Investigating the effects of repair of DNA single-strand breaks on chromatin structure

Single-strand breaks (SSBs) are one of the most common types of lesion arising within cells; formed by attack of genotoxic agents on the DNA, as well as enzymatically during normal cellular processes. Although the single-strand break repair (SSBR) pathway is relatively well characterised, and many components have been extensively studied in vitro, little is known of how this pathway operates in vivo when DNA is complexed with histone proteins to form chromatin. This compaction of the DNA into nucleosomal structures has the potential to inhibit repair, by sterically blocking access of repair factors to sites of DNA damage. Whilst previous studies have shown that repair of DNA double-strand breaks and UV-induced lesions are associated with alterations in chromatin structure, through covalent modification of histone proteins and nucleosome remodeling, few similar observations have been made concerning SSBR. Here, I have produced and employed mammalian cell lines stably expressing fluorescently-tagged histone proteins to analyse the dynamics of chromatin occurring upon DNA damage. Localised damage was introduced using micro-irradiation with a UV-A laser, and the histone proteins at the site of damage visualized in real-time using confocal microscopy. Through this method, I have identified a rearrangement of chromatin structure in the vicinity of DNA strand breaks in mammalian cells, resulting in a mobilization of histone proteins at the site of damage. Furthermore, I have shown that this alteration is partially dependent on the activities of both the SSBR factor poly(ADP-ribose) polymerase 1 (PARP-1), and the phosphoinositide 3-kinase-like kinase (PIKK) Ataxia telangiectasia mutated (ATM). I have examined a potential requirement for ATM in SSBR, and found no evidence of this, suggesting that the effects of PARP-1 and ATM on histone mobilization are reflective of the independent contributions of repair of single- and double-strand breaks respectively

An examination of the co-existence of the threespine (Gasterosteus aculeatus) and the ninespine (Pungitius pungitius) sticklebacks and its consequences for morphology and behaviour

In Britain two species of stickleback, the threespine and the ninespine are often found living side-by-side in freshwater streams. The two stickleback species share a close life-history and in particular similar dietary habits. The situation of two such ecologically similar species being able to co-exist without high levels of competition removing one or the other from the habitat begs us to question exactly what ecological process is allowing this situation to occur. Three separate populations of stickleback were observed in the study; a population of allopatric threespine sticklebacks, a ninespine population and a population of threespines that live sympatrically with ninespine fish. Morphological analysis of the three populations of sticklebacks revealed divergence amongst the sympatric threespine fish. The sympatric threespine fish were shown to have a deep mid-body depth and deeper caudal peduncle, which possibly serve to increase the fish's body acceleratory skills. In addition the sympatric threespines have a small conical shaped mouth and meristic analysis indicates an increase in the number of the fish's gill rakers. Combination of these adaptations indicates that the sympatric threespine fish are effective zooplanktivores. It was questioned whether character displacement initiated by competition from the ninespine fish in the sympatric threespines environment is the process responsible for driving the observed divergence. In turn each of the criteria of the character displacement hypothesis was tested. Investigation of the allopatric and sympatric environment indicated little difference between the sites other than the presence of the ninespine fish in the sympatric sites. Stomach contents analysis showed that the sympatric fish have increased their consumption of zooplankton. Behavioural analysis showed how the morphological adaptations have improved the sympatric fish's foraging performance in the limnetic habitat

The [Ro60/La/hY RNA] and [nucleolin/hY RNA] RNPs are not required for the reconstitution of chromosomal DNA replication in vitro.

<p>Immunodepletion of hY RNPs from cytosolic HeLa cell extracts. (A) Depletion of [Ro60/La/hY RNA] RNPs with La-specific antibodies, and (B) depletion of [nucleolin/hY RNA] RNPs with nucleolin-specific antibodies. Ro60, La and nucleolin (NCL) were analysed in the depleted extracts by Western blot analysis. Ponceau stains are shown as loading controls. For mock depletions, unspecific mouse IgG2a antibodies were used alongside purified mouse monoclonal anti-La antibodies, while pre-immune rabbit serum was used alongside the anti-nucleolin rabbit serum. (C) Functional reconstitution of chromosomal DNA replication in the immunodepleted cytosolic extracts. Late G1 phase template nuclei from mimosine-arrested human EJ30 cells were incubated in the indicated depleted extracts. Replication buffer and non-depleted cytosolic extract (cyt) served as negative and positive controls, respectively. Mock M IgG and R IgG indicate unspecific mouse and rabbit antibodies, as in panels A and B. Proportions of replicating nuclei were determined by immunoflorescence microscopy. Mean values of two independent experiments are shown.</p

Domain structure of human Y RNA.

<p>The most stable secondary structure of hY1 RNA is shown as determined by the Mfold v3.2 RNA algorithm. The four conserved key structural elements, including specific protein binding sites, are indicated.</p

Human Y RNAs are present in several distinct RNP complexes in cytosolic extract.

<p>The indicated proteins were immunoprecipitated (IP) from HeLa cytosolic extract and associated proteins and RNAs were analysed by Western blotting and qRT-PCR, respectively. (A) Protein analysis of Ro60 and La IPs. Apparent molecular weights of the precipitated protein bands are shown, the asterisk indicates the IgG heavy chain. As a reference, 10% of the input cell extract was loaded on the gel. Where indicated, extract was treated with RNase A prior to IP (RNase). (B) Protein analysis of nucleolin (NCL) IPs. (C) RNA content analysis of the Ro60, La and nucleolin (NCL) IPs. The relative amounts of all four hY RNAs and 5S rRNA in the indicated immunoprecipitates relative to mock immunoprecipitates were determined by qRT-PCR. Mean values of two independent experiments are shown.</p

Co-depletion of [Ro60/La/hY RNA] and [nucleolin/hY RNA] RNPs does not inhibit chromosomal DNA replication in vitro.

<p>[Ro60/La/hY RNA] and [nucleolin/hY RNA] RNPs were co-depleted with La- and nucleolin-specific antibodies. (A) Protein levels of nucleolin (NCL), Ro60 and La in the depleted extracts were analysed by Western blotting. Mouse IgG2a antibodies and pre-immune rabbit serum were used together for the mock depletion. (B) Analysis of hY RNA and 5S rRNA levels remaining in the depleted extract. Proportions of the indicated RNA amounts remaining in the extract after [Ro60/La/hY RNA] and [nucleolin/hY RNA] RNP co-depletion were determined by qRT-PCR. Data are shown as percentages of the mock depletion, after normalisation to HPRT mRNA. (C) Functional reconstitution of chromosomal DNA replication in the co-depleted extract. Percentages of replicating nuclei were determined as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013673#pone-0013673-g003" target="_blank">Fig. 3C</a>. Mean values of two independent experiments are shown in panels B and C.</p

Additional file 1: of A review of characteristics and outcomes of Australia’s undergraduate medical education rural immersion programs

Appendix 1: Description of papers included in the reviewa [39–41]. (DOCX 36 kb