motif A to alanine so that it is exactly similar to that of Family 4 UDGs, does not affect its uracil binding activity. This observation indicates that the KRRIH loop has an important role in the tight binding and/or uracil excision activity of MsmUdgX. Crystal structure of MsmUdgX in complex with uracil-DNA oligo and MsmUdgX H109S mutants are being studied.IV. Physiological importance of MsmUdgX in M. smegmatis MsmUdgX is a uracil DNA glycosylase superfamily protein which binds tightly to uracil (in DNA) without excising it. To elucidate its role in M. smegmatis, knockout of udgX was generated. Growth comparison of the wild type and the ΔudgX strains does not show any growth differences under the conditions tested. However, overexpression of MsmUdgX in recA deficient strains of E. coli as well as M. smegmatis leads to their retarded growth. Retarded grown is also observed in strains deficient in other DNA repair proteins that work in conjunction with RecA. These observations indicate that repair/release of MsmUdgX-uracil DNA complex might be a RecA dependent process.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, the 5-year survival rate being less than 5%. At the time of diagnosis, 90% of PDACs extend beyond the pancreas and distant metastases are often present. Due to aggressive growth, local expansion and early appearance of metastasis, primary PDAC tumours are local enough for curative surgical resection in only 10–20% of the cases. Adjuvant chemotherapy is indicated in these curative-treated cases, with slight improvement in survival. PDAC is considered to represent a heterogeneous group of biologically and prognostically different malignancies. Characterization of these subgroups is essential and there is an urgent need for more accurate biomarkers and targeted treatments in PDAC. In the current work, we immunohistochemically investigated the expression levels and prognostic values of oxidative stress markers (8-OHdG, Keap1, Prx I, II, III, V and VI), epigenetic histone modifiers (KDM4A, KDM4B, KDM4D and SIRT1–4), and cell-cycle regulators (p16, Rb, CDK4) and DNA-repair enzymes (FEN1 and MGMT) in the cohort of surgically treated PDAC patients. We found that Keap1 expression was associated with better pancreatic cancer-specific survival. Expression of antioxidative peroxiredoxins I, III, V and VI was also connected with a more favourable tumour characteristics and Prx I and VI showed prognostic value. When considering the biology of PDAC, we noticed that pivotal epigenetic regulation also occurred in exocrine pancreatic tissue adjacent to resection margins. Overexpression of the cell-cycle regulator CDK4 and the DNA-repair enzyme FEN1 in the whole population, and elevated expression level of MGMT in the most high-risk patients were connected with worse prognosis. The results of the study can be utilized in the future when individualized therapies are being designed for PDAC patients. Due to occurrence of the epigenetic regulation also in exocrine pancreatic tissue adjacent to resection margins, it could be evaluated in future for routine diagnostics and treatment optimization. The potential role of MGMT in the development of PDAC chemoresistance should be studied in the future. / Tiivistelmä Haiman duktaalinen adenokarsinooma (PDAC) on yksi aggressiivisimmista syöpäsairauksista. Viiden vuoden elossaoloennuste on vain lähellä 5 prosenttia. Diagnoosihetkellä 90% haiman adenokarsinoomista yltää haiman ulkopuolelle ja usein kasvain on jo lähettänyt etäpesäkkeitä. Kasvutaipumuksen sekä metastasoinnin takia kuratiivinen kirurginen hoito on mahdollista vain 10–20% tapauksista. Liitännäissolunsalpaajahoito on aiheellista näissä kuratiivistavoitteisesti hoidetuissa tapauksissa. Kuitenkin vaikutus kokonaiselossaoloaikaan on melko vähäinen. Uusimman tutkimustiedon valossa PDAC:aa pidetäänkin heterogeenisenä ryhmänä biologisesti ja ennusteellisesti erilaisia tautiryhmiä. Näiden tautiryhmien tunteminen ja tunnistaminen riittävän tarkkojen merkkiaineiden avulla olisi ensiarvoisen tärkeää, jotta hoitoja voitaisiin kohdentaa niistä hyötyville potilaille. Väitöskirjatutkimuksessa selvitimme immunohistokemiallisin menetelmin oksidatiivisen stressin merkkiaineiden (8-OHdG, Keap1, Prx I, II, III, V ja VI), epigeneettisten histonimodifikaattorien (KDM4A, KDM4B, KDM4D ja SIRT1–4) sekä solusyklin säätelijöiden (p16, Rb, CDK4) ja DNA-korjausentsyymien (FEN1 ja MGMT) ilmentymistä ja ennusteellista arvoa kirurgisesti hoidetuilla PDAC-potilailla. Tutkimuksessamme totesimme, että kasvainkudoksen Keap1-ilmentymä yhdistyi parempiennusteiseen taudinkuvaan. Antioksidatiivisten peroksiredoksiinien I, III, V ja VI ilmentyminen yhdistyi niin ikään suotuisampaan kasvaimen fenotyyppiin ja Prx I ja VI osoittivat ennusteellista arvoa. Havaitsimme lisäksi, että PDAC:n biologiaan keskeistesti vaikuttavaa epigeneettistä säätelyä tapahtuu myös malignin haimakudoksen viereisessä eksokriinisessä haimakudoksessa. Solusyklin säätelijä CDK4:n ja DNA-korjausentsyymi FEN1:n voimakas ilmentyminen koko tutkimuspopulaatiossa sekä kohonnut MGMT:n ilmentyminen korkeimman riskin potilailla yhdistyivät huonompaan taudin ennusteeseen. Väitöskirjatyön tutkimustuloksia voidaan tulevaisuudessa hyödyntää, kun tutkitaan yksilöllisiä hoitomuotoja PDAC-potilailla. Koska epigeneettistä säätelyä tapahtuu myös syövän viereisessä eksokriinisessa haimakudoksessa, voidaan tulevaisuudessa tämän kudoksen arviointia mahdollisesti käyttää rutiinisti diagnostiikassa sekä hoidon optimoinnissa. MGMT:n mahdollinen rooli PDAC:n kemoresistenssin kehittymisessä tulisi tulevaisuudessa selvittää.

The DNA damage response (DDR) pathway is a complicated network of interacting proteins that function to sense and remove DNA damage. Upon exposure to DNA damage, a signaling cascade is generated. The damage is either removed, restoring the original genetic sequence, or apoptosis is activated. In the absence of DDR, cells are unable to effectively process DNA damage. Unprocessed DNA damage can lead to chromosomal changes, gene mutations, and malignant transformation. Thus, the proteins involved in DDR are critical for maintaining genomic stability. One essential DDR protein is the BRCA1 Associated C-terminal Helicase, BACH1. BACH1 was initially identified through its direct association with the BRCT domain of the Breast Cancer Associated Gene, BRCA1. Similar to BRCA1, germline mutations in BACH1were identified in patients with early onset breast cancer. Interestingly, the disease-associated mutations in BACH1 were shown to have altered helicase activity in vitro, providing a direct link between BACH1 helicase activity and disease development. The correlation between BACH1 and cancer predisposition was further confirmed by the identification of BACH1 as the cancer syndrome Fanconi anemia (FA) gene product, FANCJ. Similar to other FA proteins, suppression of FANCJ leads to decreased homologous recombination, enhanced sensitivity to DNA interstrand crosslinking (ICL) agents, and chromosomal instability. In an effort to further understand the function of FANCJ in DDR, FANCJ was shown to directly associate with the mismatch repair (MMR) protein MLH1. This interaction is facilitated by lysines 141 and 142 within the helicase domain of FANCJ. Importantly, the FANCJ/MLH1 interaction is critical for ICL repair. Furthermore, in an attempt to dissect the binding site of FANCJ on MLH1, we discovered an HNPCC associated MLH1 mutation (L607H) that has intact mismatch repair, but lacks FANCJ interaction. In contrast to the MLH1 interaction, the FANCJ/BRCA1 interaction was not required for correcting the cellular defects in FANCJ null cells. Thus, in an effort to understand the functional significance of the FANCJ/BRCA1 interaction, we discovered that FANCJ promotes Pol η dependent translesion synthesis (TLS) bypass when uncoupled from BRCA1. In this thesis, we provide evidence suggesting that FANCJ and MLH1 are functionally linked and that the interaction of these proteins is critical for repair choice.