Showing papers on "Primase published in 2016"
TL;DR: It is proposed that upon phage infection, crRNA-guided StCsm binding to the emerging transcript recruits Cas10 DNase to the actively transcribed phage DNA, resulting in degradation of both the transcript and phageDNA, but not the host DNA.
209 citations
TL;DR: It is reported that PrimPol, a recently described primase-polymerase (PrimPol), plays a crucial role in the bypass of leading strand G4 structures, and implicate PrimPol in promoting restart of DNA synthesis downstream of, but closely coupled to, G4 replication impediments.
130 citations
TL;DR: Ctf4-dependent recruitment of CIP-box proteins couples other processes to DNA synthesis, including rDNA copy-number regulation, and reveals unexpected complexity of Ctf4 function, as a hub that connects multiple accessory factors to the replisome.
103 citations
TL;DR: The crystal structures of two key complexes, the human primosome and the C-terminal domain of the primase large subunit (p58C) with bound DNA/RNA duplex are reported, revealing a central role of p58C in the coordinated actions of two catalytic domains in the primosomes and ultimately could impact the design of anticancer drugs.
84 citations
TL;DR: Results indicate that PrimPol's repriming activity plays a central role in reinitiating replication downstream from CTNAs and other specific DNA lesions and indicates that its primase, rather than TLS activity, is pivotal for DNA damage tolerance.
Abstract: PrimPol is a DNA damage tolerance enzyme possessing both translesion synthesis (TLS) and primase activities To uncover its potential role in TLS-mediated IgVλ hypermutation and define its interplay with other TLS polymerases, PrimPol(-/-) and PrimPol(-/-)/Polη(-/-)/Polζ (-/-) gene knockouts were generated in avian cells Loss of PrimPol had no significant impact on the rate of hypermutation or the mutation spectrum of IgVλ However, PrimPol(-/-) cells were sensitive to methylmethane sulfonate, suggesting that it may bypass abasic sites at the IgVλ segment by repriming DNA synthesis downstream of these sites PrimPol(-/-) cells were also sensitive to cisplatin and hydroxyurea, indicating that it assists in maintaining / restarting replication at a variety of lesions To accurately measure the relative contribution of the TLS and primase activities, we examined DNA damage sensitivity in PrimPol(-/-) cells complemented with polymerase or primase-deficient PrimPol Polymerase-defective, but not primase-deficient, PrimPol suppresses the hypersensitivity of PrimPol(-/-) cells This indicates that its primase, rather than TLS activity, is pivotal for DNA damage tolerance Loss of TLS polymerases, Polη and Polζ has an additive effect on the sensitivity of PrimPol(-/-) cells Moreover, we found that PrimPol and Polη-Polζ redundantly prevented cell death and facilitated unperturbed cell cycle progression PrimPol(-/-) cells also exhibited increased sensitivity to a wide variety of chain-terminating nucleoside analogs (CTNAs) PrimPol could perform close-coupled repriming downstream of CTNAs and oxidative damage in vitro Together, these results indicate that PrimPol's repriming activity plays a central role in reinitiating replication downstream from CTNAs and other specific DNA lesions
77 citations
TL;DR: A novel multiple displacement amplification (MDA) method based on the unique DNA primase features of Thermus thermophilus (Tth) PrimPol that promises to facilitate and improve single-cell genomic analysis.
Abstract: Sequencing of a single-cell genome requires DNA amplification, a process prone to introducing bias and errors into the amplified genome. Here we introduce a novel multiple displacement amplification (MDA) method based on the unique DNA primase features of Thermus thermophilus (Tth) PrimPol. TthPrimPol displays a potent primase activity preferring dNTPs as substrates unlike conventional primases. A combination of TthPrimPol's unique ability to synthesize DNA primers with the highly processive Phi29 DNA polymerase (Φ29DNApol) enables near-complete whole genome amplification from single cells. This novel method demonstrates superior breadth and evenness of genome coverage, high reproducibility, excellent single-nucleotide variant (SNV) detection rates with low allelic dropout (ADO) and low chimera formation as exemplified by sequencing HEK293 cells. Moreover, copy number variant (CNV) calling yields superior results compared with random primer-based MDA methods. The advantages of this method, which we named TruePrime, promise to facilitate and improve single-cell genomic analysis.
70 citations
TL;DR: The structures of the enzymatic components of replisomes, and the protein-protein and protein-DNA interactions that ensure they remain intact while undergoing substantial dynamic changes as they function to copy both the leading and lagging strands simultaneously during coordinated replication are reviewed.
Abstract: DNA replication in Escherichia coli initiates at oriC, the origin of replication and proceeds bidirectionally, resulting in two replication forks that travel in opposite directions from the origin. Here, we focus on events at the replication fork. The replication machinery (or replisome), first assembled on both forks at oriC, contains the DnaB helicase for strand separation, and the DNA polymerase III holoenzyme (Pol III HE) for DNA synthesis. DnaB interacts transiently with the DnaG primase for RNA priming on both strands. The Pol III HE is made up of three subassemblies: (i) the αɛθ core polymerase complex that is present in two (or three) copies to simultaneously copy both DNA strands, (ii) the β2 sliding clamp that interacts with the core polymerase to ensure its processivity, and (iii) the seven-subunit clamp loader complex that loads β2 onto primer-template junctions and interacts with the α polymerase subunit of the core and the DnaB helicase to organize the two (or three) core polymerases. Here, we review the structures of the enzymatic components of replisomes, and the protein-protein and protein-DNA interactions that ensure they remain intact while undergoing substantial dynamic changes as they function to copy both the leading and lagging strands simultaneously during coordinated replication.
66 citations
TL;DR: The first structure of human PrimPol in ternary complex with a DNA template-primer and an incoming deoxynucleoside triphosphate (dNTP) is presented, addressing long-standing questions about how DNA primases actually initiate synthesis and how primase and polymerase activities combine in a single enzyme to carry out DNA synthesis.
Abstract: PrimPol is a novel human enzyme that contains both DNA primase and DNA polymerase activities. We present the first structure of human PrimPol in ternary complex with a DNA template-primer and an incoming deoxynucleoside triphosphate (dNTP). The ability of PrimPol to function as a DNA primase stems from a simple but remarkable feature—almost complete lack of contacts to the DNA primer strand. This, in turn, allows two dNTPs to bind initiation and elongation sites on the enzyme for the formation of the first dinucleotide. PrimPol shows the ability to synthesize DNA opposite ultraviolet (UV) lesions; however, unexpectedly, the active-site cleft of the enzyme is constrained, which precludes the bypass of UV-induced DNA lesions by conventional translesion synthesis. Together, the structure addresses long-standing questions about how DNA primases actually initiate synthesis and how primase and polymerase activities combine in a single enzyme to carry out DNA synthesis.
63 citations
TL;DR: It is shown that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis, reconciling divergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation.
63 citations
TL;DR: It is shown that the C-terminal domain of the large subunit (p58C) plays a major role in template-primer binding and also defines the elements of the DNA template and the RNA primer that interact with p58C.
54 citations
TL;DR: It is established that PolDIP2 can regulate the TLS polymerase and primer extension activities of PrimPol, further enhancing the understanding of the roles of PolD IP2 and PrimPol in eukaryotic DNA damage tolerance.
Abstract: Translesion synthesis (TLS) employs specialized DNA polymerases to bypass replication fork stalling lesions. PrimPol was recently identified as a TLS primase and polymerase involved in DNA damage tolerance. Here, we identify a novel PrimPol binding partner, PolDIP2, and describe how it regulates PrimPol's enzymatic activities. PolDIP2 stimulates the polymerase activity of PrimPol, enhancing both its capacity to bind DNA and the processivity of the catalytic domain. In addition, PolDIP2 stimulates both the efficiency and error-free bypass of 8-oxo-7,8-dihydrodeoxyguanosine (8-oxoG) lesions by PrimPol. We show that PolDIP2 binds to PrimPol's catalytic domain and identify potential binding sites. Finally, we demonstrate that depletion of PolDIP2 in human cells causes a decrease in replication fork rates, similar to that observed in PrimPol−/− cells. However, depletion of PolDIP2 in PrimPol−/− cells does not produce a further decrease in replication fork rates. Together, these findings establish that PolDIP2 can regulate the TLS polymerase and primer extension activities of PrimPol, further enhancing our understanding of the roles of PolDIP2 and PrimPol in eukaryotic DNA damage tolerance.
01 Jan 2016
TL;DR: Using degenerate oligonucleotide primers designed to amplify DNA fragments flanked by the highly conserved motifs V L D E A D and Y I H R I G, five putative RNA helicase genes are detected in the yeast Saccharomyces cerevisiae, suggesting that these genes are functionally related.
Abstract: The RNA helicase gene family encodes a group of eight homologous proteins that,share regions of sequence similarity. This group of evolutionarily conserved proteins presumably all utilize ATP (or some other nucleoside triphosphate) as an energy source for unwinding double- stranded RNA. Members of this family have been implicated in a variety of physiological functions in organisms ranging from Escherichia coli to human, such as translation initiation, mito- chondrial mRNA splicing, ribosomal assembly, and germinal line cell differentiation. We have applied polymerase chain reaction technology to search for additional members of the RNA helicase family in the yeast Saccharomyces cerevisiae. Using degenerate oligonucleotide primers designed to amplify DNA fragments flanked by the highly conserved motifs V L D E A D and Y I H R I G, we have detected five putative RNA helicase genes. Northern and Southern blot analyses demon- strated that these genes are single copy and expressed in yeast. Several members of the RNA helicase family share sequence identity ranging from 49.2% to 67.2%, suggesting that they are functionally related. The discovery of such a multitude of putative RNA helicase genes in yeast suggests that RNA helicase activities are involved in a variety of fundamentally important biological processes.
TL;DR: It is shown that MGME1 can cleave flaps to enable efficient ligation of newly replicated DNA strands in combination with POLγ and POLγ-driven strand displacement is sufficient to generate DNA- but not RNA-flap substrates suitable for MGME 1 cleavage and ligation during replication.
Abstract: Recently, MGME1 was identified as a mitochondrial DNA nuclease with preference for single-stranded DNA (ssDNA) substrates. Loss-of-function mutations in patients lead to mitochondrial disease with DNA depletion, deletions, duplications and rearrangements. Here, we assess the biochemical role of MGME1 in the processing of flap intermediates during mitochondrial DNA replication using reconstituted systems. We show that MGME1 can cleave flaps to enable efficient ligation of newly replicated DNA strands in combination with POLγ. MGME1 generates a pool of imprecisely cut products (short flaps, nicks and gaps) that are converted to ligatable nicks by POLγ through extension or excision of the 3'-end strand. This is dependent on the 3'-5' exonuclease activity of POLγ which limits strand displacement activity and enables POLγ to back up to the nick by 3'-5' degradation. We also demonstrate that POLγ-driven strand displacement is sufficient to generate DNA- but not RNA-flap substrates suitable for MGME1 cleavage and ligation during replication. Our findings have implications for RNA primer removal models, the 5'-end processing of nascent DNA at OriH, and DNA repair.
TL;DR: It is revealed that significantly cancer-related changes in human rRNA methylation were present in patients with hepatocellular carcinoma and significantly hinders DNA- and RNA-directed DNA synthesis.
Abstract: N 6-Methyladenine (m6A) is the most abundant internal modification on mammalian mRNA. Very recently, m6A has been reported as a potentially important ‘epigenetic’ mark in eukaryotes. Until now, site-specific detection of m6A is technically very challenging. Here, we first reveal that m6A significantly hinders DNA- and RNA-directed DNA synthesis. Systematic investigations of 5′-triphosphates of a variety of 5-substituted 2′-deoxyuridine analogs in primer extension have been performed. In the current study, a quantitative analysis of m6A in the RNA or DNA context has been achieved, using Bst DNA polymerase catalyzed primer extension. Molecular dynamics study predicted that m6A in template tends to enter into and be restrained in the MGR region of Bst DNA polymerase, reducing conformational flexibility of the DNA backbone. More importantly, a site-specific determination of m6A in human ribosomal RNA (rRNA) with high accuracy has been afforded. Through a cumulative analysis of methylation alterations, we first reveal that significantly cancer-related changes in human rRNA methylation were present in patients with hepatocellular carcinoma.
TL;DR: A thermostable KlenTaq DNA polymerase variant with significant reverse transcription activity that is able to discriminate 2′-O-methylated from unmethylated RNAs is presented and enables expeditious quantification of 2′, O-methylation of individual nucleotides directly from total RNA extracts by a simple qRT-PCR.
Abstract: Methylation of the 2'-hydroxyl-group of ribonucleotides is found in all major classes of RNA in eukaryotes and is one of the most abundant posttranscriptional modifications of stable RNAs. In spite of intense studies, the multiple functions of RNA 2'-O-methylation are still not understood. One major obstacle in the field are the technical demanding detection methods, which are typically laborious and do not always deliver unambiguous results. We present a thermostable KlenTaq DNA polymerase variant with significant reverse transcription activity that is able to discriminate 2'-O-methylated from unmethylated RNAs. The engineered enzyme catalyzes DNA synthesis from DNA as well as RNA templates and enables expeditious quantification of 2'-O-methylation of individual nucleotides directly from total RNA extracts by a simple qRT-PCR.
TL;DR: Current understanding of how replication fork unwinding by the CMG helicase is coupled to leading-strand synthesis by polymerase (Pol) ɛ and lagging-strands priming by Pol α/primase is reviewed, and emerging principles of replisome organization are discussed.
TL;DR: It is shown that the preferential unwinding of RNA: DNA hybrids is due to neither specific binding nor differences in the rate of strand separation, and Pif1 is capable of unwinding RNA:DNA heteroduplexes with moderately greater processivity compared with its duplex DNA:DNA counterparts.
TL;DR: The initiation of chromosomal DNA replication starts at a replication origin, which in bacteria is a discrete locus that contains DNA sequence motifs recognized by an initiator protein whose role is to assemble the replication fork machinery at this site.
Abstract: The initiation of chromosomal DNA replication starts at a replication origin, which in bacteria is a discrete locus that contains DNA sequence motifs recognized by an initiator protein whose role is to assemble the replication fork machinery at this site. In bacteria with a single chromosome, DnaA is the initiator and is highly conserved in all bacteria. As an adenine nucleotide binding protein, DnaA bound to ATP is active in the assembly of a DnaA oligomer onto these sites. Other proteins modulate DnaA oligomerization via their interaction with the N-terminal region of DnaA. Following the DnaA-dependent unwinding of an AT-rich region within the replication origin, DnaA then mediates the binding of DnaB, the replicative DNA helicase, in a complex with DnaC to form an intermediate named the prepriming complex. In the formation of this intermediate, the helicase is loaded onto the unwound region within the replication origin. As DnaC bound to DnaB inhibits its activity as a DNA helicase, DnaC must dissociate to activate DnaB. Apparently, the interaction of DnaB with primase (DnaG) and primer formation leads to the release of DnaC from DnaB, which is coordinated with or followed by translocation of DnaB to the junction of the replication fork. There, DnaB is able to coordinate its activity as a DNA helicase with the cellular replicase, DNA polymerase III holoenzyme, which uses the primers made by primase for leading strand DNA synthesis.
TL;DR: It is found that transcript RNA is more efficient in repairing a DSB in its own DNA than in a homologous but ectopic locus (in trans), and an antagonistic role of RNase H in RNA-DNA recombination is highlighted.
Abstract: The transfer of genetic information from RNA to DNA is considered an extraordinary process in molecular biology. Despite the fact that cells transcribe abundant amount of RNA with a wide range of functions, it has been difficult to uncover whether RNA can serve as a template for DNA repair and recombination. An increasing number of experimental evidences suggest a direct role of RNA in DNA modification. Recently, we demonstrated that endogenous transcript RNA can serve as a template to repair a DNA double-strand break (DSB), the most harmful DNA lesion, not only indirectly via formation of a DNA copy (cDNA) intermediate, but also directly in a homology driven mechanism in budding yeast. These results point out that the transfer of genetic information from RNA to DNA is more general than previously thought. We found that transcript RNA is more efficient in repairing a DSB in its own DNA (in cis) than in a homologous but ectopic locus (in trans). Here, we summarize current knowledge about the proces...
TL;DR: This work provides extensive quantitative evidence that the binding of pol η to PCNA and the ensuing TLS are both independent of PCNA ubiquitination and the unique properties of pols η and δ are attuned to promote an efficient and passive exchange of polymerases during TLS on the lagging strand.
Abstract: Translesion DNA synthesis (TLS) during S-phase uses specialized TLS DNA polymerases to replicate a DNA lesion, allowing stringent DNA synthesis to resume beyond the offending damage. Human TLS involves the conjugation of ubiquitin to PCNA clamps encircling damaged DNA and the role of this post-translational modification is under scrutiny. A widely-accepted model purports that ubiquitinated PCNA recruits TLS polymerases such as pol η to sites of DNA damage where they may also displace a blocked replicative polymerase. We provide extensive quantitative evidence that the binding of pol η to PCNA and the ensuing TLS are both independent of PCNA ubiquitination. Rather, the unique properties of pols η and δ are attuned to promote an efficient and passive exchange of polymerases during TLS on the lagging strand.
TL;DR: X-ray crystal structures show that Y-family human DNA polymerase η maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2′-deoxyguanosine and cyclobutane pyrimidine dimer but with rates that are 103-fold lower than for inserting the corresponding dN TPs.
TL;DR: Pfh1 is an accessory DNA helicase that interacts with the replisome and promotes replication and suppresses DNA damage at hard-to-replicate sites, and is concluded that this evolutionarily conserved helicase helps preserve genome integrity.
Abstract: Replicative DNA helicases expose the two strands of the double helix to the replication apparatus, but accessory helicases are often needed to help forks move past naturally occurring hard-to-repli ...
TL;DR: It is shown that even high-fidelity DNA polymerases are capable of efficiently using nucleotide chimera modified with a large protein like horseradish peroxidase as substrates for template-dependent DNA synthesis, despite this "cargo" being more than 100-fold larger than the natural substrates.
Abstract: DNA polymerases select the right nucleotide for the growing polynucleotide chain based on the shape and geometry of the nascent nucleotide pairs and thereby ensure high DNA replication selectivity. High-fidelity DNA polymerases are believed to possess tight active sites that allow little deviation from the canonical structures. However, DNA polymerases are known to use nucleotides with small modifications as substrates, which is key for numerous core biotechnology applications. We show that even high-fidelity DNA polymerases are capable of efficiently using nucleotide chimera modified with a large protein like horseradish peroxidase as substrates for template-dependent DNA synthesis, despite this "cargo" being more than 100-fold larger than the natural substrates. We exploited this capability for the development of systems that enable naked-eye detection of DNA and RNA at single nucleotide resolution.
TL;DR: The conformational snapshots of BaPif1 provide insights into the mechanism governing the helicase activity of Pif1, which undergoes a large conformational change upon concomitant binding of ATP and ssDNA, which is critical for P if1's activities.
TL;DR: The first crystal structure of human Polα polymerase subunit in complex with a DNA: DNA helix is presented and it is found that portion of the DNA:DNA helix in contact with the polymerase is not in a B-form but in a hybrid A-B form, which may augur the termination of primer synthesis in eukaryotes.
Abstract: The Polα/primase complex assembles the short RNA-DNA fragments for priming of lagging and leading strand DNA replication in eukaryotes. As such, the Polα polymerase subunit encounters two types of substrates during primer synthesis: an RNA:DNA helix and a DNA:DNA helix. The engagement of the polymerase subunit with the DNA:DNA helix has been suggested as the of basis for primer termination in eukaryotes. However, there is no structural information on how the Polα polymerase subunit actually engages with a DNA:DNA helix during primer synthesis. We present here the first crystal structure of human Polα polymerase subunit in complex with a DNA:DNA helix. Unexpectedly, we find that portion of the DNA:DNA helix in contact with the polymerase is not in a B-form but in a hybrid A-B form. Almost all of the contacts observed previously with an RNA primer are preserved with a DNA primer--with the same set of polymerase residues tracking the sugar-phosphate backbone of the DNA or RNA primer. Thus, rather than loss of specific contacts, the free energy cost of distorting DNA from B- to hybrid A-B form may augur the termination of primer synthesis in eukaryotes.
TL;DR: It is shown that Pif1, a 5′–3′ helicase, not only stimulates the strand displacement activity of Pol δ but it also allows efficient replication through the block, by removing bound Rap1 in front of the polymerase.
Abstract: Using an in vitro reconstituted system in this work we provide direct evidence that the yeast repressor/activator protein 1 (Rap1), tightly bound to its consensus site, forms a strong non-polar barrier for the strand displacement activity of DNA polymerase δ. We propose that relief of inhibition may be mediated by the activity of an accessory helicase. To this end, we show that Pif1, a 5'-3' helicase, not only stimulates the strand displacement activity of Pol δ but it also allows efficient replication through the block, by removing bound Rap1 in front of the polymerase. This stimulatory activity of Pif1 is not limited to the displacement of a single Rap1 molecule; Pif1 also allows Pol δ to carry out DNA synthesis across an array of bound Rap1 molecules that mimics a telomeric DNA-protein assembly. This activity of Pif1 represents a novel function of this helicase during DNA replication.
TL;DR: This review summarizes the current state of knowledge of the eukaryotic core replisome proteins, their structure, individual functions, and how they are organized at the replication fork as a machine.
Abstract: The cellular replicating machine, or "replisome," is composed of numerous different proteins. The core replication proteins in all cell types include a helicase, primase, DNA polymerases, sliding clamp, clamp loader, and single-strand binding (SSB) protein. The core eukaryotic replisome proteins evolved independently from those of bacteria and thus have distinct architectures and mechanisms of action. The core replisome proteins of the eukaryote include: an 11-subunit CMG helicase, DNA polymerase alpha-primase, leading strand DNA polymerase epsilon, lagging strand DNA polymerase delta, PCNA clamp, RFC clamp loader, and the RPA SSB protein. There are numerous other proteins that travel with eukaryotic replication forks, some of which are known to be involved in checkpoint regulation or nucleosome handling, but most have unknown functions and no bacterial analogue. Recent studies have revealed many structural and functional insights into replisome action. Also, the first structure of a replisome from any cell type has been elucidated for a eukaryote, consisting of 20 distinct proteins, with quite unexpected results. This review summarizes the current state of knowledge of the eukaryotic core replisome proteins, their structure, individual functions, and how they are organized at the replication fork as a machine.
TL;DR: The method of hydrogen/deuterium exchange mass spectrometry is used to address the importance of DnaB–DnaC complex formation as a prerequisite for helicase loading, and shows that the DnB ring opens and closes, and that specific amino acids near the N-terminus of DnC interact with a site in DnaC's C-terminal domain to trap it as an open ring.
Abstract: Helicase loading at a DNA replication origin often requires the dynamic interactions between the DNA helicase and an accessory protein In E coli, the DNA helicase is DnaB and DnaC is its loading partner We used the method of hydrogen/deuterium exchange mass spectrometry to address the importance of DnaB-DnaC complex formation as a prerequisite for helicase loading Our results show that the DnaB ring opens and closes, and that specific amino acids near the N-terminus of DnaC interact with a site in DnaB's C-terminal domain to trap it as an open ring This event correlates with conformational changes of the RecA fold of DnaB that is involved in nucleotide binding, and of the AAA+ domain of DnaC DnaC also causes an alteration of the helical hairpins in the N-terminal domain of DnaB, presumably occluding this region from interacting with primase Hence, DnaC controls the access of DnaB by primase
TL;DR: The eukaryotic core replisome shows an unanticipated architecture, with one polymerase sitting above the helicase and the other below, and structures of Mcm2 bound to an H3/H4 tetramer suggest a direct role of the replisomes in handling nucleosomes, which are important to DNA organization and gene regulation.
TL;DR: The findings elucidate the detailed molecular mechanism in solution of ssDNA binding by hSSB1, a major player in the maintenance of genomic stability and reveals that ssDNA recognition in solution is modulated by base-stacking of four key aromatic residues within the OB domain.
Abstract: 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.