RESUMO
Single-stranded DNA-binding proteins (SSBs) are essential to all living organisms as protectors and guardians of the genome. Apart from the well-characterized RPA, humans have also evolved two further SSBs, termed hSSB1 and hSSB2. Over the last few years, we have used NMR spectroscopy to determine the molecular and structural details of both hSSBs and their interactions with DNA and RNA. Here we provide a detailed overview of how to express and purify recombinant versions of these important human proteins for the purpose of detailed structural analysis by high-resolution solution-state NMR.
Assuntos
Proteínas de Ligação a DNA/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas Mitocondriais/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Clonagem Molecular , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Fermentação , Humanos , Marcação por Isótopo , Espectroscopia de Ressonância Magnética , Proteínas Mitocondriais/isolamento & purificação , Proteínas Mitocondriais/metabolismo , Ligação Proteica , RNA Bacteriano/metabolismo , Proteínas Recombinantes/químicaRESUMO
Single-stranded DNA-binding proteins (SSBs) are required for all known DNA metabolic events such as DNA replication, recombination and repair. While a wealth of structural and functional data is available on the essential human SSB, hSSB1 (NABP2/OBFC2B), the close homolog hSSB2 (NABP1/OBFC2A) remains relatively uncharacterized. Both SSBs possess a well-structured OB (oligonucleotide/oligosaccharide-binding) domain that is able to recognize single-stranded DNA (ssDNA) followed by a flexible carboxyl-tail implicated in the interaction with other proteins. Despite the high sequence similarity of the OB domain, several recent studies have revealed distinct functional differences between hSSB1 and hSSB2. In this study, we show that hSSB2 is able to recognize cyclobutane pyrimidine dimers (CPD) that form in cellular DNA as a consequence of UV damage. Using a combination of biolayer interferometry and NMR, we determine the molecular details of the binding of the OB domain of hSSB2 to CPD-containing ssDNA, confirming the role of four key aromatic residues in hSSB2 (W59, Y78, W82, and Y89) that are also conserved in hSSB1. Our structural data thus demonstrate that ssDNA recognition by the OB fold of hSSB2 is highly similar to hSSB1, indicating that one SSB may be able to replace the other in any initial ssDNA binding event. However, any subsequent recruitment of other repair proteins most likely depends on the divergent carboxyl-tail and as such is likely to be different between hSSB1 and hSSB2.
Assuntos
Dano ao DNA , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Raios Ultravioleta , Sítios de Ligação/genética , Reparo do DNA , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Interferometria/métodos , Espectroscopia de Ressonância Magnética/métodos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ligação Proteica , Domínios ProteicosRESUMO
Single-stranded DNA binding (SSB) proteins are essential to protect singe-stranded DNA (ssDNA) that exists as a result of several important DNA repair pathways in living cells. In humans, besides the well-characterised Replication Protein A (RPA) we have described another SSB termed human SSB1 (hSSB1, OBFC2B) and have shown that this protein is an important player in the maintenance of the genome. In this review we define the structural and biophysical details of how hSSB1 interacts with both DNA and other essential proteins. While the presence of the oligonucleotide/oligosaccharide (OB) domain ensures ssDNA binding by hSSB1, it has also been shown to self-oligomerise as well as interact with and being modified by several proteins highlighting the versatility that hSSB1 displays in the context of DNA repair. A detailed structural understanding of these processes will likely lead to the designs of tailored hSSB1 inhibitors as anti-cancer drugs in the near future.