Inhibition of Aurora B kinase (AURKB) enhances the effectiveness of 5-fluorouracil chemotherapy against colorectal cancer cells.

BACKGROUND: 5-Fluorouracil (5-FU) remains a core component of systemic therapy for colorectal cancer (CRC). However, response rates remain low, and development of therapy resistance is a primary issue. Combinatorial strategies employing a second agent to augment the therapeutic effect of chemotherapy is predicted to reduce the incidence of treatment resistance and increase the durability of response to therapy. METHODS: Here, we employed quantitative proteomics approaches to identify novel druggable proteins and molecular pathways that are deregulated in response to 5-FU, which might serve as targets to improve sensitivity to chemotherapy. Drug combinations were evaluated using 2D and 3D CRC cell line models and an ex vivo culture model of a patient-derived tumour. RESULTS: Quantitative proteomics identified upregulation of the mitosis-associated protein Aurora B (AURKB), within a network of upregulated proteins, in response to a 24 h 5-FU treatment. In CRC cell lines, AURKB inhibition with the dihydrogen phosphate prodrug AZD1152, markedly improved the potency of 5-FU in 2D and 3D in vitro CRC models. Sequential treatment with 5-FU then AZD1152 also enhanced the response of a patient-derived CRC cells to 5-FU in ex vivo cultures. CONCLUSIONS: AURKB inhibition may be a rational approach to augment the effectiveness of 5-FU chemotherapy in CRC.

The role of inner nuclear membrane proteins in tumourigenesis and as potential targets for cancer therapy.

Despite significant advances in our understanding of tumourigenesis and cancer therapeutics, cancer continues to account for 30% of worldwide deaths. Therefore, there remains an unmet need for the development of cancer therapies to improve patient quality of life and survival outcomes. The inner nuclear membrane has an essential role in cell division, cell signalling, transcription, cell cycle progression, chromosome tethering, cell migration and mitosis. Furthermore, expression of several inner nuclear membrane proteins has been shown to be frequently altered in tumour cells, resulting in the dysregulation of cellular pathways to promote tumourigenesis. However, to date, minimal research has been conducted to investigate how targeting these dysregulated and variably expressed proteins may provide a novel avenue for cancer therapies. In this review, we present an overview of the involvement of the inner nuclear membrane proteins within the hallmarks of cancer and how they may be exploited as potent anti-cancer therapeutics.

hSSB1 (NABP2/OBFC2B) modulates the DNA damage and androgen-induced transcriptional response in prostate cancer.

BACKGROUND: Activation and regulation of androgen receptor (AR) signaling and the DNA damage response impact the prostate cancer (PCa) treatment modalities of androgen deprivation therapy (ADT) and radiotherapy. Here, we have evaluated a role for human single-strand binding protein 1 (hSSB1/NABP2) in modulation of the cellular response to androgens and ionizing radiation (IR). hSSB1 has defined roles in transcription and maintenance of genome stability, yet little is known about this protein in PCa. METHODS: We correlated hSSB1 with measures of genomic instability across available PCa cases from The Cancer Genome Atlas (TCGA). Microarray and subsequent pathway and transcription factor enrichment analysis were performed on LNCaP and DU145 prostate cancer cells. RESULTS: Our data demonstrate that hSSB1 expression in PCa correlates with measures of genomic instability including multigene signatures and genomic scars that are reflective of defects in the repair of DNA double-strand breaks via homologous recombination. In response to IR-induced DNA damage, we demonstrate that hSSB1 regulates cellular pathways that control cell cycle progression and the associated checkpoints. In keeping with a role for hSSB1 in transcription, our analysis revealed that hSSB1 negatively modulates p53 and RNA polymerase II transcription in PCa. Of relevance to PCa pathology, our findings highlight a transcriptional role for hSSB1 in regulating the androgen response. We identified that AR function is predicted to be impacted by hSSB1 depletion, whereby this protein is required to modulate AR gene activity in PCa. CONCLUSIONS: Our findings point to a key role for hSSB1 in mediating the cellular response to androgen and DNA damage via modulation of transcription. Exploiting hSSB1 in PCa might yield benefits as a strategy to ensure a durable response to ADT and/or radiotherapy and improved patient outcomes.

Targeting the COMMD4-H2B protein complex in lung cancer.

BACKGROUND: Lung cancer is the biggest cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for 85-90% of all lung cancers. Identification of novel therapeutic targets are required as drug resistance impairs chemotherapy effectiveness. COMMD4 is a potential NSCLC therapeutic target. The aims of this study were to investigate the COMMD4-H2B binding pose and develop a short H2B peptide that disrupts the COMMD4-H2B interaction and mimics COMMD4 siRNA depletion. METHODS: Molecular modelling, in vitro binding and site-directed mutagenesis were used to identify the COMMD4-H2B binding pose and develop a H2B peptide to inhibit the COMMD4-H2B interaction. Cell viability, DNA repair and mitotic catastrophe assays were performed to determine whether this peptide can specially kill NSCLC cells. RESULTS: Based on the COMMD4-H2B binding pose, we have identified a H2B peptide that inhibits COMMD4-H2B by directly binding to COMMD4 on its H2B binding binding site, both in vitro and in vivo. Treatment of NSCLC cell lines with this peptide resulted in increased sensitivity to ionising radiation, increased DNA double-strand breaks and induction of mitotic catastrophe in NSCLC cell lines. CONCLUSIONS: Our data shows that COMMD4-H2B represents a novel potential NSCLC therapeutic target.

Mono-phosphorylation at Ser4 of barrier-to-autointegration factor (Banf1) significantly reduces its DNA binding capability by inducing critical changes in its local conformation and DNA binding surface.

Barrier-to-autointegration factor (Banf1) is a small DNA-bridging protein. The binding status of Banf1 to DNA is regulated by its N-terminal phosphorylation and dephosphorylation, which plays a critical role in cell proliferation. Banf1 can be phosphorylated at Ser4 into mono-phosphorylated Banf1, which is further phosphorylated at Thr3 to form di-phosphorylated Banf1. It was observed decades ago that mono-phosphorylated Banf1 cannot bind to DNA. However, the underlying molecular- and atomic-level mechanisms remain unclear. A clear understanding of these mechanisms will aid in interfering with the cell proliferation process for better global health. Herein, we explored the detailed atomic bases of unphosphorylated Banf1-DNA binding and how mono- and di-phosphorylation of Banf1 impair these atomic bases to eliminate its DNA-binding capability, followed by exploring the DNA-binding capability of mono- and di-phosphorylation Banf1, using comprehensive and systematic molecular modelling and molecular dynamics simulations. This work presented in detail the residue-level binding energies, hydrogen bonds and water bridges between Banf1 and DNA, some of which have not been reported. Moreover, we revealed that mono-phosphorylation of Banf1 causes its N-terminal secondary structure changes, which in turn induce significant changes in Banf1's DNA binding surface, thus eliminating its DNA-binding capability. At the atomic level, we also uncovered the alterations in interactions due to the induction of mono-phosphorylation that result in the N-terminal secondary structure changes of Banf1. Additionally, our modelling showed that phosphorylated Banf1 with their dominant N-terminal secondary structures bind to DNA with a significantly lower affinity and the docked binding pose are not stable in MD simulations. These findings help future studies in predicting effect of mutations in Banf1 on its DNA-binding capability and open a novel avenue for the development of therapeutics such as cancer drugs, targeting cell proliferation by inducing conformational changes in Banf1's N-terminal domain.

Barrier-to-autointegration-factor (Banf1) modulates DNA double-strand break repair pathway choice via regulation of DNA-dependent kinase (DNA-PK) activity.

DNA repair pathways are essential to maintain the integrity of the genome and prevent cell death and tumourigenesis. Here, we show that the Barrier-to-Autointegration Factor (Banf1) protein has a role in the repair of DNA double-strand breaks. Banf1 is characterized as a nuclear envelope protein and mutations in Banf1 are associated with the severe premature aging syndrome, Néstor-Guillermo Progeria Syndrome. We have previously shown that Banf1 directly regulates the activity of PARP1 in the repair of oxidative DNA lesions. Here, we show that Banf1 also has a role in modulating DNA double-strand break repair through regulation of the DNA-dependent Protein Kinase catalytic subunit, DNA-PKcs. Specifically, we demonstrate that Banf1 relocalizes from the nuclear envelope to sites of DNA double-strand breaks. We also show that Banf1 can bind to and directly inhibit the activity of DNA-PKcs. Supporting this, cellular depletion of Banf1 leads to an increase in non-homologous end-joining and a decrease in homologous recombination, which our data suggest is likely due to unrestrained DNA-PKcs activity. Overall, this study identifies how Banf1 regulates double-strand break repair pathway choice by modulating DNA-PKcs activity to control genome stability within the cell.

Human single-stranded DNA binding protein 1 (hSSB1, OBFC2B), a critical component of the DNA damage response.

Our genomic DNA is found predominantly in a double-stranded helical conformation. However, there are a number of cellular transactions and DNA damage events that result in the exposure of single stranded regions of DNA. DNA transactions require these regions of single stranded DNA, but they are only transient in nature as they are particularly susceptible to further damage through chemical and enzymatic degradation, metabolic activation, and formation of secondary structures. To protect these exposed regions of single stranded DNA, all living organisms have members of the Single Stranded DNA Binding (SSB) protein family, which are characterised by a conserved oligonucleotide/oligosaccharide-binding (OB) domain. In humans, three such proteins members have been identified; namely the Replication Protein A (RPA) complex, hSSB1 and hSSB2. While RPA is extremely well characterised, the roles of hSSB1 and hSSB2 have only emerged recently. In this review, we discuss the critical roles that hSSB1 plays in the maintenance of genomic stability.

hSSB1 associates with and promotes stability of the BLM helicase.

BACKGROUND: Maintenance of genome stability is critical in human cells. Mutations in or loss of genome stability pathways can lead to a number of pathologies including cancer. hSSB1 is a critical DNA repair protein functioning in the repair and signalling of stalled DNA replication forks, double strand DNA breaks and oxidised DNA lesions. The BLM helicase is central to the repair of both collapsed DNA replication forks and double strand DNA breaks by homologous recombination. RESULTS: In this study, we demonstrate that hSSB1 and BLM helicase form a complex in cells and the interaction is altered in response to ionising radiation (IR). BLM and hSSB1 also co-localised at nuclear foci following IR-induced double strand breaks and stalled replication forks. We show that hSSB1 depleted cells contain less BLM protein and that this deficiency is due to proteasome mediated degradation of BLM. Consequently, there is a defect in recruitment of BLM to chromatin in response to ionising radiation-induced DSBs and to hydroxyurea-induced stalled and collapsed replication forks. CONCLUSIONS: Our data highlights that BLM helicase and hSSB1 function in a dynamic complex in cells and that this complex is likely required for BLM protein stability and function.

ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1.

The MRE11/RAD50/NBS1 (MRN) complex plays a central role as a sensor of DNA double strand breaks (DSB) and is responsible for the efficient activation of ataxia-telangiectasia mutated (ATM) kinase. Once activated ATM in turn phosphorylates RAD50 and NBS1, important for cell cycle control, DNA repair and cell survival. We report here that MRE11 is also phosphorylated by ATM at S676 and S678 in response to agents that induce DNA DSB, is dependent on the presence of NBS1, and does not affect the association of members of the complex or ATM activation. A phosphosite mutant (MRE11S676AS678A) cell line showed decreased cell survival and increased chromosomal aberrations after radiation exposure indicating a defect in DNA repair. Use of GFP-based DNA repair reporter substrates in MRE11S676AS678A cells revealed a defect in homology directed repair (HDR) but single strand annealing was not affected. More detailed investigation revealed that MRE11S676AS678A cells resected DNA ends to a greater extent at sites undergoing HDR. Furthermore, while ATM-dependent phosphorylation of Kap1 and SMC1 was normal in MRE11S676AS678A cells, there was no phosphorylation of Exonuclease 1 consistent with the defect in HDR. These results describe a novel role for ATM-dependent phosphorylation of MRE11 in limiting the extent of resection mediated through Exonuclease 1.

hSSB1 (NABP2/ OBFC2B) is required for the repair of 8-oxo-guanine by the hOGG1-mediated base excision repair pathway.

The maintenance of genome stability is essential to prevent loss of genetic information and the development of diseases such as cancer. One of the most common forms of damage to the genetic code is the oxidation of DNA by reactive oxygen species (ROS), of which 8-oxo-7,8-dihydro-guanine (8-oxoG) is the most frequent modification. Previous studies have established that human single-stranded DNA-binding protein 1 (hSSB1) is essential for the repair of double-stranded DNA breaks by the process of homologous recombination. Here we show that hSSB1 is also required following oxidative damage. Cells lacking hSSB1 are sensitive to oxidizing agents, have deficient ATM and p53 activation and cannot effectively repair 8-oxoGs. Furthermore, we demonstrate that hSSB1 forms a complex with the human oxo-guanine glycosylase 1 (hOGG1) and is important for hOGG1 localization to the damaged chromatin. In vitro, hSSB1 binds directly to DNA containing 8-oxoguanines and enhances hOGG1 activity. These results underpin the crucial role hSSB1 plays as a guardian of the genome.

Nucleophosmin: from structure and function to disease development.

Nucleophosmin (NPM1) is a critical cellular protein that has been implicated in a number of pathways including mRNA transport, chromatin remodeling, apoptosis and genome stability. NPM1 function is a critical requirement for normal cellular biology as is underlined in cancer where NPM1 is commonly overexpressed, mutated, rearranged and sporadically deleted. Consistent with a multifunctional role within the cell, NPM1 can function not only as a proto-oncogene but also as a tumor suppressor. The aim of this review is to look at the less well-described role of NPM1 in the DNA repair pathways as well as the role of NPM1 in the regulation of apoptosis and its mutation in cancers.

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.

Néstor-Guillermo Progeria Syndrome: a biochemical insight into Barrier-to-Autointegration Factor 1, alanine 12 threonine mutation.

BACKGROUND: Premature aging syndromes recapitulate many aspects of natural aging and provide an insight into this phenomenon at a molecular and cellular level. The progeria syndromes appear to cause rapid aging through disruption of normal nuclear structure. Recently, a coding mutation (c.34G > A [p.A12T]) in the Barrier to Autointegration Factor 1 (BANF1) gene was identified as the genetic basis of Néstor-Guillermo Progeria syndrome (NGPS). This mutation was described to cause instability in the BANF1 protein, causing a disruption of the nuclear envelope structure. RESULTS: Here we demonstrate that the BANF1 A12T protein is indeed correctly folded, stable and that the observed phenotype, is likely due to the disruption of the DNA binding surface of the A12T mutant. We demonstrate, using biochemical assays, that the BANF1 A12T protein is impaired in its ability to bind DNA while its interaction with nuclear envelope proteins is unperturbed. Consistent with this, we demonstrate that ectopic expression of the mutant protein induces the NGPS cellular phenotype, while the protein localizes normally to the nuclear envelope. CONCLUSIONS: Our study clarifies the role of the A12T mutation in NGPS patients, which will be of importance for understanding the development of the disease.

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 repair. 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 heterotrimer. 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.

The expression and role of the Lem-D proteins Ankle2, Emerin, Lemd2, and TMPO in triple-negative breast cancer cell growth.

Background: Triple-negative breast cancer (TNBC) is a sub-classification of breast carcinomas, which leads to poor survival outcomes for patients. TNBCs do not possess the hormone receptors that are frequently targeted as a therapeutic in other cancer subtypes and, therefore, chemotherapy remains the standard treatment for TNBC. Nuclear envelope proteins are frequently dysregulated in cancer cells, supporting their potential as novel cancer therapy targets. The Lem-domain (Lem-D) (LAP2, Emerin, MAN1 domain, and Lem-D) proteins are a family of inner nuclear membrane proteins, which share a ~45-residue Lem-D. The Lem-D proteins, including Ankle2, Lemd2, TMPO, and Emerin, have been shown to be associated with many of the hallmarks of cancer. This study aimed to define the association between the Lem-D proteins and TNBC and determine whether these proteins could be promising therapeutic targets. Methods: GENT2, TCGA, and KM plotter were utilized to investigate the expression and prognostic implications of several Lem-D proteins: Ankle2, TMPO, Emerin, and Lemd2 in publicly available breast cancer patient data. Immunoblotting and immunofluorescent analysis of immortalized non-cancerous breast cells and a panel of TNBC cells were utilized to establish whether protein expression of the Lem-D proteins was significantly altered in TNBC. SiRNA was used to decrease individual Lem-D protein expression, and functional assays, including proliferation assays and apoptosis assays, were conducted. Results: The Lem-D proteins were generally overexpressed in TNBC patient samples at the mRNA level and showed variable expression at the protein level in TNBC cell lysates. Similarly, protein levels were generally negatively correlated with patient survival outcomes. siRNA-mediated depletion of the individual Lem-D proteins in TNBC cells induced aberrant nuclear morphology, decreased proliferation, and induced cell death. However, minimal effects on nuclear morphology or cell viability were observed following Lem-D depletion in non-cancerous MCF10A cells. Conclusion: There is evidence to suggest that Ankle2, TMPO, Emerin, and Lemd2 expressions are correlated with breast cancer patient outcomes, but larger patient sample numbers are required to confirm this. siRNA-mediated depletion of these proteins was shown to specifically impair TNBC cell growth, suggesting that the Lem-D proteins may be a specific anti-cancer target.

Targeting the hSSB1-INTS3 Interface: A Computational Screening Driven Approach to Identify Potential Modulators.

Human single-stranded DNA binding protein 1 (hSSB1) forms a heterotrimeric complex, known as a sensor of single-stranded DNA binding protein 1 (SOSS1), in conjunction with integrator complex subunit 3 (INTS3) and C9ORF80. This sensory protein plays an important role in homologous recombination repair of double-strand breaks in DNA to efficiently recruit other repair proteins at the damaged sites. Previous studies have identified elevated hSSB1-mediated DNA repair activities in various cancers, highlighting its potential as an anticancer target. While prior efforts have focused on inhibiting hSSB1 by targeting its DNA binding domain, this study seeks to explore the inhibition of the hSSB1 function by disrupting its interaction with the key partner protein INTS3 in the SOSS1 complex. The investigative strategy entails a molecular docking-based screening of a specific compound library against the three-dimensional structure of INTS3 at the hSSB1 binding interface. Subsequent assessments involve in vitro analyses of protein-protein interaction (PPI) disruption and cellular effects through co-immunoprecipitation and immunofluorescence assays, respectively. Moreover, the study includes an evaluation of the structural stability of ligands at the INTS3 hot-spot site using molecular dynamics simulations. The results indicate a potential in vitro disruption of the INTS3-hSSB1 interaction by three of the tested compounds obtained from the virtual screening with one impacting the recruitment of hSSB1 and INTS3 to chromatin following DNA damage. To our knowledge, our results identify the first set of drug-like compounds that functionally target INTS3-hSSB1 interaction, and this provides the basis for further biophysical investigations that should help to speed up PPI inhibitor discovery.

Sequence- and Structure-Dependent Cytotoxicity of Phosphorothioate and 2'-O-Methyl Modified Single-Stranded Oligonucleotides.

Single-stranded oligonucleotides (SSOs) are a rapidly expanding class of therapeutics that comprises antisense oligonucleotides, microRNAs, and aptamers, with ten clinically approved molecules. Chemical modifications such as the phosphorothioate backbone and the 2'-O-methyl ribose can improve the stability and pharmacokinetic properties of therapeutic SSOs, but they can also lead to toxicity in vitro and in vivo through nonspecific interactions with cellular proteins, gene expression changes, disturbed RNA processing, and changes in nuclear structures and protein distribution. In this study, we screened a mini library of 277 phosphorothioate and 2'-O-methyl-modified SSOs, with or without mRNA complementarity, for cytotoxic properties in two cancer cell lines. Using circular dichroism, nucleic magnetic resonance, and molecular dynamics simulations, we show that phosphorothioate- and 2'-O-methyl-modified SSOs that form stable hairpin structures through Watson-Crick base pairing are more likely to be cytotoxic than those that exist in an extended conformation. In addition, moderate and highly cytotoxic SSOs in our dataset have a higher mean purine composition than pyrimidine. Overall, our study demonstrates a structure-cytotoxicity relationship and indicates that the formation of stable hairpins should be a consideration when designing SSOs toward optimal therapeutic profiles.

Kruppel-associated Box (KRAB)-associated co-repressor (KAP-1) Ser-473 phosphorylation regulates heterochromatin protein 1ß (HP1-ß) mobilization and DNA repair in heterochromatin.

The DNA damage response encompasses a complex series of signaling pathways that function to regulate and facilitate the repair of damaged DNA. Recent studies have shown that the repair of transcriptionally inactive chromatin, named heterochromatin, is dependent upon the phosphorylation of the co-repressor, Krüppel-associated box (KRAB) domain-associated protein (KAP-1), by the ataxia telangiectasia-mutated (ATM) kinase. Co-repressors, such as KAP-1, function to regulate the rigid structure of heterochromatin by recruiting histone-modifying enzymes, such HDAC1/2, SETDB1, and nucleosome-remodeling complexes such as CHD3. Here, we have characterized a phosphorylation site in the HP1-binding domain of KAP-1, Ser-473, which is phosphorylated by the cell cycle checkpoint kinase Chk2. Expression of a nonphosphorylatable S473A mutant conferred cellular sensitivity to DNA-damaging agents and led to defective repair of DNA double-strand breaks in heterochromatin. In addition, cells expressing S473A also displayed defective mobilization of the HP1-ß chromodomain protein. The DNA repair defect observed in cells expressing S473A was alleviated by depletion of HP1-ß, suggesting that phosphorylation of KAP-1 on Ser-473 promotes the mobilization of HP1-ß from heterochromatin and subsequent DNA repair. These results suggest a novel mechanism of KAP-1-mediated chromatin restructuring via Chk2-regulated HP1-ß exchange from heterochromatin, promoting DNA repair.

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.

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.