Role of in Human Disease Research 3.0.

has become a commonly used animal model for biomedical research in a variety of areas [...

A simple bypass assay for DNA polymerases shows that cancer-associated hypermutating variants exhibit differences in vitro.

Errors made by DNA polymerases contribute to both natural variation and, in extreme cases, genome instability and its associated diseases. Recently, the importance of polymerase misincorporation in disease has been highlighted by the identification of cancer-associated polymerase variants with mutations in the exonuclease domain. A subgroup of these variants have a hypermutation phenotype in tumours, and when modelled in yeast, they show mutation rates in excess of that seen with polymerase with simple loss of proofreading activity.

Association of Mutations in Replicative DNA Polymerase Genes with Human Disease: Possible Application of Models for Studies.

Replicative DNA polymerases, such as DNA polymerase α-primase, δ and ε, are multi-subunit complexes that are responsible for the bulk of nuclear DNA replication during the S phase. Over the last decade, extensive genome-wide association studies and expression profiling studies of the replicative DNA polymerase genes in human patients have revealed a link between the replicative DNA polymerase genes and various human diseases and disorders including cancer, intellectual disability, microcephalic primordial dwarfism and immunodeficiency. These studies suggest the importance of dissecting the mechanisms involved in the functioning of replicative DNA polymerases in understanding and treating a range of human diseases.

The role of insulators and transcription in 3D chromatin organization of flies.

The DNA in many organisms, including humans, is shown to be organized in topologically associating domains (TADs). In , several architectural proteins are enriched at TAD borders, but it is still unclear whether these proteins play a functional role in the formation and maintenance of TADs. Here, we show that depletion of BEAF-32, Cp190, Chro, and Dref leads to changes in TAD organization and chromatin loops.

Expression of the cancer-associated DNA polymerase ε P286R in fission yeast leads to translesion synthesis polymerase dependent hypermutation and defective DNA replication.

Somatic and germline mutations in the proofreading domain of the replicative DNA polymerase ε (POLE-exonuclease domain mutations, POLE-EDMs) are frequently found in colorectal and endometrial cancers and, occasionally, in other tumours. POLE-associated cancers typically display hypermutation, and a unique mutational signature, with a predominance of C > A transversions in the context TCT and C > T transitions in the context TCG. To understand better the contribution of hypermutagenesis to tumour development, we have modelled the most recurrent POLE-EDM (POLE-P286R) in Schizosaccharomyces pombe.

Drosophila telomere capping protein HOAP interacts with DSB sensor proteins Mre11 and Nbs.

In eukaryotes, specific DNA-protein structures called telomeres exist at linear chromosome ends. Telomere stability is maintained by a specific capping protein complex. This capping complex is essential for the inhibition of the DNA damage response (DDR) at telomeres and contributes to genome integrity.

The DNA polymerases of .

DNA synthesis during replication or repair is a fundamental cellular process that is catalyzed by a set of evolutionary conserved polymerases. Despite a large body of research, the DNA polymerases of have not yet been systematically reviewed, leading to inconsistencies in their nomenclature, shortcomings in their functional (Gene Ontology, GO) annotations and an under-appreciation of the extent of their characterization. Here, we describe the complete set of DNA polymerases in , applying nomenclature already in widespread use in other species, and improving their functional annotation.

Diseases Associated with Mutation of Replication and Repair Proteins.

Alterations in proteins that function in DNA replication and repair have been implicated in the development of human diseases including cancer, premature ageing, skeletal disorders, mental retardation, microcephaly, and neurodegeneration. Drosophila has orthologues of most human replication and repair proteins and high conservation of the relevant cellular pathways, thus providing a versatile system in which to study how these pathways are corrupted leading to the diseased state. In this chapter I will briefly review the diseases associated with defects in replication and repair proteins and discuss how past and future studies on the Drosophila orthologues of such proteins can contribute to the dissection of the mechanisms involved in disease development.

Novel roles of HP1a and Mcm10 in DNA replication, genome maintenance and photoreceptor cell differentiation.

Both Mcm10 and HP1a are known to be required for DNA replication. However, underlying mechanism is not clarified yet especially for HP1. Knockdown of both HP1a and Mcm10 genes inhibited the progression of S phase in Drosophila eye imaginal discs.

DNA polymerase α interacts with PrSet7 and mediates H4K20 monomethylation in Drosophila.

In human cells, appropriate monomethylation of histone H4 lysine 20 by PrSet7 (also known as SET8 and SETD7) is important for the correct transcription of specific genes and timely progression through the cell cycle. Over-methylation appears to be prevented through the interaction of PrSet7 with proliferating cell nuclear antigen (PCNA), which targets PrSet7 for destruction through the pathway mediated by CRL4(C) (dt2) (the cullin ring finger ligase-4 complex containing Cdt2). However, the factors involved in positive regulation of PrSet7 histone methylation remain undefined.

Drosophila Mcm10 is required for DNA replication and differentiation in the compound eye.

Mini chromosome maintenance 10 (Mcm10) is an essential protein, which is conserved from S. cerevisiae to Drosophila and human, and is required for the initiation of DNA replication. Knockdown of Drosophila Mcm10 (dMcm10) by RNA interference in eye imaginal discs induces abnormal eye morphology (rough eye phenotype), and the number of ommatidia is decreased in adult eyes.

Functional analysis of Drosophila DNA polymerase ε p58 subunit.

DNA polymerase ε (polε) plays a central role in DNA replication in eukaryotic cells, and has been suggested to the main synthetic polymerase on the leading strand. It is a hetero-tetrameric enzyme, comprising a large catalytic subunit (the A subunit ~250 kDa), a B subunit of ~60 kDa in most species (~80 kDa in budding yeast) and two smaller subunits (each ~20 kDa). In Drosophila, two subunits of polε (dpolε) have been identified.

Drosophila RecQ4 is directly involved in both DNA replication and the response to UV damage in S2 cells.

The RecQ4 protein shows homology to both the S.cerevisiae DNA replication protein Sld2 and the DNA repair related RecQ helicases. Experimental data also suggest replication and repair functions for RecQ4, but the precise details of its involvement remain to be clarified.

Forced binding of the origin of replication complex to chromosomal sites in Drosophila S2 cells creates an origin of replication.

Origins of replication in higher eukaryotes appear to lack specific sequence characteristics and those mapped often appear to be spread over several kilobases. This has complicated the study of site-specific events at origins of replication in vivo. Here we show that fusion of a Gal4-binding domain to proteins of the origin of replication complex (Orc) is sufficient to direct initiation to Gal4-binding sites inserted in the Drosophila S2 cell chromosome.

Decreased MCM2-6 in Drosophila S2 cells does not generate significant DNA damage or cause a marked increase in sensitivity to replication interference.

A reduction in the level of some MCM proteins in human cancer cells (MCM5 in U20S cells or MCM3 in Hela cells) causes a rapid increase in the level of DNA damage under normal conditions of cell proliferation and a loss of viability when the cells are subjected to replication interference. Here we show that Drosophila S2 cells do not appear to show the same degree of sensitivity to MCM2-6 reduction. Under normal cell growth conditions a reduction of >95% in the levels of MCM3, 5, and 6 causes no significant short term alteration in the parameters of DNA replication or increase in DNA damage.

GINS inactivation phenotypes reveal two pathways for chromatin association of replicative alpha and epsilon DNA polymerases in fission yeast.

The tetrameric GINS complex, consisting of Sld5-Psf1-Psf2-Psf3, plays an essential role in the initiation and elongation steps of eukaryotic DNA replication, although its biochemical function is unclear. Here we investigate the function of GINS in fission yeast, using fusion of Psf1 and Psf2 subunits to a steroid hormone-binding domain (HBD) to make GINS function conditional on the presence of beta-estradiol. We show that inactivation of Psf1-HBD causes a tight but rapidly reversible DNA replication arrest phenotype.

DNAReplication: a database of information and resources for the eukaryotic DNA replication community.

DNAReplication (at http://www.dnareplication.net) has been set up as a freely available single resource to facilitate access to information on eukaryotic DNA replication.

The human TPR protein TTC4 is a putative Hsp90 co-chaperone which interacts with CDC6 and shows alterations in transformed cells.

Background: The human TTC4 protein is a TPR (tetratricopeptide repeat) motif-containing protein. The gene was originally identified as being localized in a genomic region linked to breast cancer and subsequent studies on melanoma cell lines revealed point mutations in the TTC4 protein that may be associated with the progression of malignant melanoma.

Methodology/principle Findings: Here we show that TTC4 is a nucleoplasmic protein which interacts with HSP90 and HSP70, and also with the replication protein CDC6.

The Drosophila Df31 protein interacts with histone H3 tails and promotes chromatin bridging in vitro.

Df31 is a small hydrophilic protein from Drosophila melanogaster that can act as a histone chaperone in vitro. The protein is also detected as an integral component of chromatin, present at approximately the same level as histone H1. We have developed a simple assay to measure protein binding to oligonucleosomes and used it to characterise the DF31-oligonucleosome interaction.

Differential requirements for MCM proteins in DNA replication in Drosophila S2 cells.

Background: The MCM2-7 proteins are crucial components of the pre replication complex (preRC) in eukaryotes. Since they are significantly more abundant than other preRC components, we were interested in determining whether the entire cellular content was necessary for DNA replication in vivo.

Methodology/principle Findings: We performed a systematic depletion of the MCM proteins in Drosophila S2 cells using dsRNA-interference.

Drosophila CAP-D2 is required for condensin complex stability and resolution of sister chromatids.

The precise mechanism of chromosome condensation and decondensation remains a mystery, despite progress over the last 20 years aimed at identifying components essential to the mitotic compaction of the genome. In this study, we analyse the localization and role of the CAP-D2 non-SMC condensin subunit and its effect on the stability of the condensin complex. We demonstrate that a condensin complex exists in Drosophila embryos, containing CAP-D2, the anticipated SMC2 and SMC4 proteins, the CAP-H/Barren and CAP-G (non-SMC) subunits.

The Drosophila Cdc6/18 protein has functions in both early and late S phase in S2 cells.

The Cdc6/18 protein has been mainly characterised for its role in the initiation of DNA replication. Several studies exist, however, which suggest that it may also have a role in controlling the G2/M transition. Here we present studies on the Drosophila Cdc6 (DmCdc6) protein that support this dual function for the protein.

Enigmatic variations: divergent modes of regulating eukaryotic DNA replication.

Proteins involved in DNA replication are conserved from yeast to mammals, suggesting that the mechanism was established at an early stage of eukaryotic evolution. In spite of this common origin, recent findings have revealed surprising variations in how replication initiation is controlled, implying that a conserved mechanism has not necessarily resulted in regulatory conservation.

Depletion of Drad21/Scc1 in Drosophila cells leads to instability of the cohesin complex and disruption of mitotic progression.

Background: The coordination of cell cycle events is necessary to ensure the proper duplication and dissemination of the genome. In this study, we examine the consequences of depleting Drad21 and SA, two non-SMC subunits of the cohesin complex, by dsRNA-mediated interference in Drosophila cultured cells.

Results: We have shown that a bona fide cohesin complex exists in Drosophila embryos.