Reduced excision repair cross-complementing 1 expression associates with enhanced papilloma formation and fibroblast transformation after genetic disruption of secreted protein acidic and rich in cysteine

SPARC (secreted protein acidic and rich in cysteine)/ osteonectin/BM-40 is a matricellular protein implicated in development, cell transformation and tumorigenesis. We have examined the role of SPARC in cell transformation induced chemically with 7,12-dimethylbenz[a]anthracene (DMBA) and 12- tetradecanoylphorbol-13-acetate (TPA) in embryonic fibroblasts and in the skin of mice. Embryonic fibroblasts from SPARCnull mice showed increases in cell proliferation, enhanced sensitivity to DMBA and a higher number of DMBA/TPA-induced transformation foci. The number of DMBA-DNA adducts was 9 times higher in SPARCnull fibroblasts and their stability was lower than wild-type fibroblasts, consistent with a reduction in excision repair cross-complementing 1 the nucleotide excision repair enzyme in these cells. The SPARCnull mice showed an increase in both the speed and number of papillomas forming after topical administration of DMBA/TPA to the skin. These papillomas showed reduced growth and reduced progression to a more malignant phenotype, indicating that the effect of SPARC on tumorigenesis depends upon the transformation stage and/or tissue context. These data reinforce a growing number of observations in which SPARC has shown opposite effects on different tumor types/stages.

Multiple human single-stranded DNA binding proteins function in genome maintenance : structural, biochemical and functional analysis

DNA exists predominantly in a duplex form that is preserved via specific base pairing. This base pairing affords a considerable degree of protection against chemical or physical damage and preserves coding potential. However, there are many situations, e.g. during DNA damage and programmed cellular processes such as DNA replication and transcription, in which the DNA duplex is separated into two singlestranded DNA (ssDNA) strands. This ssDNA is vulnerable to attack by nucleases, binding by inappropriate proteins and chemical attack. It is very important to control the generation of ssDNA and protect it when it forms, and for this reason all cellular organisms and many viruses encode a ssDNA binding protein (SSB). All known SSBs use an oligosaccharide/oligonucleotide binding (OB)-fold domain for DNA binding. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating strand-exchange proteins and helicases, and mediation of protein–protein interactions. Recently two additional human SSBs have been identified that are more closely related to bacterial and archaeal SSBs. Prior to this it was believed that replication protein A, RPA, was the only human equivalent of bacterial SSB. RPA is thought to be required for most aspects of DNA metabolism including DNA replication, recombination and repair. This review will discuss in further detail the biological pathways in which human SSBs function.

Human single-stranded DNA binding proteins are essential for maintaining genomic stability

The double-stranded conformation of cellular DNA is a central aspect of DNA stabilisation and protection. The helix preserves the genetic code against chemical and enzymatic degradation, metabolic activation, and formation of secondary structures. However, there are various instances where single-stranded DNA is exposed, such as during replication or transcription, in the synthesis of chromosome ends, and following DNA damage. In these instances, single-stranded DNA binding proteins are essential for the sequestration and processing of single-stranded DNA. In order to bind single-stranded DNA, these proteins utilise a characteristic and evolutionary conserved single-stranded DNA-binding domain, the oligonucleotide/oligosaccharide-binding (OB)-fold. In the current review we discuss a subset of these proteins involved in the direct maintenance of genomic stability, an important cellular process in the conservation of cellular viability and prevention of malignant transformation. We discuss the central roles of single-stranded DNA binding proteins from the OB-fold domain family in DNA replication, the restart of stalled replication forks, DNA damage repair, cell cycle-checkpoint activation, and telomere maintenance.

PRMT2 and RORγ expression are associated with breast cancer survival outcomes

Protein arginine methyltransferases (PRMTs) methylate arginine residues on histones and target transcription factors that play critical roles in many cellular processes, including gene transcription, mRNA splicing, proliferation, and differentiation. Recent studies have linked PRMT-dependent epigenetic marks and modifications to carcinogenesis and metastasis in cancer. However, the role of PRMT2-dependent signaling in breast cancer remains obscure. We demonstrate PRMT2 mRNA expression was significantly decreased in breast cancer relative to normal breast. Gene expression profiling, Ingenuity and protein-protein interaction network analysis after PRMT2-short interfering RNA transfection into MCF-7 cells, revealed that PRMT2-dependent gene expression is involved in cell-cycle regulation and checkpoint control, chromosomal instability, DNA repair, and carcinogenesis. For example, PRMT2 depletion achieved the following: 1) increased p21 and decreased cyclinD1 expression in (several) breast cancer cell lines, 2) decreased cell migration, 3) induced an increase in nucleotide excision repair and homologous recombination DNA repair, and 4) increased the probability of distance metastasis free survival (DMFS). The expression of PRMT2 and retinoid-related orphan receptor-γ (RORγ) is inversely correlated in estrogen receptor-positive breast cancer and increased RORγ expression increases DMFS. Furthermore, we found decreased expression of the PRMT2-dependent signature is significantly associated with increased probability of DMFS. Finally, weighted gene coexpression network analysis demonstrated a significant correlation between PRMT2-dependent genes and cell-cycle checkpoint, kinetochore, and DNA repair circuits. Strikingly, these PRMT2-dependent circuits are correlated with pan-cancer metagene signatures associated with epithelial-mesenchymal transition and chromosomal instability. This study demonstrates the role and significant correlation between a histone methyltransferase (PRMT2)-dependent signature, RORγ, the cell-cycle regulation, DNA repair circuits, and breast cancer survival outcomes.

Biochemical characterization of Aprataxin, the protein deficient in Ataxia with Oculomotor Apraxia type 1

Neurodegenerative disorders are heterogenous in nature and include a range of ataxias with oculomotor apraxia, which are characterised by a wide variety of neurological and ophthalmological features. This family includes recessive and dominant disorders. A subfamily of autosomal recessive cerebellar ataxias are characterised by defects in the cellular response to DNA damage. These include the well characterised disorders Ataxia-Telangiectasia (A-T) and Ataxia-Telangiectasia Like Disorder (A-TLD) as well as the recently identified diseases Spinocerebellar ataxia with axonal neuropathy Type 1 (SCAN1), Ataxia with Oculomotor Apraxia Type 2 (AOA2), as well as the subject of this thesis, Ataxia with Oculomotor Apraxia Type 1 (AOA1). AOA1 is caused by mutations in the APTX gene, which is located at chromosomal locus 9p13. This gene codes for the 342 amino acid protein Aprataxin. Mutations in APTX cause destabilization of Aprataxin, thus AOA1 is a result of Aprataxin deficiency. Aprataxin has three functional domains, an N-terminal Forkhead Associated (FHA) phosphoprotein interaction domain, a central Histidine Triad (HIT) nucleotide hydrolase domain and a C-terminal C2H2 zinc finger. Aprataxins FHA domain has homology to FHA domain of the DNA repair protein 5’ polynucleotide kinase 3’ phosphatase (PNKP). PNKP interacts with a range of DNA repair proteins via its FHA domain and plays a critical role in processing damaged DNA termini. The presence of this domain with a nucleotide hydrolase domain and a DNA binding motif implicated that Aprataxin may be involved in DNA repair and that AOA1 may be caused by a DNA repair deficit. This was substantiated by the interaction of Aprataxin with proteins involved in the repair of both single and double strand DNA breaks (XRay Cross-Complementing 1, XRCC4 and Poly-ADP Ribose Polymerase-1) and the hypersensitivity of AOA1 patient cell lines to single and double strand break inducing agents. At the commencement of this study little was known about the in vitro and in vivo properties of Aprataxin. Initially this study focused on generation of recombinant Aprataxin proteins to facilitate examination of the in vitro properties of Aprataxin. Using recombinant Aprataxin proteins I found that Aprataxin binds to double stranded DNA. Consistent with a role for Aprataxin as a DNA repair enzyme, this binding is not sequence specific. I also report that the HIT domain of Aprataxin hydrolyses adenosine derivatives and interestingly found that this activity is competitively inhibited by DNA. This provided initial evidence that DNA binds to the HIT domain of Aprataxin. The interaction of DNA with the nucleotide hydrolase domain of Aprataxin provided initial evidence that Aprataxin may be a DNA-processing factor. Following these studies, Aprataxin was found to hydrolyse 5’adenylated DNA, which can be generated by unscheduled ligation at DNA breaks with non-standard termini. I found that cell extracts from AOA1 patients do not have DNA-adenylate hydrolase activity indicating that Aprataxin is the only DNA-adenylate hydrolase in mammalian cells. I further characterised this activity by examining the contribution of the zinc finger and FHA domains to DNA-adenylate hydrolysis by the HIT domain. I found that deletion of the zinc finger ablated the activity of the HIT domain against adenylated DNA, indicating that the zinc finger may be required for the formation of a stable enzyme-substrate complex. Deletion of the FHA domain stimulated DNA-adenylate hydrolysis, which indicated that the activity of the HIT domain may be regulated by the FHA domain. Given that the FHA domain is involved in protein-protein interactions I propose that the activity of Aprataxins HIT domain may be regulated by proteins which interact with its FHA domain. We examined this possibility by measuring the DNA-adenylate hydrolase activity of extracts from cells deficient for the Aprataxin-interacting DNA repair proteins XRCC1 and PARP-1. XRCC1 deficiency did not affect Aprataxin activity but I found that Aprataxin is destabilized in the absence of PARP-1, resulting in a deficiency of DNA-adenylate hydrolase activity in PARP-1 knockout cells. This implies a critical role for PARP-1 in the stabilization of Aprataxin. Conversely I found that PARP-1 is destabilized in the absence of Aprataxin. PARP-1 is a central player in a number of DNA repair mechanisms and this implies that not only do AOA1 cells lack Aprataxin, they may also have defects in PARP-1 dependant cellular functions. Based on this I identified a defect in a PARP-1 dependant DNA repair mechanism in AOA1 cells. Additionally, I identified elevated levels of oxidized DNA in AOA1 cells, which is indicative of a defect in Base Excision Repair (BER). I attribute this to the reduced level of the BER protein Apurinic Endonuclease 1 (APE1) I identified in Aprataxin deficient cells. This study has identified and characterised multiple DNA repair defects in AOA1 cells, indicating that Aprataxin deficiency has far-reaching cellular consequences. Consistent with the literature, I show that Aprataxin is a nuclear protein with nucleoplasmic and nucleolar distribution. Previous studies have shown that Aprataxin interacts with the nucleolar rRNA processing factor nucleolin and that AOA1 cells appear to have a mild defect in rRNA synthesis. Given the nucleolar localization of Aprataxin I examined the protein-protein interactions of Aprataxin and found that Aprataxin interacts with a number of rRNA transcription and processing factors. Based on this and the nucleolar localization of Aprataxin I proposed that Aprataxin may have an alternative role in the nucleolus. I therefore examined the transcriptional activity of Aprataxin deficient cells using nucleotide analogue incorporation. I found that AOA1 cells do not display a defect in basal levels of RNA synthesis, however they display defective transcriptional responses to DNA damage. In summary, this thesis demonstrates that Aprataxin is a DNA repair enzyme responsible for the repair of adenylated DNA termini and that it is required for stabilization of at least two other DNA repair proteins. Thus not only do AOA1 cells have no Aprataxin protein or activity, they have additional deficiencies in PolyADP Ribose Polymerase-1 and Apurinic Endonuclease 1 dependant DNA repair mechanisms. I additionally demonstrate DNA-damage inducible transcriptional defects in AOA1 cells, indicating that Aprataxin deficiency confers a broad range of cellular defects and highlighting the complexity of the cellular response to DNA damage and the multiple defects which result from Aprataxin deficiency. My detailed characterization of the cellular consequences of Aprataxin deficiency provides an important contribution to our understanding of interlinking DNA repair processes.

Single nucleotide polymorphism (SNP) - genotyping of Community Acquired Methicillin-Resistant Staphylococcus aureus, including the subtyping of PVL toxin producers using Real-Time PCR

Staphylococcus aureus is a common pathogen that causes a variety of infections including soft tissue infections, impetigo, septicemia toxic shock and scalded skin syndrome. Traditionally, Methicillin-Resistant Staphylococcus aureus (MRSA) was considered a Hospital-Acquired (HA) infection. It is now recognised that the frequency of infections with MRSA is increasing in the community, and that these infections are not originating from hospital environments. A 2007 report by the Centers for Disease Control and Prevention (CDC) stated that Staphylococcus aureus is the most important cause of serious and fatal infections in the USA. Community-Acquired MRSA (CA-MRSA) are genetically diverse and distinct, meaning they are able to be identified and tracked by way of genotyping. Genotyping of MRSA using Single nucleotide polymorphisms (SNPs) is a rapid and robust method for monitoring MRSA, specifically ST93 (Queensland Clone) dissemination in the community. It has been shown that a large proportion of CA-MRSA infections in Queensland and New South Wales are caused by ST93. The rationale for this project was that SNP analysis of MLST genes is a rapid and cost-effective method for genotyping and monitoring MRSA dissemination in the community. In this study, 16 different sequence types (ST) were identified with 41% of isolates identified as ST93 making it the predominate clone. Males and Females were infected equally with an average patient age of 45yrs. Phenotypically, all of the ST93 had an identical antimicrobial resistance pattern. They were resistant to the β-lactams – Penicillin, Flu(di)cloxacillin and Cephalothin but sensitive to all other antibiotics tested. Virulence factors play an important role in allowing S. aureus to cause disease by way of colonising, replication and damage to the host. One virulence factor of particular interest is the toxin Panton-Valentine leukocidin (PVL), which is composed of two separate proteins encoded by two adjacent genes. PVL positive CA-MRSA are shown to cause recurrent, chronic or severe skin and soft tissue infections. As a result, it is important that PVL positive CA-MRSA is genotyped and tracked. Especially now that CA-MRSA infections are more prevalent than HA-MRSA infections and are now deemed endemic in Australia. 98% of all isolates in this study tested positive for the PVL toxin gene. This study showed that PVL is present in many different community based ST, not just ST93, which were all PVL positive. With this toxin becoming entrenched in CA-MRSA, genotyping would provide more accurate data and a way of tracking the dissemination. PVL gene can be sub-typed using an allele-specific Real-Time PCR (RT-PCR) followed by High resolution meltanalysis. This allows the identification of PVL subtypes within the CA-MRSA population and allow the tracking of these clones in the community.

Genotyping of enterococcus faecalis and enterococcus faecium isolates using a set of eight single nucleotide polymorphisms (SNPs)

A SNP genotyping method was developed for E. faecalis and E. faecium using the 'Minimum SNPs' program. SNP sets were interrogated using allele-specific real-time PCR. SNP-typing sub-divided clonal complexes 2 and 9 of E. faecalis and 17 of E. faecium, members of which cause the majority of nosocomial infections globally.

Comparison of Nucleotide Sequences between Northern and Southern Philippine Isolates of Rice Grassy Stunt Virus Indicates Occurrence of Natural Genetic Reassortment

Rice grassy stunt virus is a member of the genus Tenuivirus, is persistently transmitted by a brown planthopper, and has occurred in rice plants in South, Southeast, and East Asia (similar to North and South America). We determined the complete nucleotide (nt) sequences of RNAs 1 (9760 nt), 2 (4069 nt), 3 (3127 nt), 4 (2909 nt), 5 (2704 nt), and 6 (2590 nt) of a southern Philippine isolate from South Cotabato and compared them with those of a northern Philippine isolate from Laguna (Toriyama et al., 1997, 1998). The numbers of nucleotides in the terminal untranslated regions and open reading frames were identical between the two isolates except for the 5′ untranslated region of the complementary strand of RNA 4. Overall nucleotide differences between the two isolates were only 0.08% in RNA 1, 0.58% in RNA 4, and 0.26% in RNA 5, whereas they were 2.19% in RNA 2, 8.38% in RNA 3, and 3.63% in RNA 6. In the intergenic regions, the two isolates differed by 9.12% in RNA 2, 11.6% in RNA 3, and 6.86% in RNA 6 with multiple consecutive nucleotide deletion/insertions, whereas they differed by only 0.78% in RNA 4 and 0.34% in RNA 5. The nucleotide variation in the intergenic region of RNA 6 within the South Cotabato isolate was only 0.33%. These differences in accumulation of mutations among individual RNA segments indicate that there was genetic reassortment in the two geographical isolates; RNAs 1, 4, and 5 of the two isolates came from a common ancestor, whereas RNAs 2, 3, and 6 were from two different ancestors.

Complete Nucleotide Sequence of Rice Tungro Spherical Virus Genome of a Highly Virulent Strain Vt6

The complete nucleotide sequence of rice tungro spherical virus (RTSV) strain Vt6, originally from Mindanao, the Philippines, with higher virulence to resistant rice cultivars, was determined and compared with the published sequence for the Philippine-type strain A (RTSV-A-Shen). It was reported that RTSV-A was not able to infect a rice resistant cultivar TKM 6 (10). RTSV-Vt6 and RTSV-A-Shen share 90% and 95% homology at nucleotide and amino-acid levels, respectively. The N-terminal leader sequence of RTSV-Vt6 contained a 39-amino acids-region (positions 65 to 103) which was totally different from that of RTSV-A-Shen; the difference resulted from frame shifting by nucleotide insertions and deletions. To confirm the amino-acid sequence differences of the leader polypeptide, the same region was cloned and sequenced using a newly obtained variant of RTSV-type 6, which had been collected in the field of IRRI, and seven field isolates from Mindanao, the Philippines. Since all the sequences of the target region are identical to that of the Vt6 leader polypeptide, the sequence difference in the leader region seems not to correlate with the virulence of Vt6.

Highly discriminatory single-nucleotide polymorphism interrogation of Escherichia coli by use of allele-specific real-time PCR and eBURST analysis

In total, 782 Escherichia coli strains originating from various host sources have been analyzed in this study by using a highly discriminatory single-nucleotide polymorphism (SNP) approach. A set of eight SNPs, with a discrimination value (Simpson's index of diversity [D]) of 0.96, was determined using the Minimum SNPs software, based on sequences of housekeeping genes from the E. coli multilocus sequence typing (MLST) database. Allele-specific real-time PCR was used to screen 114 E. coli isolates from various fecal sources in Southeast Queensland (SEQ). The combined analysis of both the MLST database and SEQ E. coli isolates using eight high-D SNPs resolved the isolates into 74 SNP profiles. The data obtained suggest that SNP typing is a promising approach for the discrimination of host-specific groups and allows for the identification of human-specific E. coli in environmental samples. However, a more diverse E. coli collection is required to determine animal- and environment-specific E. coli SNP profiles due to the abundance of human E. coli strains (56%) in the MLST database.