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.

Physical and functional interaction of the archaeal single-stranded DNA-binding protein SSB with RNA polymerase

Archaeal transcription utilizes a complex multisubunit RNA polymerase and the basal transcription factors TBP and TF(II)B, closely resembling its eukaryal counterpart. We have uncovered a tight physical and functional interaction between RNA polymerase and the single-stranded DNA-binding protein SSB in Sulfolobus solfataricus. SSB stimulates transcription from promoters in vitro under TBP-limiting conditions and supports transcription in the absence of TBP. SSB also rescues transcription from repression by reconstituted chromatin. We demonstrate the potential for promoter melting by SSB, suggesting a plausible basis for the stimulation of transcription. This stimulation requires both the single-stranded DNA-binding domain and the acidic C-terminal tail of the SSB. The tail forms a stable interaction with RNA polymerase. These data reveal an unexpected role for single-stranded DNA-binding proteins in transcription in archaea.

Single-stranded DNA-binding protein hSSB1 is critical for genomic stability

Single-strand DNA (ssDNA)-binding proteins (SSBs) are ubiquitous and essential for a wide variety of DNA metabolic processes, including DNA replication, recombination, DNA damage detection and repair1. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating nucleases, helicases and strand-exchange proteins, activating transcription and mediating protein–protein interactions. In eukaryotes, the major SSB, replication protein A (RPA), is a heterotrimer1. Here we describe a second human SSB (hSSB1), with a domain organization closer to the archaeal SSB than to RPA. Ataxia telangiectasia mutated (ATM) kinase phosphorylates hSSB1 in response to DNA double-strand breaks (DSBs). This phosphorylation event is required for DNA damage-induced stabilization of hSSB1. Upon induction of DNA damage, hSSB1 accumulates in the nucleus and forms distinct foci independent of cell-cycle phase. These foci co-localize with other known repair proteins. In contrast to RPA, hSSB1 does not localize to replication foci in S-phase cells and hSSB1 deficiency does not influence S-phase progression. Depletion of hSSB1 abrogates the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets after ionizing radiation. Cells deficient in hSSB1 exhibit increased radiosensitivity, defective checkpoint activation and enhanced genomic instability coupled with a diminished capacity for DNA repair. These findings establish that hSSB1 influences diverse endpoints in the cellular DNA damage response.

Restoration of native folding of single-stranded DNA sequences through reverse mutations: an indication of a new epigenetic mechanism

We used in vivo (biological), in silico (computational structure prediction), and in vitro (model sequence folding) analyses of single-stranded DNA sequences to show that nucleic acid folding conservation is the selective principle behind a high-frequency single-nucleotide reversion observed in a three-nucleotide mutated motif of the Maize streak virus replication associated protein (Rep) gene. In silico and in vitro studies showed that the three-nucleotide mutation adversely affected Rep nucleic acid folding, and that the single-nucleotide reversion [C(601)A] restored wild-type-like folding. In vivo support came from infecting maize with mutant viruses: those with Rep genes containing nucleotide changes predicted to restore a wild-type-like fold [A(601)/G(601)] preferentially accumulated over those predicted to fold differently [C(601)/T(601)], which frequently reverted to A(601) and displaced the original population. We propose that the selection of native nucleic acid folding is an epigenetic effect, which might have broad implications in the evolution of plants and their viruses.

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.

Human single-stranded DNA binding protein 1 (hSSB1/NABP2) is required for the stability and repair of stalled replication forks

Aberrant DNA replication is a primary cause of mutations that are associated with pathological disorders including cancer. During DNA metabolism, the primary causes of replication fork stalling include secondary DNA structures, highly transcribed regions and damaged DNA. The restart of stalled replication forks is critical for the timely progression of the cell cycle and ultimately for the maintenance of genomic stability. Our previous work has implicated the single-stranded DNA binding protein, hSSB1/NABP2, in the repair of DNA double-strand breaks via homologous recombination. Here, we demonstrate that hSSB1 relocates to hydroxyurea (HU)-damaged replication forks where it is required for ATR and Chk1 activation and recruitment of Mre11 and Rad51. Consequently, hSSB1-depleted cells fail to repair and restart stalled replication forks. hSSB1 deficiency causes accumulation of DNA strand breaks and results in chromosome aberrations observed in mitosis, ultimately resulting in hSSB1 being required for survival to HU and camptothecin. Overall, our findings demonstrate the importance of hSSB1 in maintaining and repairing DNA replication forks and for overall genomic stability.

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.

hSSB1 interacts directly with the MRN complex stimulating its recruitment to DNA double-strand breaks and its endo-nuclease activity

hSSB1 is a recently discovered single-stranded DNA binding protein that is essential for efficient repair of DNA double-strand breaks (DSBs) by the homologous recombination pathway. hSSB1 is required for the efficient recruitment of the MRN complex to sites of DSBs and for the efficient initiation of ATM dependent signalling. Here we explore the interplay between hSSB1 and MRN. We demonstrate that hSSB1 binds directly to NBS1, a component of the MRN complex, in a DNA damage independent manner. Consistent with the direct interaction, we observe that hSSB1 greatly stimulates the endo-nuclease activity of the MRN complex, a process that requires the C-terminal tail of hSSB1. Interestingly, analysis of two point mutations in NBS1, associated with Nijmegen breakage syndrome, revealed weaker binding to hSSB1, suggesting a possible disease mechanism.

hSSB1 rapidly binds at the sites of DNA double-strand breaks and is required for the efficient recruitment of the MRN complex

hSSB1 is a newly discovered single-stranded DNA (ssDNA)-binding protein that is essential for efficient DNA double-strand break signalling through ATM. However, the mechanism by which hSSB1 functions to allow efficient signalling is unknown. Here, we show that hSSB1 is recruited rapidly to sites of double-strand DNA breaks (DSBs) in all interphase cells (G1, S and G2) independently of, CtIP, MDC1 and the MRN complex (Rad50, Mre11, NBS1). However expansion of hSSB1 from the DSB site requires the function of MRN. Strikingly, silencing of hSSB1 prevents foci formation as well as recruitment of MRN to sites of DSBs and leads to a subsequent defect in resection of DSBs as evident by defective RPA and ssDNA generation. Our data suggests that hSSB1 functions upstream of MRN to promote its recruitment at DSBs and is required for efficient resection of DSBs. These findings, together with previous work establish essential roles of hSSB1 in controlling ATM activation and activity, and subsequent DSB resection and homologous recombination (HR).

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.

Putative full-length clones of the genomic DNA segments of subterranean clover stunt virus and identification of the segment coding for the viral coat protein

Subterranean clover stunt disease is an economically important aphid-borne virus disease affecting certain pasture and grain legumes in Australia. The virus associated with the disease, subterranean clover stunt virus (SCSV), was previously found to be representative of a new type of single-stranded DNA virus. Analysis of the virion DNA and restriction mapping of double-stranded cDNA synthesized from virion DNA suggested that SCSV has a segmented genome composed of 3 or 4 different species of circular ssDNA each of about 850-880 nucleotides. To further investigate the complexity of the SCSV genome, we have isolated the replicative form DNA from infected pea and from it prepared putative full-length clones representing the SCSV genome segments. Analysis of these clones by restriction mapping indicated that clones representing at least 4 distinct genomic segments were obtained. This method is thus suitable for generating an extensive genomic library of novel ssDNA viruses containing multiple genome segments such as SCSV and banana bunchy top virus. The N-terminal amino acid sequence and amino acid composition of the coat protein of SCSV were determined. Comparison of the amino acid sequence with partial DNA sequence data, and the distinctly different restriction maps obtained for the full-length clones suggested that only one of these clones contained the coat protein gene. The results confirmed that SCSV has a functionally divided genome composed of several distinct ssDNA circles each of about 1 kb.

LacO-LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19lacO3/lacOs), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline™ adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOs and lacO3 were 5.7 ± 0.3 × 10 -11 M and 4.1 ± 0.2 × 10-11 M respectively, which compare favorably with literature reports of 5 × 10-10 - 1 × 10-9 M for native laCO1 and 1-1.2 × 10-10 M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction.

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.