hSSB2 (NABP1) is required for the recruitment of RPA during the cellular response to DNA UV damage.

Maintenance of genomic stability is critical to prevent diseases such as cancer. As such, eukaryotic cells have multiple pathways to efficiently detect, signal and repair DNA damage. One common form of exogenous DNA damage comes from ultraviolet B (UVB) radiation. UVB generates cyclobutane pyrimidine dimers (CPD) that must be rapidly detected and repaired to maintain the genetic code. The nucleotide excision repair (NER) pathway is the main repair system for this type of DNA damage. Here, we determined the role of the human Single-Stranded DNA Binding protein 2, hSSB2, in the response to UVB exposure. We demonstrate that hSSB2 levels increase in vitro and in vivo after UVB irradiation and that hSSB2 rapidly binds to chromatin. Depletion of hSSB2 results in significantly decreased Replication Protein A (RPA32) phosphorylation and impaired RPA32 localisation to the site of UV-induced DNA damage. Delayed recruitment of NER protein Xeroderma Pigmentosum group C (XPC) was also observed, leading to increased cellular sensitivity to UVB. Finally, hSSB2 was shown to have affinity for single-strand DNA containing a single CPD and for duplex DNA with a two-base mismatch mimicking a CPD moiety. Altogether our data demonstrate that hSSB2 is involved in the cellular response to UV exposure.

A structural analysis of DNA binding by hSSB1 (NABP2/OBFC2B) in solution.

Single-stranded DNA binding proteins (SSBs) play an important role in DNA processing events such as replication, recombination and repair. Human single-stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) contains a single oligosaccharide/oligonucleotide binding (OB) domain followed by a charged C-terminus and is structurally homologous to the SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus Recent work has revealed that hSSB1 is critical to homologous recombination and numerous other important biological processes such as the regulation of telomeres, the maintenance of DNA replication forks and oxidative damage repair. Since the ability of hSSB1 to directly interact with single-stranded DNA (ssDNA) is paramount for all of these processes, understanding the molecular details of ssDNA recognition is essential. In this study, we have used solution-state nuclear magnetic resonance in combination with biophysical and functional experiments to structurally analyse ssDNA binding by hSSB1. We reveal that ssDNA recognition in solution is modulated by base-stacking of four key aromatic residues within the OB domain. This DNA binding mode differs significantly from the recently determined crystal structure of the SOSS1 complex containing hSSB1 and ssDNA. Our findings elucidate the detailed molecular mechanism in solution of ssDNA binding by hSSB1, a major player in the maintenance of genomic stability.

Biophysical Characterisation and Quantification of Nucleic Acid-Protein Interactions: EMSA, MST and SPR.

Cell viability is only possible due to a dynamic range of essential nucleic acid-protein complex formation. DNA replication and repair, gene expression, transcription and protein synthesis are well-known processes mediated by nucleic acids (DNA and RNA) - protein interactions. Novel nucleic acid- protein complexes have been identified in the past few years aided by the development of numerous new techniques such as RNA capture or Tandem RNA Affinity Purification (TRAP). However, the biophysical and biochemical details of these interactions are mostly unknown. Here, we present three techniques (Electrophoretic Mobility Shift Assays, Microscale Thermophoresis and Surface Plasmon Resonance) that are commonly used to quantify and characterize DNA-protein and RNA-protein interactions and discuss their main advantages and limitations.

The structural basis of DNA binding by the single-stranded DNA-binding protein from Sulfolobus solfataricus.

Canonical single-stranded DNA-binding proteins (SSBs) from the oligosaccharide/oligonucleotide-binding (OB) domain family are present in all known organisms and are critical for DNA replication, recombination and repair. The SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus (SsoSSB) has a 'simple' domain organization consisting of a single DNA-binding OB fold coupled to a flexible C-terminal tail, in contrast with other SSBs in this family that incorporate up to four OB domains. Despite the large differences in the domain organization within the SSB family, the structure of the OB domain is remarkably similar all cellular life forms. However, there are significant differences in the molecular mechanism of ssDNA binding. We have determined the structure of the SsoSSB OB domain bound to ssDNA by NMR spectroscopy. We reveal that ssDNA recognition is modulated by base-stacking of three key aromatic residues, in contrast with the OB domains of human RPA and the recently discovered human homologue of SsoSSB, hSSB1. We also demonstrate that SsoSSB binds ssDNA with a footprint of five bases and with a defined binding polarity. These data elucidate the structural basis of DNA binding and shed light on the molecular mechanism by which these 'simple' SSBs interact with ssDNA.

Virus de la Inmunodeficiencia Humana y Linfoma en un Hospital Universitario de Corrientes Argentina / HIV and Lymphoma in a University Hospital in Corrientes Argentina

Los pacientes infectados por el HIV presentan una mayor morbimortalidad por cáncer como Linfoma y Sarcoma de Kaposi, constituyendo en este grupo entre la 2da y 3ra causa de muerte, a pesar de la TARV . Varios estudios han demostrado la disminución de infecciones oportunistas, Sarcoma de Kaposi, Linfoma primario cerebral,en menor proporción de otros Linfomas NoH como el Linfoma Burkit, y un aumento de neoplasias no asociados al SIDA como el Linfoma Hodgkin.Objetivo: Analizar las características clínicas, tratamiento, sobrevida de pacientes HIV seropositivos con linfoma,diagnosticados y tratados en el Servicio de Clínica Médica del Hospital Escuela José F. de San Martín en la ciudad de Corrientes- Argentina. Evaluar la prevalencia se seropositividad en nuestra población de pacientes con Linfoma, y determinar el pronóstico al momento del diagnóstico.Material y Método: Se realizó un estudio descriptivo observacional mediante un análisis retrospectivo de las historias clínicas de siete pacientes con diagnóstico de Linfoma y serología (+) para HIV, tratados en el servicio de Clínica Médica del Hospital Escuela de Corrientes desde Enero 2003 a Enero 2013.Resultados: Entre 76 casos de Linfoma, 18 fueron tipo Hodgkin y 58 tipo NH. Del total 7 tenían serología positiva para HIV (12,5%) ,1 de 18 con enfermedad de Hodgkin y 6 de 58 LNH, es decir 5.5% y 10.34% respectivamente...