Starting DNA Synthesis: Initiation Processes during the Replication of Chromosomal DNA in Humans.

The initiation reactions of DNA synthesis are central processes during human chromosomal DNA replication. They are separated into two main processes: the initiation events at replication origins, the start of the leading strand synthesis for each replicon, and the numerous initiation events taking place during lagging strand DNA synthesis. In addition, a third mechanism is the re-initiation of DNA synthesis after replication fork stalling, which takes place when DNA lesions hinder the progression of DNA synthesis. The initiation of leading strand synthesis at replication origins is regulated at multiple levels, from the origin recognition to the assembly and activation of replicative helicase, the Cdc45-MCM2-7-GINS (CMG) complex. In addition, the multiple interactions of the CMG complex with the eukaryotic replicative DNA polymerases, DNA polymerase α-primase, DNA polymerase δ and ε, at replication forks play pivotal roles in the mechanism of the initiation reactions of leading and lagging strand DNA synthesis. These interactions are also important for the initiation of signalling at unperturbed and stalled replication forks, "replication stress" events, via ATR (ATM-Rad 3-related protein kinase). These processes are essential for the accurate transfer of the cells' genetic information to their daughters. Thus, failures and dysfunctions in these processes give rise to genome instability causing genetic diseases, including cancer. In their influential review "Hallmarks of Cancer: New Dimensions", Hanahan and Weinberg (2022) therefore call genome instability a fundamental function in the development process of cancer cells. In recent years, the understanding of the initiation processes and mechanisms of human DNA replication has made substantial progress at all levels, which will be discussed in the review.

Replication Protein A, the Main Eukaryotic Single-Stranded DNA Binding Protein, a Focal Point in Cellular DNA Metabolism.

Replication protein A (RPA) is a heterotrimeric protein complex and the main single-stranded DNA (ssDNA)-binding protein in eukaryotes. RPA has key functions in most of the DNA-associated metabolic pathways and DNA damage signalling. Its high affinity for ssDNA helps to stabilise ssDNA structures and protect the DNA sequence from nuclease attacks. RPA consists of multiple DNA-binding domains which are oligonucleotide/oligosaccharide-binding (OB)-folds that are responsible for DNA binding and interactions with proteins. These RPA-ssDNA and RPA-protein interactions are crucial for DNA replication, DNA repair, DNA damage signalling, and the conservation of the genetic information of cells. Proteins such as ATR use RPA to locate to regions of DNA damage for DNA damage signalling. The recruitment of nucleases and DNA exchange factors to sites of double-strand breaks are also an important RPA function to ensure effective DNA recombination to correct these DNA lesions. Due to its high affinity to ssDNA, RPA's removal from ssDNA is of central importance to allow these metabolic pathways to proceed, and processes to exchange RPA against downstream factors are established in all eukaryotes. These faceted and multi-layered functions of RPA as well as its role in a variety of human diseases will be discussed.

Lagging Strand Initiation Processes in DNA Replication of Eukaryotes-Strings of Highly Coordinated Reactions Governed by Multiprotein Complexes.

In their influential reviews, Hanahan and Weinberg coined the term 'Hallmarks of Cancer' and described genome instability as a property of cells enabling cancer development. Accurate DNA replication of genomes is central to diminishing genome instability. Here, the understanding of the initiation of DNA synthesis in origins of DNA replication to start leading strand synthesis and the initiation of Okazaki fragment on the lagging strand are crucial to control genome instability. Recent findings have provided new insights into the mechanism of the remodelling of the prime initiation enzyme, DNA polymerase α-primase (Pol-prim), during primer synthesis, how the enzyme complex achieves lagging strand synthesis, and how it is linked to replication forks to achieve optimal initiation of Okazaki fragments. Moreover, the central roles of RNA primer synthesis by Pol-prim in multiple genome stability pathways such as replication fork restart and protection of DNA against degradation by exonucleases during double-strand break repair are discussed.

SV40 T antigen helicase domain regions responsible for oligomerisation regulate Okazaki fragment synthesis initiation.

The initiation of Okazaki fragment synthesis during cellular DNA replication is a crucial step for lagging strand synthesis, which is carried out by the primase function of DNA polymerase α-primase (Pol-prim). Since cellular replication protein A (RPA) prevents primase from starting RNA synthesis on single-stranded DNA (ssDNA), primase requires auxiliary factors, such as the simian virus 40 (SV40) T antigen (Tag), for the initiation reaction on RPA-bound ssDNA. Here, we investigated the ability of Tag variants and Tag protein complexes to bind to ssDNA and their resulting effects on the stimulation of Pol-prim on free and RPA-bound ssDNA. Atomic force microscopy imaging showed that while Tag131-627 (V350E/P417D) and Tag131-627 (L286D/R567E) (abbreviated as M1 and M2, respectively) could bind to ssDNA as monomers, these monomeric Tags could come together and bind to ssDNA as dimers as well. In a model assay for the initiation of Okazaki fragment synthesis, full-length Tag SV40 Tag1-708 and monomeric M2 stimulated DNA synthesis of Pol-prim on ssDNA and on RPA-bound ssDNA. In contrast, neither monomeric M1 nor M1-M2 dimers could stimulate Pol-prim, on ssDNA or on RPA-bound ssDNA. Overall, we show that a lack of stimulatory activity of monomeric M1 and M1-M2 dimers suggests that residues V350 and P417 are not only important for interactions between Tag molecules but also for protein-protein interactions within Okazaki fragment initiation complexes. Thus, we highlight that mutations in M1 are dominant negative with regard to Okazaki fragment initiation.

Alkoxylalkyl Esters of Nucleotide Analogs Inhibit Polyomavirus DNA Replication and Large T Antigen Activities.

Polyomavirus infections occur commonly in humans and are normally nonfatal. However, in immunocompromised individuals, they are intractable and frequently fatal. Due to a lack of approved drugs to treat polyomavirus infections, cidofovir, a phosphonate nucleotide analog approved to treat cytomegalovirus infections, has been repurposed as an antipolyomavirus agent. Cidofovir has been modified in various ways to improve its efficacies as a broad-spectrum antiviral agent. However, the actual mechanisms and targets of cidofovir and its modified derivatives as antipolyomavirus agents are still under research. Here, polyomavirus large tumor antigen (Tag) activities were identified as the viral target of cidofovir derivatives. The alkoxyalkyl ester derivatives of cidofovir efficiently inhibit polyomavirus DNA replication in cell-free human extracts and a viral in vitro replication system utilizing only purified proteins. We present evidence that DNA helicase and DNA binding activities of polyomavirus Tags are diminished in the presence of low concentrations of alkoxyalkyl ester derivatives of cidofovir, suggesting that the inhibition of viral DNA replication is at least in part mediated by inhibiting single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) binding activities of Tags. These findings show that the alkoxyalkyl ester derivatives of cidofovir are effective in vitro without undergoing further conversions, and we conclude that the inhibitory mechanisms of nucleotide analog-based drugs are more complex than previously believed.

Determination of the number of RAD51 molecules in different human cell lines.

Knowledge about precise numbers of specific molecules is necessary for understanding and verification of biological pathways. The RAD51 protein is central in the repair of DNA double-strand breaks (DSBs) by homologous recombination repair and understanding its role in cellular pathways is crucial to design mechanistic DNA repair models. Here, we determined the number of RAD51 molecules in several human cell lines including primary fibroblasts. We showed that between 20000 to 100000 of RAD51 molecules are available per human cell that theoretically can be used for simultaneously loading at least 7 DSBs. Interestingly, the amount of RAD51 molecules does not significantly change after the induction of DNA damage using bleomycin or γ-irradiation in cells but an accumulation of RAD51 on the chromatin occurs. Furthermore, we generated an EGFP-RAD51 fusion under the control of HSV thymidine kinase promoter sequences yielding moderate protein expression levels comparable to endogenously expressed RAD51. Initial characterizations suggest that these low levels of ectopically expressed RAD51 are compatible with cell cycle progression of human cells. Hence, we provide parameters for the quantitative understanding and modeling of RAD51-involving processes.

ATPase activity of human binding immunoglobulin protein (BiP) variants is enhanced by signal sequence and physiological concentrations of Mn2.

B-cell immunoglobulin binding protein (BiP) is an essential endoplasmic reticulum (ER) chaperone normally found in the ER lumen. However, BiP also has other extracellular and intracellular functions. As it is unclear whether peripheral BiP has a signal and/or ER retention sequence, here we produced and biochemically characterised four variants of BiP. The variants differed depending on the presence or the absence of signal and ER retention peptides. Proteins were purified using nickel affinity chromatography, and variant size and quality were confirmed using SDS/PAGE gels. The thermal denaturing temperature of these proteins was found to be 46-47 °C. In addition, we characterised nucleotide binding properties in the absence and the presence of divalent cations. Interestingly, in the absence of cations, ADP has a higher binding affinity to BiP than ATP. The presence of divalent cations results in a decrease of the Kd values of both ADP and ATP, indicating higher affinities of both nucleotides for BiP. ATPase assays were carried out to study the enzyme activity of these variants and to characterise the kinetic parameters of BiP variants. Variants with the signal sequence had higher specific activities than those without. Both Mg2+ and Mn2+ efficiently stimulated the ATPase activity of these variants at low micromolar concentrations, whereas calcium failed to stimulate BiP ATPase. Our novel findings indicate the potential functionality of BiP variants that retain a signal sequence, and also reveal the effect of physiological concentrations of cations on the nucleotide binding properties and enzyme activities of all variants.

Optimizing fluorescent protein expression for quantitative fluorescence microscopy and spectroscopy using herpes simplex thymidine kinase promoter sequences.

The modulation of expression levels of fluorescent fusion proteins (FFPs) is central for recombinant DNA technologies in modern biology as overexpression of proteins contributes to artifacts in biological experiments. In addition, some microscopy techniques such as fluorescence correlation spectroscopy (FCS) and single-molecule-based techniques are very sensitive to high expression levels of FFPs. To reduce the levels of recombinant protein expression in comparison with the commonly used, very strong CMV promoter, the herpes simplex virus thymidine kinase (TK) gene promoter, and mutants thereof were analyzed. Deletion mutants of the TK promoter were constructed and introduced into the Gateway® system for ectopic expression of enhanced green fluorescent protein (eGFP), monomeric cherry (mCherry), and FFPs containing these FPs. Two promoter constructs, TK2ST and TKTSC, were established, which have optimal low expression levels suitable for FCS studies in U2OS, HeLa CCL2, NIH 3T3, and BALB/c cells. Interestingly, when tested in these four cell lines, promoter constructs having a deletion within TK gene 5'-UTR showed significantly higher protein expression levels than the equivalent constructs lacking this deletion. This suggests that a negative regulatory element is localized within the TK gene 5'-UTR.

Interplay of DNA damage and cell cycle signaling at the level of human replication protein A.

Replication protein A (RPA) is the main human single-stranded DNA (ssDNA)-binding protein. It is essential for cellular DNA metabolism and has important functions in human cell cycle and DNA damage signaling. RPA is indispensable for accurate homologous recombination (HR)-based DNA double-strand break (DSB) repair and its activity is regulated by phosphorylation and other post-translational modifications. HR occurs only during S and G2 phases of the cell cycle. All three subunits of RPA contain phosphorylation sites but the exact set of HR-relevant phosphorylation sites on RPA is unknown. In this study, a high resolution capillary isoelectric focusing immunoassay, used under native conditions, revealed the isoforms of the RPA heterotrimer in control and damaged cell lysates in G2. Moreover, the phosphorylation sites of chromatin-bound and cytosolic RPA in S and G2 phases were identified by western and IEF analysis with all available phosphospecific antibodies for RPA2. Strikingly, most of the RPA heterotrimers in control G2 cells are phosphorylated with 5 isoforms containing up to 7 phosphates. These isoforms include RPA2 pSer23 and pSer33. DNA damaged cells in G2 had 9 isoforms with up to 14 phosphates. DNA damage isoforms contained pSer4/8, pSer12, pThr21, pSer23, and pSer33 on RPA2 and up to 8 unidentified phosphorylation sites.

PARG is dispensable for recovery from transient replicative stress but required to prevent detrimental accumulation of poly(ADP-ribose) upon prolonged replicative stress.

Poly(ADP-ribosyl)ation is involved in numerous bio-logical processes including DNA repair, transcription and cell death. Cellular levels of poly(ADP-ribose) (PAR) are regulated by PAR polymerases (PARPs) and the degrading enzyme PAR glycohydrolase (PARG), controlling the cell fate decision between life and death in response to DNA damage. Replication stress is a source of DNA damage, leading to transient stalling of replication forks or to their collapse followed by the generation of double-strand breaks (DSB). The involvement of PARP-1 in replicative stress response has been described, whereas the consequences of a deregulated PAR catabolism are not yet well established. Here, we show that PARG-deprived cells showed an enhanced sensitivity to the replication inhibitor hydroxyurea. PARG is dispensable to recover from transient replicative stress but is necessary to avoid massive PAR production upon prolonged replicative stress, conditions leading to fork collapse and DSB. Extensive PAR accumulation impairs replication protein A association with collapsed forks resulting in compromised DSB repair via homologous recombination. Our results highlight the critical role of PARG in tightly controlling PAR levels produced upon genotoxic stress to prevent the detrimental effects of PAR over-accumulation.

Basic fibroblast growth factor modifies the hypoxic response of human bone marrow stromal cells by ERK-mediated enhancement of HIF-1α activity.

Human bone marrow stromal cells (hBMSCs, also known as bone marrow-derived mesenchymal stem cells) are promising tools for the cellular therapy of human pathologies related to various forms of hypoxia. Although the current concepts of their clinical use include the expansion of hBMSC in standard cell culture conditions, the effect of the mitogen-driven ex vivo expansion on the adaptation to the hypoxic environment is unknown. Here, we provide data that the basic fibroblast growth factor (FGF2) enhances the induction of a wide range of hypoxia-related adaptive genes in hypoxic hBMSCs. We identified that the FGF2 signal is transmitted by the ERK pathway similar to that of hypoxia that also utilises the distal elements of the same signalling machinery including the extracellular signal-regulated kinase 1/2 (ERK1/2) and mitogen-activated protein kinase kinases (MEK1/2) in hBMSCs. We found that the simultaneous activation of ERK1/2 by FGF2 and hypoxia transforms the activation dynamics from oscillatory into sustained one. Activated ERKs co-localise with stabilised hypoxia inducible factor-1α (HIF-1α) followed by the reduction of its nuclear mobility as well as increased DNA binding capacity leading to the up-regulation of hypoxia-adaptive genes. Our findings indicate that the status of the ERK pathway has significant impacts on the molecular adaptation of hBMSCs to the hypoxic milieu.

Segregation of replicative DNA polymerases during S phase: DNA polymerase ε, but not DNA polymerases α/δ, are associated with lamins throughout S phase in human cells.

DNA polymerases (Pol) α, δ, and ε replicate the bulk of chromosomal DNA in eukaryotic cells, Pol ε being the main leading strand and Pol δ the lagging strand DNA polymerase. By applying chromatin immunoprecipitation (ChIP) and quantitative PCR we found that at G(1)/S arrest, all three DNA polymerases were enriched with DNA containing the early firing lamin B2 origin of replication and, 2 h after release from the block, with DNA containing the origin at the upstream promoter region of the MCM4 gene. Pol α, δ, and ε were released from these origins upon firing. All three DNA polymerases, Mcm3 and Cdc45, but not Orc2, still formed complexes in late S phase. Reciprocal ChIP of the three DNA polymerases revealed that at G(1)/S arrest and early in S phase, Pol α, δ, and ε were associated with the same nucleoprotein complexes, whereas in late S phase Pol ε and Pol α/δ were largely associated with distinct complexes. At G(1)/S arrest, the replicative DNA polymerases were associated with lamins, but in late S phase only Pol ε, not Pol α/δ, remained associated with lamins. Consistently, Pol ε, but not Pol δ, was found in nuclear matrix fraction throughout the cell cycle. Therefore, Pol ε and Pol α/δ seem to pursue their functions at least in part independently in late S phase, either by physical uncoupling of lagging strand maturation from the fork progression, or by recruitment of Pol δ, but not Pol ε, to post-replicative processes such as translesion synthesis or post-replicative repair.

Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy.

Eukaryotic DNA replication is a dynamic process requiring the co-operation of specific replication proteins. We measured the mobility of eGFP-Cdc45 by Fluorescence Correlation Spectroscopy (FCS) in vivo in asynchronous cells and in cells synchronized at the G1/S transition and during S phase. Our data show that eGFP-Cdc45 mobility is faster in G1/S transition compared to S phase suggesting that Cdc45 is part of larger protein complex formed in S phase. Furthermore, the size of complexes containing Cdc45 was estimated in asynchronous, G1/S and S phase-synchronized cells using gel filtration chromatography; these findings complemented the in vivo FCS data. Analysis of the mobility of eGFP-Cdc45 and the size of complexes containing Cdc45 and eGFP-Cdc45 after UVC-mediated DNA damage revealed no significant changes in diffusion rates and complex sizes using FCS and gel filtration chromatography analyses. This suggests that after UV-damage, Cdc45 is still present in a large multi-protein complex and that its mobility within living cells is consistently similar following UVC-mediated DNA damage.

Stimulation of BK virus DNA replication by NFI family transcription factors.

BK polyomavirus (BKV) establishes persistent, low-level, and asymptomatic infections in most humans and causes polyomavirus-associated nephropathy (PVAN) and other pathologies in some individuals. The activation of BKV replication following kidney transplantation, leading to viruria, viremia, and, ultimately, PVAN, is associated with immune suppression as well as inflammation and stress from ischemia-reperfusion injury of the allograft, but the stimuli and molecular mechanisms leading to these pathologies are not well defined. The replication of BKV DNA in cell cultures is regulated by the viral noncoding control region (NCCR) comprising the core origin and flanking sequences, to which BKV T antigen (Tag), cellular proteins, and small regulatory RNAs bind. Six nuclear factor I (NFI) binding sites occur in sequences flanking the late side of the core origin (the enhancer) of the archetype virus, and their mutation, either individually or in toto, reduces BKV DNA replication when placed in competition with templates containing intact BKV NCCRs. NFI family members interacted with the helicase domain of BKV Tag in pulldown assays, suggesting that NFI helps recruit Tag to the viral core origin and may modulate its function. However, Tag may not be the sole target of the replication-modulatory activities of NFI: the NFIC/CTF1 isotype stimulates BKV template replication in vitro at low concentrations of DNA polymerase-α primase (Pol-primase), and the p58 subunit of Pol-primase associates with NFIC/CTF1, suggesting that NFI also recruits Pol-primase to the NCCR. These results suggest that NFI proteins (and the signaling pathways that target them) activate BKV replication and contribute to the consequent pathologies caused by acute infection.

Modification of histones by sugar ß-N-acetylglucosamine (GlcNAc) occurs on multiple residues, including histone H3 serine 10, and is cell cycle-regulated.

The monosaccharide, ß-N-acetylglucosamine (GlcNAc), can be added to the hydroxyl group of either serines or threonines to generate an O-linked ß-N-acetylglucosamine (O-GlcNAc) residue (Love, D. C., and Hanover, J. A. (2005) Sci. STKE 2005 312, 1-14; Hart, G. W., Housley, M. P., and Slawson, C. (2007) Nature 446, 1017-1022). This post-translational protein modification, termed O-GlcNAcylation, is reversible, analogous to phosphorylation, and has been implicated in many cellular processes. Here, we present evidence that in human cells all four core histones of the nucleosome are substrates for this glycosylation in the relative abundance H3, H4/H2B, and H2A. Increasing the intracellular level of UDP-GlcNAc, the nucleotide sugar donor substrate for O-GlcNAcylation enhanced histone O-GlcNAcylation and partially suppressed phosphorylation of histone H3 at serine 10 (H3S10ph). Expression of recombinant H3.3 harboring an S10A mutation abrogated histone H3 O-GlcNAcylation relative to its wild-type version, consistent with H3S10 being a site of histone O-GlcNAcylation (H3S10glc). Moreover, O-GlcNAcylated histones were lost from H3S10ph immunoprecipitates, whereas immunoprecipitation of either H3K4me3 or H3K9me3 (active or inactive histone marks, respectively) resulted in co-immunoprecipitation of O-GlcNAcylated histones. We also examined histone O-GlcNAcylation during cell cycle progression. Histone O-GlcNAcylation is high in G(1) cells, declines throughout the S phase, increases again during late S/early G(2), and persists through late G(2) and mitosis. Thus, O-GlcNAcylation is a novel histone post-translational modification regulating chromatin conformation during transcription and cell cycle progression.

Inhibition of human BK polyomavirus replication by small noncoding RNAs.

Small noncoding RNAs regulate a variety of cellular processes, including genomic imprinting, chromatin remodeling, replication, transcription, and translation. Here, we report small replication-regulating RNAs (srRNAs) that specifically inhibit DNA replication of the human BK polyomavirus (BKV) in vitro and in vivo. srRNAs from FM3A murine mammary tumor cells were enriched by DNA replication assay-guided fractionation and hybridization to the BKV noncoding control region (NCCR) and synthesized as cDNAs. Selective mutagenesis of the cDNA sequences and their putative targets suggests that the inhibition of BKV DNA replication is mediated by srRNAs binding to the viral NCCR, hindering early steps in the initiation of DNA replication. Ectopic expression of srRNAs in human cells inhibited BKV DNA replication in vivo. Additional srRNAs were designed and synthesized that specifically inhibit simian virus 40 (SV40) DNA replication in vitro. These observations point to novel mechanisms for regulating DNA replication and suggest the design of synthetic agents for inhibiting replication of polyomaviruses and possibly other viruses.

Host-specific replication of BK virus DNA in mouse cell extracts is independently controlled by DNA polymerase alpha-primase and inhibitory activities.

The activation of the human polyomavirus BK causes polyomavirus-associated nephropathy in immunocompromised humans. Studies of the virus have been restricted since the virus DNA replication is species specific. Cell-based and cell-free DNA replication systems, including the BK virus (BKV) monopolymerase DNA replication system using purified proteins, reproduce the species specificity (28). Therefore, the major host proteins comprising this assay, DNA polymerase alpha-primase (Pol-prim) and replication protein A (RPA), were intensively studied here. We demonstrate that Pol-prim plays a major role in the species specificity of BKV DNA replication. Both large subunits p180 and p68 of the enzyme complex have central functions in modulating the host specificity. Recently, an inhibitory activity of BKV DNA replication was described (C. Mahon, B. Liang, I. Tikhanovich, J. R. Abend, M. J. Imperiale, H. P. Nasheuer, and W. R. Folk, J. Virol. 83:5708-5717, 2009), but neither mouse Pol-prim nor mouse RPA diminishes cell-free BKV DNA replication. However, the inhibition of BKV DNA replication in mouse extracts depends on sequences flanking the core origin. In the presence of human Pol-prim, the inhibitory effect of mouse cell factors is abolished with plasmid DNAs containing the murine polyomavirus early promoter region, whereas the late enhancer region and the core origin are supplied from BKV. Thus, BKV replication is regulated by both Pol-prim, as a core origin species-specific factor, and inhibitory activities, as origin-flanking sequence-dependent factor(s).

Eukaryotic single-stranded DNA binding proteins: central factors in genome stability.

The single-stranded DNA binding proteins (SSBs) are required to maintain the integrity of the genome in all organisms. Replication protein A (RPA) is a nuclear SSB protein found in all eukaryotes and is required for multiple processes in DNA metabolism such as DNA replication, DNA repair, DNA recombination, telomere maintenance and DNA damage signalling. RPA is a heterotrimeric complex, binds ssDNA with high affinity, and interacts specifically with multiple proteins to fulfil its function in eukaryotes. RPA is phosphorylated in a cell cycle and DNA damage-dependent manner with evidence suggesting that phosphorylation has an important function in modulating the cellular DNA damage response. Considering the DNA-binding properties of RPA a mechanism of "molecular counting" to initiate DNA damage-dependent signalling is discussed. Recently a human homologue to the RPA2 subunit, called RPA4, was discovered and RPA4 can substitute for RPA2 in the RPA complex resulting in an "alternative" RPA (aRPA), which can bind to ssDNA with similar affinity as canonical RPA. Additional human SSBs, hSSB1 and hSSB2, were recently identified, with hSSB1 being localized in the nucleus and having implications in DNA repair. Mitochondrial SSBs (mtSSBs) have been found in all eukaryotes studied. mtSSBs are related to prokaryotic SSBs and essential to main the genome stability in eukaryotic mitochondria. Recently human mtSSB was identified as a novel binding partner of p53 and that it is able to stimulate the intrinsic exonuclease activity of p53. These findings and recent results associated with mutations in RPA suggest a link of SSBs to cancer.

The use of transgenic mice in cancer and genome stability research.

The development of effective cancer therapeutics is an important goal of modern biomedical sciences. To identify potential cancer therapeutic targets, the processes involved in tumorigenesis must be understood at all levels, which requires the development of model systems accurately mimicing tumor development. Cancer is the general name given to a variety of complex diseases characterised by uncontrolled cell proliferation. Cancer development is dependent not only on the changes occurring within the transformed cells, but also on the interactions of the cells with their microenvironment. The majority of our current understanding of carcinogenesis comes from the in vitro analysis of late-stage tumor tissue removed from cancer patients. While this has elucidated many genomic changes experienced by cancer cells, it provides little information about the factors influencing early-stage cancer development in vivo. Also certain hallmarks of cancer, such as metastasis and angiogenesis, are impossible to study in vitro. The mouse has become an important model for studying the in vivo aspects of human cancer development. Transgenic mouse models have been engineered to develop cancers, which accurately mimic their human counterparts, and have potential applications to test the effectiveness of novel cancer therapeutics. One of the most promising transgenic mouse models of human cancer arises from mice engineered with genomic instability. These transgenic models have been shown to develop human-like cancers and have the potential to provide insights into the molecular events occurring in earliest stages of tumorigenesis in vivo.