The most critical biochemistry in an organism supports the central dogma of molecular biology: transcription of DNA to RNA and translation of RNA to peptide sequence. Proteins are then responsible for catalyzing, regulating and ensuring the fidelity of transcription and translation. At the heart of these processes lie selective biomolecular interactions and specific dynamics that are necessary for complex formation and catalytic activity. Through advanced biophysical and computational methods, it has become possible to probe these macromolecular dynamics and interactions at the molecular and atomic levels to tease out their underlying physical bases. To the end of a more thorough understanding of these physical bases, we have performed studies to probe the motions and interactions intrinsic to the function of biomolecular complexes: modeling the dual-base flipping strategy of alkylpurine glycosylase D, dynamically tracing evolution and epistasis in the 3-ketosteroid family of nuclear receptors, discovering the allosteric and conformational aspects of transcription regulation in liver receptor homologue 1, leveraging specific contacts in tyrosyl-DNA phosphodiesterase 2 for the development of novel inhibitor scaffolds, and detailing the experimentally observed connection between solvation and sequence-specific binding affinity in PU.1-DNA complexes at the atomic level. While each study seeks to solve system-specific problems, the collection outlines a general and broadly applicable description of the biophysical motivations of biochemical processes.

DNA in eukaryotes is packaged into nucleosomes, which present steric impediments to many of the factors and enzymes that act on DNA, including DNA repair enzymes. Within the nucleosome, DNA remains vulnerable to oxidative damage that can result from normal cellular metabolism, ionizing radiation, and various chemical agents. Oxidatively damaged DNA is repaired in a stepwise fashion via the base excision repair (BER) pathway. Other DNA repair pathways, including Nucleotide Excision Repair (NER), Mismatch Repair (MMR), Homologous Recombination (HR), and Non-homologous End-Joining (NHEJ) are all thought to require nucleosome remodeling or disruption. In contrast, it was reported that the first step of BER does not require or induce nucleosome disruption. For example, the human DNA glycosylase hNTH1 (human Endonuclease III) was discovered to excise thymine glycol lesions from nucleosomes without nucleosome disruption, and could excise optimally oriented lesions with an efficiency approaching that seen for naked DNA (Prasad, Wallace, and Pederson 2007). To determine if the properties of hNTH1 are shared by other human DNA glycosylases, we compared hNTH1 with NEIL1, a human DNA glycoylase that also excises thymine glycol from DNA, with respect to their activities on nucleosome substrates. We found that the cellular concentrations and apparent kcat/KM ratios for hNTH1 and NEIL1 are similar. However, NEIL1 and hNTH1 differ in that NEIL1 binds undamaged DNA far more avidly than hNTH1. After adjustment for non-specific DNA binding, hNTH1 and NEIL1 proved to have similar intrinsic activities towards nucleosome substrates. We next wanted to examine the effects of nucleosomes on enzymes that catalyze the remaining steps in BER. We therefore assembled the entire four-step BER reaction with model, lesion-containing nucleosomes. The rates of substrate processing during the first three steps in BER, catalyzed by a DNA glycosylase, AP endonuclease, and DNA Polymerase Pol), varied with the helical orientation of the substrate relative to the underlying histone octamer. In contrast, the rate of action by DNA Ligase III- (in association with XRCC1) was independent of lesion orientation. These results are consistent with structural studies of BER enzymes and the previously proposed DNA unwrapping model for how BER enzymes gain access to lesions in nucleosomes (Prasad, Wallace, and Pederson 2007). During these investigations, we also discovered a synergistic interaction between Pol and Ligase III- complexed with XRCC1 that enhances the repair of lesions in nucleosomes. Together, our results support the hypothesis that DNA glycosylases have evolved to function in specific cellular environments (e.g. NEIL1 may function exclusively during DNA replication), but also possess DNA binding motifs and mechanisms of substrate recognition that impart a similar intrinsic activity on nucleosomes. In addition to hNTH1 and NEIL1, we have discovered that lesion orientation is also an important factor to the activities of APE and Pol and that the complete BER reaction can occur without requiring or inducing nucleosome disruption. Finally, protein-protein interactions between XRCC1 and Pol may be important for the efficient in vivo repair of lesions in nucleosomes.

The aim of this study was to observe levels of oxidative DNA damage in patients with multiple injuries in correlation with the nutritional support that the patients have received during their hospital stay. Oxidative DNA damage was evaluated in two periods of time, first evaluation was performed during standard nutritional support according to the ESPEN guidelines. Second evaluation was performed after a change in nutrition according to individual parameters of metabolism and utilization of nutritional components based on indirect calorimetric measurements. This study included 6 patients with multiple injuries hospitalized in the Intensive Care Unit 1 at the Department of Surgery, University Hospital in Hradec Kralove. In this experiment DNA isolated from peripheral lymphocytes was used to evaluate oxidative DNA damage. This DNA was analyzed using the Comet Assay method. The enzymatic version of the Comet Assay was used to determine the oxidative damage of purines and pyrimidines, and the alkaline version was used for detection of single strand breaks. Mann-Whitney test was used for statistic evaluation the difference between both measuremetns, correlation analysis for relations between Comet Assay results and clinical parameters. Significant correlations between a total amount of nutrients given...

DNA repair is a vital process of a living organism. Inherited or acquired defects in DNA repair systems and cellular surveillance mechanisms are expected to be important, if not crucial factors in the development of human cancers. DNA repair is a multigene and multifactorial process which is most comprehensively characterized by the phenotypic evaluation of DNA repair capacity (DRC). DRC represents a complex marker with high informative value, as it comprises all genetic, epigenetic and non-genetic factors, by which it is modulated. Accordingly, DRC reflects the actual capability of the cell, tissue or organism to protect its DNA integrity. The present PhD study was focused on investigating DRC, which specifically involves base and nucleotide excision repair pathways, in human populations with different characteristics. The main aim was to answer substantial questions on the possible use of DRC as biomarkers in epidemiological studies. The study was in fact designed to understand the extent of physiological variability of DRC in a population, its modulation by genetic and non-genetic factors, tentative adaptability to high genotoxic stress and, finally, its involvement in cancer aetiology. In order to explore these issues, DRC, in respect to genetic and environmental variability, was investigated...

Apesar de intensivos estudos e substanciais progressos no entendimento dos fatores de risco e suscetibilidade ao câncer de mama (CM), esta neoplasia permanece como importante causa de morte entre mulheres. Idade, história familiar, menarca precoce, menopausa tardia, ocorrência da primeira gravidez após os 30 anos e da nuliparidade constituem fatores de risco. Além disso, polimorfismos nos genes envolvidos no reparo de danos no DNA, como os genes XRCC1 e XRCC3, podem contribuir para o aumento da suscetibilidade ao CM. Os objetivos do presente trabalho foram avaliar pelo Teste do MN e Ensaio Cometa os danos basais e a resposta celular aos danos induzidos, in vitro, no DNA pelo quimioterápico Etoposido em pacientes com CM, virgens de qualquer tipo tratamento e em mulheres saudáveis utilizadas como controles e além disso, estabelecer as freqüências dos polimorfismos nos genes XRCC1 e XRCC3 na amostra de pacientes com CM e em mulheres saudáveis e associação destes dois polimorfismos com a suscetibilidade ao CM. No Teste do MN, foi observada uma sensibilidade maior do grupo de pacientes aos danos induzidos pelo Etoposido. O Ensaio Cometa mostrou que pacientes e mulheres saudáveis respondem de modo semelhante ao tratamento com o Etoposido. Também foi observado que pacientes acima de 45 anos apresentaram um grau maior de sensibilidade aos danos induzidos pelo Etoposido na concentração de 25 M quando comparadas com pacientes abaixo de 45 anos avaliadas no Ensaio Cometa. Quanto ao hábito tabagista, este se mostrou um fator de contribuição ao aumento de sensibilidade a indução de danos pelo Etoposido no Ensaio Cometa no grupo de mulheres saudáveis, para os tratamentos com esta droga nas concentrações de 10 e 25 M. Na análise molecular, o alelo variante 241Met do gene XRCC3 mostrou-se mais freqüente no grupo de pacientes tanto na amostra estudada na análise citogenética quanto na amostra estudada na análise molecular, sugerindo uma diminuição da capacidade de reparo destas pacientes, o que poderia conferir um risco aumentado ao CM. Quanto ao hábito tabagista, somente as pacientes não fumantes, portadoras do alelo 241Met do gene XRCC3, possuem um risco aumentado para o CM. Não foi encontrada associação do polimorfismo Arg399Gln do gene XRCC1 com o risco ao CM mesmo quando associado à fatores de risco como hábito tabagista e a presença de familiares com câncer. / In spite of intensive studies and substantial improvements in the understanding of the risk factors and breast cancer (BC) susceptibility, this neoplasia remains as an important cause of death among women worldwide. Age, family history of cancer, early menarche, late menopause, the first pregnancy after the age of 30 years and nulliparity are BC risk factors. Furthermore genetic polymorphisms in repair genes like XRCC1 and XRCC3 could contribute to increase BC risk. The aims of the present study were to evaluate, by Micronucleus Test and Comet Assay, the basal damage and the cellular response to DNA damage induced by Etoposide, in vitro, in BC patients without chemotherapy treatment and in healthy women. Also establish the frequencies of polymorphisms of XRCC1 and XRCC3 genes in this sample and the association of these two polymorphisms with the susceptibility to BC. In the Micronucleus Test it was observed increased sensibility to DNA damage induced by Etoposide in patients group. Patients and healthy women exhibited the same repair capacity to DNA damage induced by Etoposide when evaluated by Comet Assay. Patients > 45 years old showed more sensibility to DNA damage induced by Etoposide (25 M) when were compared with patients 45 years old in Comet Assay. Tobacco habits contributed to increased sensibility to damage induced by Etoposide in Comet Assay in healthy women group when treated with Etoposide in 10 and 25 M. In the molecular analysis, the XRCC3 241Met allele was more frequent in patients group in both analysis (cytogenetic and molecular) suggesting a low repair capacity of DNA damage and consequently increase risk to BC. Non-smokers patients, carriers of XRCC3 241Met allele showed an increased risk to BC. The polymorphism Arg399Gln in XRCC1 gene was not associated with BC risk even if associated with risk factors like tobacco habit and family history of cancer.

Lors de la vie, des mécanismes de réparation de l’ADN sont mis en oeuvre lors d’agressions, pour protéger le génome. La réparation par excision de nucléotides (NER) est l’un de ces mécanismes. Des mutations des facteurs NER sont à l’origine de 3 maladies génétiques humaines: Xeroderma pigmentosum (XP), la trichothiodystrophie (TTD) et le syndrome de Cockayne (CS). Certains de leurs signes cliniques ne sont pas expliqués par un défaut de réparation de l’ADN. Des études suggèrent que ces facteurs interviennent dans d’autres processus, notamment lors de l’expression des gènes. Durant ma thèse, je me suis intéressé aux rôles des facteurs NER dans la transcription. En effet, j’ai montré que ces facteurs, dit de réparation, étaient recrutés avec la machinerie transcriptionnelle au niveau du promoteur et du terminateur de gènes activés. Ils influencent l’environnement chromatinien des gènes activés (boucles de chromatine et modifications post-­‐ traductionnelles des histones). Ma thèse apporte une meilleure compréhension du processus de transcription des gènes activés, permettant de mieux comprendre certaines anomalies associées aux yndromes XP, CS et TTD. / Throughout life, the mechanisms of DNA repair are implemented in attacks to protect the integrity of our DNA. The nucleotide excision repair (NER) is one of these mechanisms. Mutations targeting genes of NER factors (XPA-­‐G, TTD-­‐A, CSA and CSB) are responsible for three human genetic diseases : Xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS). Some of them clinical features cannot be explained by a defect in DNA repair only. Previous studies suggest that these factors could be involved in other functions, including gene expression. In my thesis, I am interested in the roles of NER factors during the transcription process. Indeed, we have shown that these “repair” factors, were recruited with the transcription al machinery at the promoter and terminator of activated genes during transcription. They influence the chromatin environment of activated genes (chromatin loops and post-­‐translational modifications of histones).My thesis provides a better understanding of the transcription process of activated genes and allows a better understanding of some syndromes associated with XP, CS and TTD.

Aicardi-Goutières Syndrome (AGS) is a single gene, autoimmune disorder, with variable onset in the first year of life. Its clinical features exhibit similarities to several autoimmune diseases and congenital viral infections. AGS can result from mutations in ADAR1, TREX1 and SAMHD1 as well as any of the three genes that encode the protein subunits of the RNase H2 enzyme. It is hypothesised that impairment of nucleic acid metabolism results in abnormal nucleic acid species within the cell. This in turn is thought to cause the aberrant immune response that leads to AGS. The RNase H2 complex contains the catalytic RNASEH2A subunit and the auxiliary RNASEH2B and RNASEH2C subunits, which are thought to provide structural support and facilitate interactions with additional cellular proteins. RNase H2 can cleave the RNA strand of an RNA:DNA hybrid as well as 5’ of a single ribonucleotide embedded in dsDNA. Therefore, RNase H2 may have roles in several cellular processes, including DNA replication and repair, transcription, and viral infection. The aim of this PhD project was to investigate the physiological functions of RNase H2. The localisation of the RNase H2 proteins was investigated using EGFP-tagging and fluorescent microscopy. The interaction between the PIP-box of RNASEH2B and PCNA was found to localise RNase H2 and not RNase H1 to nuclear replication foci during S-phase. This suggests that RNase H2 is the dominant RNase H activity during DNA replication. Stable cell lines expressing EGFP-RNASEH2B and an alternative isoform, EGFP-RNASEH2Balt, were generated and used to perform a protein-protein interaction screen by GFP-Trap and mass spectrometry. The results indicate putative physical interactions between RNASEH2B and other factors involved in DNA replication and repair. Further evidence for a role in DNA repair was revealed when mammalian RNase H2 null cells were treated with hydroxyurea. Low doses of hydroxyurea increased ribonucleotide incorporation into genomic DNA and impaired S-phase progression. In contrast to wild-type cells, RNase H2 null cell proliferation also failed to recover from this replicative stress after HU withdrawal. However, the ribonucleotide content of genomic DNA from these cells did return to pre-hydroxyurea treatment levels. This suggests that an alternative repair pathway exists in mammalian cells, which can remove ribonucleotides from DNA in the absence of RNase H2, but that this pathway is also harmful to the cells. There is evidence that TREX1 facilitates viral infection while SAMHD1 has been shown to restrict viral infection. Therefore, experiments were performed to investigate if RNase H2 could be a viral facilitator or restriction factor. Ribonucleotides can be incorporated into viral DNA, so RNase H2 could act as a restriction factor by nicking and damaging the pre-integration complex. However, RNase H2 could also function as a facilitator of infection by processing viral RNA:DNA hybrid by-products and thus prevent the host immune response. The data obtained during this PhD project provides further evidence that RNase H2 is involved in DNA replication and repair and has contributed to the understanding of the function of RNase H2 in the cell. However, it is still unknown how mutations in RNase H2 lead to the pathology of AGS.

Maintaining genomic integrity is crucial for cell survival. In Escherichia coli, Rec-Amediated homologous recombination (HR) plays an essential role in the repair of DNA double-strand breaks (DSB) and the SOS response through a series of highly dynamic interactions with the chromosome. A greater understanding of the mechanism of homologous recombination requires quantitative analysis of genomic studies in live cells. The aim of this thesis was to investigate the dynamics of the RecA-DNA interactions in vivo following the induction of a site-specific DSB in the chromosome of E. coli. This DSB is caused by the cleavage of a DNA hairpin by the hairpin-specific endonuclease, SbcCD. The DNA hairpin is formed only on the lagging strand template of replication by a 246 bp-interrupted palindrome. As a result cleavage only occurs on one sister chromosome, leaving one unbroken chromosome to serve as a template for repair by HR. Here, this system has been used as a basis to develop a method that combines chromatin immunoprecipitation with quantitative PCR (ChIP-qPCR) and next-generation sequencing (ChIP-Seq) to quantify RecA protein binding during the active repair of a single chromosomal DSB. This study reports that DSB-dependent RecA binding is stimulated in response to the eight base DNA sequence Chi (5’-GCTGGTGG-3’). Increasing the number of Chi sites close to the DSB stimulates more RecA loading to DNA, with ChIP-Seq analysis also revealing a role for subsequent Chi sites in RecA binding during DSBR. If the Chi sites close to the DSB are removed then Chi-dependent RecA binding to DNA can be observed at distances greater than 100 kb from the DSB, suggesting that these subsequent Chi sites can be engaged in DSBR. Through collaboration, these in vivo data were combined with stochastic modeling to determine that, in vivo, Chi is recognised by the RecBCD complex with an efficiency of 20- 35%. The genomic analysis also revealed two unexpected aspects of RecA protein binding. First, ChIP-Seq analyses identified that following a DSB at lacZ there is RecA enrichment detected in the terminus region of the E. coli chromosome. This RecA binding is Chi-dependent, indicating a role for HR. Second, DSB-independent binding was observed at the RNA encoding genes dispersed throughout the chromosome. A temporal analysis of RecA dynamics was also performed. These analyses revealed that RecA binding to DNA near the DSB is extremely dynamic, cycling between periods of high RecA enrichment and periods of low RecA enrichment. This is the first in vivo study of DSB-dependent RecA-DNA distribution and dynamics in recombination proficient E. coli cells.

DNA repair is not a single mechanism found within cells. There exists numerous different DNA repair mechanisms that function within every type of cell. The majority of these mechanisms risk accumulating mutations. However, there are a few repair mechanisms that are known to be error-free and one of these is direct reversal repair. This study focused on two proteins highly involved in direct reversal DNA repair--ALKBH2 and ALKBH3. Previous studies have shown that in mice, these two proteins play a significant role in preventing and repairing DNA damage due to methylation as well as decreasing the frequency of mutagenic alkyl adducts. The goal of this study was to characterize the roles of the direct reversal repair proteins in human cells. We expected to see a similar phenotype to that of the Alkbh2 and Alkbh3-deficient mice. Telomerase immortalized human skin fibroblasts were targeted for the ALKBH2 and ALKBH3 alleles using a RNA-guided CRISPR-Cas9 construct that was designed to induce double stranded DNA breaks within the exons and disrupt the open reading frame, eliminating protein activity. Isolated clones were analyzed using fragment analysis and DNA sequencing to characterize any alterations in the open reading frame of the genes. Through sequencing analysis, results showed that one clone was successfully targeted for one of the ALKBH3 alleles with a single nucleotide insertion in its sequence, causing a disruption of the open reading frame. Though the ultimate goal of the experiment was not attained, we concluded that HTERTG fibroblasts can be expanded to serve as a model in which to construct targeted human cell lines that have near normal karyotypes.

Double-strand breaks (DSBs) are highly cytotoxic DNA lesions that are usually repaired by two major mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR). HR is initiated by 5'-3' resection, generating 3' single stranded DNA tails coated by Replication protein A (RPA), which can be used in later steps for homology search and repair. The 5'-3' resection step is a critical determinant of repair pathway choice that commits cells to HR instead of NHEJ, and it's also required for DNA damage checkpoint activation. Studies in the budding yeast Saccharomyces cerevisiae have shown that the conserved Mre11-Rad50-Xrs2 (MRX) complex, together with Sae2, initiates end resection while more extensive processing of 5' strands requires the 5'-3' exonuclease Exo1, or the combined activities of the Sgs1 helicase and Dna2 endonuclease. In this thesis we will discuss the function of RPA and Sae2 based on our experimental observations. RPA is an essential eukaryotic single-stranded DNA binding protein with a central role in DNA metabolism. It has been shown in vitro that RPA directly participates in end resection by stimulating the Sgs1 helicase and Dna2 endonuclease. To investigate the role of RPA for end resection in vivo, we used a heat-inducible degron allele (td-RFA1) that allows rapid conditional depletion of RPA in Saccharomyces cerevisiae. Complete loss of RPA resulted in a defect in both the Exo1 and Sgs1-Dna2 extensive resection mechanisms, while resection initiation by MRX-Sae2 was unaffected. Interestingly, Dna2 was unable to localize to DSBs in the absence of RPA, whereas Exo1 localization was unaffected indicating that the role of RPA in the resection pathways is distinct. The short single-stranded DNA tails formed in the absence of RPA were unstable, represented by 3' strand loss and formation of foldback hairpin structures. Thus, RPA is required to generate ssDNA, and also to protect ssDNA from degradation and inappropriate annealing that could lead to genome rearrangements. While Mre11 possesses 3'-5' dsDNA exonuclease and ssDNA endonuclease activities, Sae2 was reported to activate its endonuclease activity, which initiates end resection. We identified mre11-P110L and four more mutants from a screen that bypass Sae2 for camptothecin (CPT) and MMS resistance. None of them restored endonuclease activity, neither did they improve resection. Persistent Mre11 foci and hyper-checkpoint signaling caused by sae2Δ upon DNA damage was suppressed by mre11-P110L. These findings demonstrate that the DNA damage sensitivity of sae2Δ is not caused by defective resection, but by failure to remove MRX from ends and switch off checkpoint.