Identification of novel DNA repair proteins via primary sequence, secondary structure, and homology

<p>Abstract</p> <p>Background</p> <p>DNA repair is the general term for the collection of critical mechanisms which repair many forms of DNA damage such as methylation or ionizing radiation. DNA repair has mainly been studied in experimental and clinical situations, and relatively few information-based approaches to new extracting DNA repair knowledge exist. As a first step, automatic detection of DNA repair proteins in genomes via informatics techniques is desirable; however, there are many forms of DNA repair and it is not a straightforward process to identify and classify repair proteins with a single optimal method. We perform a study of the ability of homology and machine learning-based methods to identify and classify DNA repair proteins, as well as scan vertebrate genomes for the presence of novel repair proteins. Combinations of primary sequence polypeptide frequency, secondary structure, and homology information are used as feature information for input to a Support Vector Machine (SVM).</p> <p>Results</p> <p>We identify that SVM techniques are capable of identifying portions of DNA repair protein datasets without admitting false positives; at low levels of false positive tolerance, homology can also identify and classify proteins with good performance. Secondary structure information provides improved performance compared to using primary structure alone. Furthermore, we observe that machine learning methods incorporating homology information perform best when data is filtered by some clustering technique. Analysis by applying these methodologies to the scanning of multiple vertebrate genomes confirms a positive correlation between the size of a genome and the number of DNA repair protein transcripts it is likely to contain, and simultaneously suggests that all organisms have a non-zero minimum number of repair genes. In addition, the scan result clusters several organisms' repair abilities in an evolutionarily consistent fashion. Analysis also identifies several functionally unconfirmed proteins that are highly likely to be involved in the repair process. A new web service, INTREPED, has been made available for the immediate search and annotation of DNA repair proteins in newly sequenced genomes.</p> <p>Conclusion</p> <p>Despite complexity due to a multitude of repair pathways, combinations of sequence, structure, and homology with Support Vector Machines offer good methods in addition to existing homology searches for DNA repair protein identification and functional annotation. Most importantly, this study has uncovered relationships between the size of a genome and a genome's available repair repetoire, and offers a number of new predictions as well as a prediction service, both which reduce the search time and cost for novel repair genes and proteins.</p

Analysis of the Genome of the Sexually Transmitted Insect Virus Helicoverpa zea Nudivirus 2

The sexually transmitted insect virus Helicoverpa zea nudivirus 2 (HzNV-2) was determined to have a circular double-stranded DNA genome of 231,621 bp coding for an estimated 113 open reading frames (ORFs). HzNV-2 is most closely related to the nudiviruses, a sister group of the insect baculoviruses. Several putative ORFs that share homology with the baculovirus core genes were identified in the viral genome. However, HzNV-2 lacks several key genetic features of baculoviruses including the late transcriptional regulation factor, LEF-1 and the palindromic hrs, which serve as origins of replication. The HzNV-2 genome was found to code for three ORFs that had significant sequence homology to cellular genes which are not generally found in viral genomes. These included a presumed juvenile hormone esterase gene, a gene coding for a putative zinc-dependent matrix metalloprotease, and a major facilitator superfamily protein gene; all of which are believed to play a role in the cellular proliferation and the tissue hypertrophy observed in the malformation of reproductive organs observed in HzNV-2 infected corn earworm moths, Helicoverpa zea

Rad54: the Swiss Army knife of homologous recombination?

Homologous recombination (HR) is a ubiquitous cellular pathway that mediates transfer of genetic information between homologous or near homologous (homeologous) DNA sequences. During meiosis it ensures proper chromosome segregation in the first division. Moreover, HR is critical for the tolerance and repair of DNA damage, as well as in the recovery of stalled and broken replication forks. Together these functions preserve genomic stability and assure high fidelity transmission of the genetic material in the mitotic and meiotic cell divisions. This review will focus on the Rad54 protein, a member of the Snf2-family of SF2 helicases, which translocates on dsDNA but does not display strand displacement activity typical for a helicase. A wealth of genetic, cytological, biochemical and structural data suggests that Rad54 is a core factor of HR, possibly acting at multiple stages during HR in concert with the central homologous pairing protein Rad51

Reconstitution of recombination-associated DNA synthesis with human proteins.

The repair of DNA breaks by homologous recombination is a high-fidelity process, necessary for the maintenance of genome integrity. Thus, DNA synthesis associated with recombinational repair must be largely error-free. In this report, we show that human DNA polymerase delta (δ) is capable of robust DNA synthesis at RAD51-mediated recombination intermediates dependent on the processivity clamp PCNA. Translesion synthesis polymerase eta (η) also extends these substrates, albeit far less processively. The single-stranded DNA binding protein RPA facilitates recombination-mediated DNA synthesis by increasing the efficiency of primer utilization, preventing polymerase stalling at specific sequence contexts, and overcoming polymerase stalling caused by topological constraint allowing the transition to a migrating D-loop. Our results support a model whereby the high-fidelity replicative DNA polymerase δ performs recombination-associated DNA synthesis, with translesion synthesis polymerases providing a supportive role as in normal replication

AIP1 is a novel Agenet/Tudor domain protein from Arabidopsis that interacts with regulators of DNA replication, transcription and chromatin remodeling

Background: DNA replication and transcription are dynamic processes regulating plant development that are dependent on the chromatin accessibility. Proteins belonging to the Agenet/Tudor domain family are known as histone modification "readers" and classified as chromatin remodeling proteins. Histone modifications and chromatin remodeling have profound effects on gene expression as well as on DNA replication, but how these processes are integrated has not been completely elucidated. It is clear that members of the Agenet/Tudor family are important regulators of development playing roles not well known in plants. Methods: Bioinformatics and phylogenetic analyses of the Agenet/Tudor Family domain in the plant kingdom were carried out with sequences from available complete genomes databases. 3D structure predictions of Agenet/Tudor domains were calculated by I-TASSER server. Protein interactions were tested in two-hybrid, GST pulldown, semi-in vivo pulldown and Tandem Affinity Purification assays. Gene function was studied in a T-DNA insertion GABI-line. Results: In the present work we analyzed the family of Agenet/Tudor domain proteins in the plant kingdom and we mapped the organization of this family throughout plant evolution. Furthermore, we characterized a member from Arabidopsis thaliana named AIP1 that harbors Agenet/Tudor and DUF724 domains. AIP1 interacts with ABAP1, a plant regulator of DNA replication licensing and gene transcription, with a plant histone modification "reader" (LHP1) and with non modified histones. AIP1 is expressed in reproductive tissues and its down-regulation delays flower development timing. Also, expression of ABAP1 and LHP1 target genes were repressed in flower buds of plants with reduced levels of AIP1. Conclusions: AIP1 is a novel Agenet/Tudor domain protein in plants that could act as a link between DNA replication, transcription and chromatin remodeling during flower development

ATM in focus:a damage sensor and cancer target

The ability of a cell to conserve and maintain its native DNA sequence is fundamental for the survival and normal functioning of the whole organism and protection from cancer development. Here we review recently obtained results and current topics concerning the role of the ataxia-telangiectasia mutated (ATM) protein kinase as a damage sensor and its potential as therapeutic target for treating cancer. This monograph discusses DNA repair mechanisms activated after DNA double-strand breaks (DSBs), i.e. non-homologous end joining, homologous recombination and single strand annealing and the role of ATM in the above types of repair. In addition to DNA repair, ATM participates in a diverse set of physiological processes involving metabolic regulation, oxidative stress, transcriptional modulation, protein degradation and cell proliferation. Full understanding of the complexity of ATM functions and the design of therapeutics that modulate its activity to combat diseases such as cancer necessitates parallel theoretical and experimental efforts. This could be best addressed by employing a systems biology approach, involving mathematical modelling of cell signalling pathways

Structural and Functional Characterization of Deinococcal DNA Damage Response A (DdrA)

Deinococci exhibit a remarkable resilience toward DNA damage through the actions of several unique proteins, including DdrA. Although DdrA is critical for damage resistance, little is known about its mechanism of action. Despite sharing sequence similarity with Rad52, DdrA has been reported to lack single-stranded DNA annealing activity. In order to better characterize DdrA, structural studies were undertaken with the primary objective of gaining insight into the mechanism by which DdrA functions. Significant progress was made toward elucidating the X-ray crystal structure; in particular, identifying suitable DdrA domain boundaries for successful expression, purification and crystallization. In addition, we demonstrate for the first time that DdrA mediates ssDNA annealing to levels comparable to Rad52 in vitro. Residues (K22 and K105) critical for ssDNA binding and annealing were identified and further used to demonstrate that DdrA mediates resistance to extreme levels of DNA damage through its ability to anneal ssDNA in vivo

Vaccinia protein C16 blocks innate immune sensing of DNA by binding the Ku complex

VACV gene C16L encodes a 37-kDa protein that is highly conserved in orthopoxviruses and functions as an immunomodulator. Intranasal infection of mice with a virus lacking C16L (vΔC16) induced less weight loss, fewer signs of illness and increased infiltration of leukocytes to the lungs compared with wild-type virus. To understand C16’s mechanism of action, tandem affinity purification and mass spectrometry were used to identify C16 binding partners. This revealed that Ku70, Ku80 and PHD2 interact with C16 in cells. Ku70 and Ku80 constitute the Ku heterodimer, a well characterised DNA repair complex. MEFs lacking Ku, or the other component of the DNA-dependent protein kinase (DNA-PK) complex, the catalytic subunit of DNA-PK (DNA-PKcs), were shown to be deficient in the upregulation of IRF-3-dependent genes such as Cxcl10, Il6 and Ifnb in response to transfection of DNA, but not poly (I:C). Furthermore, following infection of MEFs with VACV strain MVA the activation of Cxcl10 or Il6 transcription was dependent on DNA-PK. Therefore, DNA-PK is a DNA sensor capable of detecting poxvirus DNA and activating IRF-3-dependent innate immunity. C16 inhibited the binding of Ku to DNA, and therefore inhibited DNA-mediated induction of Cxcl10 and Il-6 in MEFs. The role of C16 in vivo was also examined: infection with vΔC16 led to increased production of Cxcl10 and Il-6 following intranasal infection of mice compared with wild-type virus. C16 is therefore an inhibitor of DNA-PK-mediated DNA sensing and innate immune activation. C16 was also shown to bind to PHD2, an enzyme involved in regulation of hypoxic signalling. VACV was found to activate the transcription of hypoxia-related genes, and C16 expression in cells was also capable of doing this. The role of hypoxic signalling in VACV infection remains poorly understood

An investigation into the biosynthesis of proximicins

PhD ThesisThe proximicins are a family of three compounds – A-C – produced by two marine Actinomycete Verrucosispora strains – V. maris AB18-032 and V. sp. str. 37 - and are characterised by the presence of 2,4-disubstituted furan rings. Proximicins demonstrate cell-arresting and antimicrobial ability, making them interesting leads for clinical drug development. Proximicin research has been largely overshadowed by other Verrucosispora strain secondary metabolites (SM), and despite the publication of the V. maris AB18-032 draft, the enzymatic machinery responsible for their production has not been established. It has been noted in related research into a pyrrole-containing homolog – congocidine –due to the structural similarity exhibited, proximicins likely utilise a similar biosynthetic route. The initial aim of this research was to confirm the presumed pathway to proximicin biosynthesis. Following the sequencing, assembly and annotation of the second proximicin producer, Verrucosispora sp. str. MG37, and genome mining of V. maris AB18-032, no common clusters mimicked that of congocidine, casting doubt on the previously assumed analogous biosynthetic routes. A putative proximicin biosynthesis (ppb) cluster was identified, containing non-ribosomal peptide synthetase (NRPS) enzymes, exhibiting some homology with congocidine. NRPSsystems represent a network of interacting proteins, which act as a SM assembly line: crucially, adenylation (A)- domain enzymes act as the ‘gate-keeper’, determining which precursors are included into the elongating peptide. To elucidate the route to proximicins, activity characterisation of the four A-domains present in ppb cluster was attempted. The A-domain Ppb120 was shown to possess novel activity, demonstrating a high promiscuity towards heterocycle containing precursors, in addition to the absence of an apparent essential domain. This discovery refutes previous work outlining the core residues which dictate A-domain activity, while also presenting a facile route to novel heterocycle-containing compounds. Despite extensive work, A-domains ppb195 and ppb210, were ineffectively purified in the active form – informing future work into A-domains activity characterisation. Finally, the ppb220 A-domain which lies at the border of ppb, was inactive suggesting over-estimation of the cluster margins. To confirm ppb220 redundancy and confirm ppb boundaries, CRISPR/Cas gene editing studies were done. The gene responsible for the orange pigment of Verrucosispora strains was initially targeted and successfully deleted, and ppb studies commenced. The research here refutes the previously presumed route to proximicin biosynthesis; the ppb cluster instead comprises enzymes exhibiting unique activity and structure. The findings represent the foundations for allowing exploitation of chemistry exhibited within the proximicin family. The novelty exhibited can be utilised in the search for antimicrobial clinical leads, by allowing the production of compounds containing previously inaccessible heterocycle chemistry

Papilloomiviiruse transkriptsiooni ja regulaatorvalgu E2 uurimine

Väitekirja elektrooniline versioon ei sisalda publikatsioonePapilloomiviirused on viiruste perekond, mis nakatab nii inimeste kui loomade epiteelrakke ja põhjustab papilloome ehk näsakasvajaid, mis võivad teatud tingimustes areneda halvaloomulisteks kasvajateks. Inimese papilloomiviiruseid on teada üle 200 tüübi ja nad jagatakse kaheks grupiks nende koespetsiifilisuse alusel: limaskesti nakatavad papilloomiviirused ja naharakke nakatavad papilloomiviirused. Naha papilloomiviirused on siiani pälvinud vähem tähelepanu, sest nende võimalik seos nahakasvajatega on tulnud ilmsiks alles viimasel kümnendil. Meie keskendusime oma töös naha papilloomiviirusele HPV5, mida on viimasel ajal hakatud seostama nahakasvajate tekkega, aga mida on siiani ikkagi suhteliselt vähe uuritud. Meie töö tulemusena valmis HPV5 transkriptsiooni kaart, mille iseloomustamiseks me kasutasime inimese sääreluu kasvaja rakuliini U2OS. Selle rakuliini abil õnnestus meil kirjeldada 14 erinevat viiruse mRNAd. Edasises töös keskendusime viirusvalgu E2 uurimisele, mis on põhiline viiruse elutsükli regulaator. E2 valgul on lisaks täispikale valgule veel kaks isovormi, valgu C-terminaalset osa sisaldav E2C ja alternatiivse splaissinguga saadud E8/E2. Me uurisime E2 paiknemist rakus ja leidsime, et lühike E8 valgu järjestus on piisav selleks, et valk suunata raku tuuma. Meile teadaolevalt on see järjestus lühim tuuma suunav järjestus, mis on siiani teada. Tänu proteoomika arengule on siiani leitud üle 200 E2 partnervalgu, millest enamuse funktsioon viiruse elutsüklis on siiani teadmata. Meie leidsime uue E2-ga seonduva rakulise valgu, milleks on tuumamüosiin 1. See on esimene müosiini perekonda kuuluv E2 partnervalk ja katsed näitasid, et tuumamüosiin 1 mõjutab HPV5 DNA paljunemist rakus.Cutaneous papillomaviruses infect human cutaneous epithelium and in most cases these infections pass without symptoms, but in some instances, they can cause lesions and induce squamous cell carcinomas. One of the most prevalent virus types detected in skin cancer is HPV5. We tried to bring through our work more understanding about the properties of these viruses and focused our attention to HPV5 and to the first part of its life-cycle in the cells. We wanted to characterize HPV5 more thoroughly at RNA level and managed to identify HPV transcription map. We also studied papillomavirus main regulator protein E2, which influences all main viral life events. We studied E2 localization in the cell and identified short nuclear targeting signal, that sends proteins to the nucleus. We also found new E2 interaction partner nuclear myosin 1, which influences HPV5 replication