Estudou-se o comportamento frente a radiação UV de células humanas XP12RO, deficientes em reparo-excisão de dímeros de pirimidina. Cinéticas de incorporação de precursor radiativo de DNA em tempos crescentes apos a exposição a UV mostraram uma rápida inibição da taxa de síntese, até se alcançar um platô bem abaixo do valor obtido para células não irradiadas. Tanto o tempo para que este platô fosse alcançado quanto o valor basal de síntese obtido dependiam da dose de UV fornecida. Este tempo era compatível com o necessário para que a maquinaria de replicação percorresse a distância média interdímeros esperada para a dose de UV aplicada. Verificou-se também que o DNA recém-sintetizado após UV apresentava um peso molecular e uma taxa de elongação bem menores que nos controles não irradiados, sugerindo todos estes resultados que o dímero se constitue num bloqueio temporário para a replicação de DNA. Utilizando uma metodologia baseada no tratamento do DNA nativo com S1 endonuclease de Aspergillus oryzae, específica para DNA simples-fita, foi possível detectar a existência de lacunas de DNA replicado após UV, lacunas estas que desaparecem gradativamente com o passar do tempo pós-irradiação. DNA não irradiado manteve-se refratário à enzima, nas mesmas condições. A digestão enzimática acarretava o aparecimento de duas populações distintas de DNA, uma de alto peso molecular e outra de peso molecular bem menor, ambas se equivalendo em quantidade. Este fenômeno pôde ser observado em uma ampla faixa de doses de UV, tanto em células XP12RO como em outras linhagens celulares, e mesmo sob condições diversas de proliferação celular. Além disso, o desaparecimento das lacunas, no caso de células de roedor previamente irradiadas com UV, era retardado pela presença de cafeína, um conhecido inibidor de reparo pós-replicação (RPR) nestas linhagens. Foi efetuada uma análise da progressão da forquilha de replicação e da distribuição de lacunas do DNA replicado após UV, através de ensaios enzimáticos combinados com bandeamento de DNA em gradientes isopícnicos de CsCl. Os resultados assim obtidos levaram-nos a considerar um modelo de replicação a partir de molde lesado onde síntese descontínua (3\'-5\') propicia a formação de lacunas, enquanto que síntese contínua (5\'-3\') é retardada temporariamente pela presença da lesão, sem contudo acarretar a formação de descontinuidades físicas no DNA replicado. A mesma metodologia de digestão de DNA com S1 endonuclease permitiu verificar a ocorrência de uma nítida relação causal entre a frequência de lacunas e a frequência correspondente de dímeros, em crescentes doses de UV, sugerindo fortemente que dímeros estão opostos às lacunas no DNA recém sintetizado. Além disso, um tratamento estatístico da cinética de clivagens enzimáticas observada para as lacunas tornou possível calcular a extensão física destas, detectando-se a presença de duas populações distintas, onde 65% correspondem a 1250 nucleotídeos e 35% correspondem a 150 nucleotídeos. Finalmente, foi verificado que DNA recém-sintetizado longos tempos após UV apresenta um drástico declínio da frequência de lacunas, não obstante a frequência de dímeros permanecer essencialmente inalterada. Estes resultados favorecem a hipótese de ocorrer um processo induzido de RPR, o qual permitiria à maquinaria de replicação transpor eficientemente os dímeros presentes, apesar destes não terem propriedades codificadoras. / The synthesis of DNA in human XP12RO cells, deficient in excision repair of pyrimidine dimers was studied. The rate of incorporation of radioactive precursors into DNA was measured at different times after irradiation. The DNA synthesis decreases shortly after irradiation, reaching a lateau whose value and time to be attained was dependent on the UV dose. This time period was the one expected for the replication machinery to coyer the interdimer distance at the UV dose applied. It was also verified that the newly synthesized DNA after UV irradiation presented much smaller molecular weight and elongation rate, when compared with the non-irradiated controls. These results suggest that the dimer imposes a delay to DNA replication machinery. Using a methodology based on the treatment of native DNA with S1-endonuclease from Aspergillus orizae, specific for single-stranded DNA, it was possible to detect gaps in the DNA replicated after UV treatment. Thesee gaps disapeared gradually with time after irradiation. The nonirradiated DNA remained refractory to the enzyme, under the same experimental conditions. The enzymatic digestion originated approximately equal alounts of two distinct double-stranded DNA populations, one of high molecular weight and other of much smaller molecular weight. This fenomenon could be seen on a wide range of UV doses, in XP12RO cells as well as in other cells lines, and did not depend on the particular conditions of cell proliferation. Furthermore, the gap disappearence, in the case of rodent cells previously irradiated with UV, was delayed by the presence of caffeine, a known post-replication repair (PRR) inhibitor in these cell lines. An analysis of the progression of the replication fork and of the distribution of gaps in the DNA replicated after UV irradiation was carried out through enzymatic assays combined with DNA banding in isopicnic CsCl gradients. The results thus obtained led us to consider a model for replication on damaged template, whereby gaps are formed only in the strand replicating opposite the fork movement (3\'-5\'). The strand replicating in the same direction as the fork movement (5\'-3\') is temporarily delayed by the presence of the lesion, without originating gaps in the replicative DNA. The same methodology of DNA digestion with S1-endonuclease permitted us to verify the occurrence of a nitid relationship between the gap frequency and the corresponding dimer frequency, for different doses of UV, strongly suggesting that the dimers are opposite the gaps in the newly-synthesized DNA. Furthermore, an statistical analysis of the dependende of DNA cleavage by S1-endonuclease on the enzyme concentration rendered it possible to calculate the size of the gaps. Two distinct populations were detected, 65% corresponding to 1250 nucleotides and 35% corresponding to 150 nucleotides. Finally, it was verified that the nascent DNA synthesized long periods after UV are essentially free of gaps although the dimer frequency remained almost unaltered. These results favour the hypothesis of the occurrence of an induced process of PRR, which would permit the replication machinery to efficiently bypass the dimers, in spite of the fact that these lesions do not exhibit codifying properties.

Abstract Replicative DNA polymerases (pols) synthesize chromosomal DNA with high accuracy and speed during cell division. In eukaryotes the process involves three family B pols (α, δ, ε), whereas in Archaea, two types of pols, families B and D, are involved. In this study the B-subunits of replicative pols were analysed at the DNA, RNA and protein levels. By cloning the cDNAs for the B-subunits of human and mouse pol ε we were able to show that the encoded proteins are not only homologous to budding yeast pol ε, but also to the second largest subunit of pol α. Later studies have revealed that the B-subunits are conserved from Archaea to human, and also that they belong to the large calcineurin-like phosphoesterase superfamily consisting of a wide variety of hydrolases. At the mRNA level, the expression of the human pol ε B-subunit was strongly dependent on cell proliferation as has been observed for the A-subunit of pol ε and also for other eukaryotic replicative pols. By analysing the promoter of the POLE2 gene encoding the human pol ε B-subunit we show that the gene is regulated by two E2F-pocket protein complexes associated with the Sp1 and NF-1 transcription factors. Comparison of the promoters of the human pol ε and the pol α B-subunit indicates that the genes for the B-subunits may be generally regulated through E2F-complexes whereas adjustment of the basal activity may be achieved by distinct transcription factors. To clarify the function of the B-subunits, we screened through the expression of 13 different recombinant B-subunits. Although they were mainly expressed as insoluble proteins in E. coli, we were able to optimize the expression and purification for the B-subunit (DP1) of Methanococcus jannaschii pol D (MjaDP1). We show that MjaDP1 alone was a manganese dependent 3'-5' exonuclease with a preference for mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreader of archaeal pol D. So far, pol D is the only pol family utilising an enzyme of the calcineurin-like phosphoesterase superfamily as a proofreader.

Abstract The maintenance of integrity of the genome is essential for a cell. DNA repair and faithful DNA replication ensure the stability of the genome. DNA polymerases (pols) are the enzymes that synthesise DNA, a process important both in DNA replication and repair. In DNA replication DNA polymerases duplicate the genome during S phase prior to cell division. Pols α, δ, and ε are implicated in chromosomal DNA replication, but their exact function in replication is not yet completely clear. The mechanisms of different repair pathways and proteins involved are not yet completely characterised either. The deeper understanding of DNA repair and replication mechanisms is crucial for our understanding on the function of the cell. The mechanism of repair of DNA double strand breaks (DSBs) by non-homologous end joining (NHEJ) was studied with an in vitro assay. DNA polymerase activity was found to be involved in NHEJ and important in stabilising DNA ends. Antibodies against pol α, but not pol β or ε, decreased NHEJ significantly, which indicates the involvement of pol α in NHEJ. In addition, the removal of proliferating cell nuclear antigen (PCNA) slightly decreased NHEJ activity. The division of labour between pols α, δ, and ε during DNA replication was studied. Results from UV-crosslinking, chromatin association, replication in isolated nuclei, and immunoelectron microscopy (IEM) studies showed that there are temporal differences between the activities and localisations of the pols during S phase. Pol α was active throughout S phase, pol ε was more active at early S phase, whereas the activity of pol δ increased as S phase advanced. These results suggest that pols δ and ε function independently during DNA replication. Pol ε could be crosslinked to nascent RNA, and this labelling was not linked to DNA replication, but rather to transcription. Immunoprecipitation studies indicated that pol ε, but not pols α and δ, associated with RNA polymerase II (RNA pol II). Only the hyperphosphorylated, transcriptionally active RNA pol II was found to associate with pol ε. A large proportion of pol ε and RNA pol II colocalised in cells as determined with immunoelectron microscopy. The interaction between pol ε and RNA pol II suggests that they are involved in a global regulation of transcription and DNA replication.

Deux processus majeures de la vie cellulaire, la transcription et la réplication, nécessitent l'ouverture de la double hélice d'ADN et agissent différemment sur les deux brins, ce qui génère des taux de mutation différents (asymétrie de mutation), et aboutit à des compositions en nucléotides différentes des deux brins (asymétrie de composition). Nous nous proposons de modéliser le programme spatio-temporel de réplication et son impact sur l'évolution des séquences d'ADN. Dans le génome humain, nous montrons que les asymétries de composition et de mutation peuvent être décomposées en deux contributions, l'une associée à la transcription et l'autre à la réplication. Celle associée à la réplication est proportionnelle à la polarité des fourches de réplication, elle-même proportionnelle à la dérivée du “timing” de réplication. La polarité des fourches de réplication délimite, le long des chromosomes humains, des domaines de réplication longs de plusieurs Mpb où le “timing” de réplication a une forme de U. Ces domaines de réplication sont également observés dans la lignée germinale, où ils sont révélés par une asymétrie de composition en forme de N, indiquant la conservation de ce programme de réplication sur plusieurs centaines de millions d'années. Les bords de ces domaines de réplication sont constituées d'euchromatine, permissive à la transcription et à l'initiation de la réplication. L'analyse de données d'interaction à longue portée de la chromatine suggère que ces domaines correspondent à des unités structurelles de la chromatine, au coeur d'une organisation hautement parallélisée de la réplication dans le génome humain. / Two key cellular processes, namely transcription and replication, require the opening of the DNA double helix and act differently on the two DNA strands, generating different mutational patterns (mutational asymmetry) that may result, after long evolutionary time, in different nucleotide compositions on the two DNA strands (compositional asymmetry). Here, we propose to model the spatio-temporal program of DNA replication and its impact on the DNA sequence evolution. The mutational and compositional asymmetries observed in the human genome are shown to decompose into transcription- and replication-associated components. The replication-associated asymmetry is related to the replication fork polarity, which is also shown to be proportional to the derivative of the mean replication timing. The large-scale variation of the replication fork polarity delineate Mbp scale replication domains where the replication timing is shaped as a U. Such replication domains are also observed in the germline, where they are revealed by a N-shaped compositional asymmetry, which indicates the conservation of this replication program over several hundred million years. The replication domains borders are enriched in open chromatin markers, and correspond to regions permissive to transcription and replication initiation. The analysis of chromatin interaction data suggests that these replication domains correspond to self-interacting chromatin structural units, at the heart of a highly parallelized organization of the replication program in the human genome.

Abstract Genetic information is stored in the base sequence of DNA. As DNA is often damaged by radiation or reactive chemicals, cells have developed mechanisms to correct the DNA lesions. These mechanisms involve recognition of damage, DNA repair and cell cycle delay until DNA is restored. Failures in the proper processing of DNA lesions may lead to mutations, premature aging, or diseases such as cancer. In this thesis study the human topoisomerase IIβ binding protein 1 (TopBP1) was identified as the homolog of budding yeast Dpb11 and fission yeast Cut5. TopBP1 was found to be necessary for DNA replication and to associate with replicative DNA polymerase ε. TopBP1 localised to the sites of DNA damage and stalled replication forks, which suggests a role in the DNA damage response. TopBP1 interacted with the checkpoint protein Rad9, which is a part of a protein complex whose function includes tethering proteins to sites of DNA damage. This supports a role for TopBP1 in the early steps of checkpoint activation after DNA damage. TopBP1 also interacted with the tumour suppressor protein p53 in a phosphorylation dependent manner. In addition, the data support a role for TopBP1 outside of S-phase. During M-phase, TopBP1 was found to localise to centrosomes along with the tumour suppressor proteins Brca1 and p53. Analysis of the expression of TopBP1 in mouse tissues suggested that TopBP1 may also play a role during meiosis. The localisation pattern of TopBP1 in mouse meiotic spermatocytes resembled that of many proteins functioning during meiotic recombination. For example, co-localisation of ATR kinase and TopBP1 was observed during meiotic prophase I. In accordance with the findings from mouse studies, the analysis of a cut5 mutant during yeast meiosis showed that Cut5 is essential for the meiotic checkpoint. These results strongly suggest that TopBP1 operates in replication and has checkpoint functions during both the mitotic and meiotic cell cycles.

Abstract DNA replication is a process in which a cell duplicates its genome before cell division, and must proceed accurately and in organized manner to guarantee maintenance of the integrity of the genetic information. DNA polymerases are enzymes that catalyse the synthesis of the new DNA strand by utilizing the parental strand as a template. In addition to chromosomal replication, DNA synthesis and therefore DNA polymerases are also needed in other processes like DNA repair and DNA recombination. The DNA polymerase is an essential DNA polymerase in eukaryotes and is required for chromosomal DNA replication. It has also been implicated in DNA repair, recombination, and in transcriptional and cell cycle control. The regulation of the human enzyme was explored by analysing its expression, phosphorylation and protein-protein interactions. Expression of both the A and B subunits of the human DNA polymerase ε was strongly growth-regulated. After serum-stimulation of quiescent fibroblasts, the steady-state mRNA levels were up-regulated at least 5-fold. In actively cycling cells, however, the steady-state mRNA and protein levels fluctuated less than 2-fold, being highest in G1/S phase. The promoter of the B subunit gene was analysed in detail. The 75 bp core promoter was essentially dependent on the Sp1 transcription factor. Furthermore, mitogenic control of the promoter required an intact E2F binding element, and binding of E2F2, E2F4 and p107 was demonstrated in vitro. A down-regulation element, located immediately downstream from the core promoter, bound E2F1, NF-1 and pRb transcription factors. A model of the promoter function is presented. Topoisomerase IIβ binding protein 1 (TopBP1) was found to be associated with human DNA polymerase ε. TopBP1 contains eight BRCT domains and is homologous to Saccharomyces cerevisiae Dpb11, Schizosaccharomyces pombe Cut5, Drosophila melanogaster Mus101 and the human Breast Cancer susceptibility protein 1 (BRCA1). TopBP1 is a phosphoprotein, whose expression is induced at the G1/S border and is required for chromosomal DNA replication. It co-localizes in S phase with BRCA1 into discrete foci, which do not represent sites of ongoing DNA replication. However, if DNA is damaged or replication is blocked in S phase cells, TopBP1 and BRCA1 re-localize into proliferating cell nuclear antigen (PCNA) containing foci that represent stalled replication forks. Finally, phosphorylation of DNA polymerase ε was described and at least three immunologically distinct and differentially phosphorylated forms were shown to exist. Phosphorylation is on serine and threonine residues and shows a cell cycle dependent fluctuation, but is not affected by DNA damage or by inhibition of DNA replication. BRCA1 co-immunoprecipitates with a hypophosphorylated form of DNA polymerase ε. In contrast, TopBP1 was shown to be associated with a hyperphosphorylated form.

Abstract Analysis of the primary structure of DNA polymerase ε B subunit defined similarities to B subunits of eukaryotic DNA polymerases α, δ and ε as well as the small subunits of DNA polymerase DI of Euryarchaeota. Multiple sequence alignment of these proteins revealed the presence of 12 conserved motifs and defined a novel protein superfamily. The members of the B subunit family share a common domain architecture, suggesting a similar fold, and arguing for a conserved function among these proteins. The contribution of human DNA polymerase ε to nuclear DNA replication was studied using the antibody K18 that specifically inhibits the activity of this enzyme in vitro. This antibody significantly inhibited DNA synthesis both when microinjected into nuclei of exponentially growing human fibroblasts and in isolated HeLa cell nuclei, but did not inhibit SV40 DNA replication in vitro. These results suggest that the human DNA polymerase ε contributes substantially to the replicative synthesis of DNA and emphasises the differences between cellular replication and viral model systems. The human DNA polymerases ε and δ were found capable of gap-filling DNA synthesis during nucleotide excision repair in vitro. Both enzymes required PCNA and the clamp loader RFC, and in addition, polymerase δ required Fen-1 to prevent excessive displacement synthesis. Nucleotide excision repair of a defined DNA lesion was completely reconstituted utilising largely recombinant proteins, only ligase I and DNA polymerases δ and ε provided as highly purified human enzymes. This system was also utilised to study the role of the transcription factor II H during repair. Human non-homologous end joining of model substrates with different DNA end configurations was studied in HeLa cell extracts. This process depended partially on DNA synthesis as an aphidicolin-dependent DNA polymerase was required for the formation of a subset of end joining products. Experiments with neutralising antibodies reveal that DNA polymerase α but not DNA polymerases β or ε, may represent this DNA polymerase activity. Our results indicate that DNA synthesis contributes to the stability of DNA ends, and influences both the efficiency and outcome of the end joining event. Furthermore, our results suggest a minor role of PCNA in non-homologous end joining.

The CMG (Cdc45, MCM, GINS) helicase is required for cellular proliferation and functions to unwind double-stranded DNA to allow the replication machinery to duplicate the genome. Cancer cells mismanage helicase activation through a variety of mechanisms, leading to the potential for the development of novel anti-cancer treatments. Mammalian cells load an excess of MCM complexes that act as reserves for new replication origins to be created when replication forks stall due to stress conditions, such as drug treatment. Targeting the helicase through inhibition of the MCM complex has sensitized cancer cells to drugs that inhibit DNA replication, such as aphidicolin and hydroxyurea. However, these drugs are not used in the clinical management of cancer. We hypothesized that the effectiveness of the clinically relevant drugs gemcitabine and 5-FU against pancreatic cancer cells, and oxaliplatin and etoposide against colorectal cells, could be increased through co-suppression of the MCM complex. The oncogene c-Myc also leads to the mismanagement of CMG helicases in part due to a non-transcriptional role in overactivating replication origins and causing DNA damage. We sought to elucidate the mechanism by which Myc causes overactivation of CMG helicases. Herein we demonstrate that co-suppression of reserve MCM complexes in pancreatic or colorectal cancer cell lines treated with clinically applicable chemotherapeutic compounds causes significant loss of proliferative capacity compared with cells containing the full complement of reserve MCMs. This is in part due to an inability to recover DNA replication following drug exposure, leading to an increase in apoptosis. Targeting of Myc to genomic sites induced large-scale decondensation of higher order chromatin that was required for CMG helicase assembly and activation at reserve MCM complexes. The physiological mediators of Myc, GCN5 and Tip60, are required for the chromatin unfolding and Cdc45 recruitment. We conclude that depletion of the reserve MCM complexes causes chemosensitization of multiple human tumor cell types to several chemotherapeutic drugs used in the clinical management of human cancer. This argues for the development and use of anti-MCM drugs in combination with chemotherapeutic compounds, which has the potential to increase the therapeutic index of existing clinical compounds. We have also identified a previously unknown role for Myc in normal cell cycle progression whereby DNA replication initiation is regulated through the assembly and activation of CMG helicases on Myc-mediated open chromatin regions. Our results also provide new mechanistic insight into Myc oncogenic transformation in which overstimulation of DNA replication could result in genomic instability and provide an explanation for Myc driven oncogenic transformation.

Abstract DNA polymerase ε from HeLa cells has been purified as a heterodimer of a 261 kDa catalytic subunit and a tightly associated smaller polypeptide, the B-subunit. The cDNAs encoding the B-subunits of both human and mouse Pol ε were cloned and shown to encode proteins with a predicted molecular weight of 59 kDa. These subunits are 90 % identical and share 22 % identity with the 80 kDa B-subunit of Saccharomyces cerevisiae Pol ε. The gene for the human Pol ε B-subunit was localized to chromosome 14q21-q22 by fluorescence in situ hybridization. Primary structure analysis of the Pol ε B-subunits demonstrated that they are similar to the B-subunits of Pol α, Pol δ and archaeal DNA polymerases, and comprise a novel protein family of DNA polymerase associated-B-subunits. The family members have 12 conserved motifs distributed in the C-terminal parts, which apparently form crucial structural and functional sites. Secondary structure predictions indicate that the B-subunits share a similar fold, and phylogenetic analysis demonstrated that the B-subunits of Pol α and ε form one subfamily, while the B-subunits of Pol δ and the archaeal proteins form a second subfamily. The corresponding eukaryotic and archaeal catalytic subunits are not related, but all have the characteristics of replicative DNA polymerases. This indicates that the B-subunits of replicative DNA polymerases from archaea to eukaryotes belong to the same protein family and perform similar functions. In S. cerevisiae, Pol ε associates with the checkpoint protein Dpb11. In this study, a human protein, TopBP1, with structural similarity to the budding yeast Dpb11, fission yeast Cut5 and the breast cancer susceptibility gene product Brca1 was identified. The human TOPBP1 gene localizes to chromosome 3q21-q23 and encodes a phosphoprotein of 180 kDa. TopBP1 has eight BRCT domains and is also closely related to the recently identified Drosophila melanogaster Mus101. TopBP1 expression is induced at the G1/S boundary and it performs an important role in DNA replication, as evidenced by inhibition of DNA synthesis by TopBP1 antiserum in isolated nuclei. TopBP1 also associates with Pol ε and localizes, together with Brca1 to distinct foci in S-phase, but not to sites of ongoing DNA replication. Inhibition of DNA replication leads to re-localization of TopBP1 and Brca1 to stalled replication forks. DNA damage induces formation of distinct TopBP1 foci that co-localize with Brca1 in S-phase, but not in G1-phase. The role of TopBP1 in the DNA damage response is also supported by the interaction between TopBP1 and the human checkpoint protein hRad9. These results implicate TopBP1 in replication and checkpoint functions.

Tumorigenesis is a multifaceted set of events consisting of the deregulation of several cell-autonomous and tissue microenvironmental processes that ultimately leads to the acquisition of malignant disease. Transforming growth factor beta (TGFß) and its family members are regulatory cytokines that function to ensure proper organismal development and the maintenance of homeostasis by controlling cellular differentiation, proliferation, adhesion, and survival, as well as by modulating components of the cellular microenvironment and immune system. The pleiotropic control by TGFß of these cell intrinsic and extrinsic factors is intimately linked to the prevention of tumor formation, the specifics of which are dependent on the various cellular and/or molecular signaling contexts that exist for TGFß. The diverse roles and the various levels of signal control for TGFß lend themselves to certain characteristics that are more advantageous for cancers to usurp in order to promote tumorigenesis, while other anti-tumorigenic roles for TGFß are more beneficial to tumor development if they are circumvented or disabled. Transforming growth factor ß1 (TGF-ß1) exerts its anti-tumor effects in large part by potently inhibiting cell cycle progression at any point in G1 phase to control the proliferation of a variety of cell lineages. Loss of sensitivity to TGF-ß1-induced cell cycle arrest is a crucial event during early tumorigenesis. Indeed, cancer cells of almost all tumor types display insensitivity to TGF-ß1 inhibition. As such, the pursuit of the molecular details underlying the TGF-ß1 growth arrest pathway is important for our understanding of cell cycle regulation, and significantly, how disruption of these mechanisms contributes to TGF-ß1 insensitivity and tumorigenesis. TGF-ß1 inhibition of the cell cycle in G1 phase has been shown to involve two main transcriptionally based molecular events, including the induction of cyclin-dependent kinase (CDK) inhibitors and the suppression of the c-Myc protein. Both mechanisms contribute to the maintenance of the retinoblastoma (Rb) protein in its hypophosphorylated and antiproliferative form, thus preventing progression through the cell cycle. However, this type of regulation does not offer answers to all of the questions regarding TGF-ß1 arrest. While these transcriptional mechanisms provide explanations for TGF-ß1 arrest throughout most of G1, inhibition late in G1 by TGF-ß1 however, does not require any acute regulation of transcription. In addition, the chance to utilize canonical TGF-ß1 arrest mechanisms at this time has already passed (i.e. Rb is already hyperphosphorylated by late-G1). Previous work from our group shows instead that late-G1 TGF-ß1 cell cycle arrest requires an intact direct interaction between the N-terminus of Rb (RbN) and the C terminus of Mcm7, a subunit of the Cdc45-MCM-GINS (CMG) replicative helicase. Our studies show that TGF-ß1 exposure in late-G1 prevents the disassociation of Rb with fully assembled helicases, which remain inactive. In addition, it was found that early-G1 treatment with TGF-ß1 also targets CMG components, namely MCM protein accumulation (and therefore hexamer formation) in G1 is blocked. However, the residue(s) of RbN involved as well as the molecular mechanisms Rb utilizes for late-G1 TGF-ß1 arrest are not described, nor is it evident from this work if TGF-ß1 affects other genes involved in CMG assembly and/or activation. In the following study we explore these unanswered questions for TGF-ß1 growth arrest as a means to understand novel aspects of cell cycle regulation that must be abrogated during tumorigenesis. Our hypothesis is that CMG helicase control on some level is critical for all TGF-ß1-induced inhibition of cell cycle progression throughout the entire G1 phase. In Chapter 2 herein we have investigated the details and mechanistic implications of the Rb/RbN inhibitory-interaction with the CMG helicase that is required for late-G1 TGF-ß1 arrest. We show that N-terminal exons of Rb that are lost in partially penetrant hereditary retinoblastomas inhibit DNA replication and elongation using a bipartite mechanism. Specifically, Rb exon 7 is necessary and sufficient to inhibit CMG helicase activation, while an independent loop domain within RbN that forms a projection blocks DNA polymerase α (Pol-α) and Ctf4 recruitment without affecting polymerases δ and ε or the CMG helicase. Individual disruption of exon 7 or the projection in RbN or Rb, as occurs in inherited cancers, partially impairs the ability of Rb/RbN to inhibit DNA replication and block G1-S cell cycle transit. Importantly, their combined loss abolishes these functions of Rb. Thus, TGF-ß1 cell cycle arrest in late-G1 requires the growth suppressive role of Rb in which replicative complexes are blocked directly via independent and additive N-terminal domains. TGF-ß1-induced arrest in late-G1 also requires the presence of Smad3 and Smad4, suggesting that a novel transcription-independent role may exist for Smad signaling proteins in blocking cell cycle transit directly in Rb-CMG inhibitory complexes. TGF-ß1 is thought to require a functional Rb protein to inhibit the cell cycle at any point in G1 phase. Intriguingly, while cells lacking Rb (and the inhibitory N-terminal domains) lose sensitivity to TGF-ß1 arrest in late-G1, these same cells remain sensitive to TGF-ß1 inhibition in early-G1. This Rb-independent TGF-ß1 growth arrest also occurs in the absence of c-Myc and MCM suppression, as well as without CyclinE-Cdk2 inhibition, but requires Smad3 and Smad4 respectively. Here (Chapter 3) we have identified the mechanism by which TGF-ß1 achieves Smad-dependent G1 arrest in the absence of these common mediators. TGF-ß1 inhibits the assembly of CMG replicative helicases by suppressing the recruitment of the MCM complex to chromatin. Accordingly, the entire heterohexamer fails to load onto DNA. Cdc6 phosphorylation in its amino terminus is known to be required for Cdt1-dependent loading of the MCM complex. We show that in Rb-lacking cells early-G1 TGF-ß1 treatment blocks the phosphorylation of Cdc6 at serine 54, without affecting total Cdc6 protein levels, to prevent MCM heterohexamer formation on DNA. Consistent with TGF-ß1 signals targeting this recruitment and loading step, Cdt1 overexpression promotes S-phase entry in the presence of TGF-ß1, circumventing the need for Cdc6 phosphorylation. Importantly, Cdt1 requires an intact C-terminal MCM-binding domain in order to overcome this TGF-ß1-induced cell cycle arrest mechanism. These data indicate that early-G1 TGF-ß1 arrest can occur by perturbing Cdc6 phosphorylation to block Cdt1-mediated MCM recruitment and loading, leading to inhibition of CMG assembly and S-phase entry despite the lack of Rb and normal c-Myc and CyclinE-Cdk2 activities. We conclude that the main event governing TGF-ß1-induced cell cycle arrest at any point in G1 is the inhibition of the assembly and/or activation of the replicative CMG helicase. However, TGF-ß1 growth arrest has a temporal dependence on the presence of the Rb protein. In normal cells containing Rb, the accumulation of MCM subunit proteins is blocked by TGF-ß1 in early-G1 and accordingly MCM heterohexamers are unable to form. However, if cells are allowed to transit to late-G1 when MCM complexes have already assembled on origins, but before functional CMG helicases have formed at G1-S, exposure to TGF-ß1 signaling prevents CMG activation via interactions with critical inhibitory domains within RbN. Cells lacking Rb (and these residues) are not sensitive to TGF-ß1 in late-G1. Surprisingly, these cells remain sensitive to TGF-ß1 early in G1 phase despite a lack of c-Myc/MCM protein suppression and CyclinE-Cdk2 inhibition. In these cells the recruitment and loading of the MCM complex is blocked to facilitate a TGF-ß1-mediated G1 arrest. It is only when this mechanism is overcome by Cdt1 overexpression that TGF-ß1 is unable to elicit cell cycle arrest in these cells. These data provide molecular explanations for studies reporting instances of TGF-ß1 arrest without canonical effectors, such as Rb, c-Myc loss, or CDK inhibitors. Additionally, this work argues for the development of novel cancer therapeutics targeting CMG helicase assembly or activation, the regulation of which is likely lost in a variety of TGF-ß1-insensitive and/or Rb-deficient malignancies. Indeed, reintroduction of these tumor suppressive pathways has shown efficacy in blocking growth of tumors or cancer cells lacking the same mechanisms. Our studies of Rb/RbN inhibition of DNA replication also provide proof of principle for this type of therapy, as well as the framework for how the CMG might be targeted by exploring further and perhaps mimicking Rb exon7-mediated CMG inhibition biochemically.