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Single molecule biophysics experiments for the study of DNA-protein interactions usually require production of a homogeneous population of long DNA molecules with controlled sequence content and/or internal tertiary structures. Traditionally, Lambda phage DNA has been used for this purpose, but it is difficult to customize. In this article, we provide a detailed and simple protocol for cloning large (~25kbp) plasmids with bespoke sequence content, which can be used to generate custom DNA constructs for a range of single-molecule experiments. In particular, we focus on a procedure for making long single-stranded DNA (ssDNA) molecules, ssDNA-dsDNA hybrids and long DNA constructs with flaps, which are especially relevant for studying the activity of DNA helicases and translocases. Additionally, we describe how the modification of the free ends of such substrates can facilitate their binding to functionalized surfaces allowing immobilization and imaging using dual optical tweezers and confocal microscopy. Finally, we provide examples of how these DNA constructs have been applied to study the activity of human DNA helicase B (HELB). The techniques described herein are simple, versatile, adaptable, and accessible to any laboratory with access to standard molecular biology methods.

Streptococcus pneumoniae is a major human pathogen that can cause severe invasive diseases such as pneumonia, septicaemia and meningitis. Young children are at a particularly high risk, with an estimated 3–4 million cases of severe disease and between 300 000 and 500 000 deaths attributable to pneumococcal disease each year. The haemolytic toxin pneumolysin (Ply) is a primary virulence factor for this bacterium, yet despite its key role in pathogenesis, immune evasion and transmission, the regulation of Ply production is not well defined. Using a genome-wide association approach, we identified a large number of potential affectors of Ply activity, including a gene acquired horizontally on the antibiotic resistance-conferring Integrative and Conjugative Element (ICE) ICESp23FST81. This gene encodes a novel modular protein, ZomB, which has an N-terminal UvrD-like helicase domain followed by two Cas4-like domains with potent ATP-dependent nuclease activity. We found the regulatory effect of ZomB to be specific for the ply operon, potentially mediated by its high affinity for the BOX repeats encoded therein. Using a murine model of pneumococcal colonization, we further demonstrate that a ZomB mutant strain colonizes both the upper respiratory tract and lungs at higher levels when compared to the wild-type strain. While the antibiotic resistance-conferring aspects of ICESp23FST81 are often credited with contributing to the success of the S. pneumoniae lineages that acquire it, its ability to control the expression of a major virulence factor implicated in bacterial transmission is also likely to have played an important role.

Promutagenic O6-alkylguanine adducts in DNA are repaired in humans by O6-methylguanine-DNA-methyltransferase (MGMT) in an irreversible reaction. Here we describe the synthesis of a phosphoramidite that allows the preparation of oligodeoxyribonucleotides (ODNs) containing a novel tricyclic thio analogue of O6-methylguanine in which the third ring bridges the 6-thio group and C7 of a 7-deazapurine. These ODNs are very poor substrates for MGMT and poorly recognised by the alkyltransferase-like protein, Atl1. Examination of the active sites of both MGMT and Atl1 suggest large steric clashes hindering binding of the analogue. Such analogues, if mutagenic, are likely to be highly toxic.

Human DNA helicase B (HELB) is a poorly characterized helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single-molecule approaches to characterize the biochemical activities of HELB protein with a particular focus on its interactions with Replication Protein A (RPA) and RPA–single-stranded DNA (ssDNA) filaments. HELB is a monomeric protein that binds tightly to ssDNA with a site size of ∼20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5′ to 3′ direction accompanied by the formation of DNA loops. HELB also displays classical helicase activity, but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA, which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from ssDNA. This activity, which can allow other proteins access to ssDNA intermediates despite their shielding by RPA, may underpin the diverse roles of HELB in cellular DNA transactions., [Significance] Single-stranded DNA (ssDNA) is a key intermediate in many cellular DNA transactions, including DNA replication, repair, and recombination. Nascent ssDNA is rapidly bound by the Replication Protein A (RPA) complex, forming a nucleoprotein filament that both stabilizes ssDNA and mediates downstream processing events. Paradoxically, however, the very high affinity of RPA for ssDNA may block the recruitment of further factors. In this work, we show that RPA–ssDNA nucleoprotein filaments are specifically targeted by the human HELB helicase. Recruitment of HELB by RPA–ssDNA activates HELB translocation activity, leading to processive removal of upstream RPA complexes. This RPA clearance activity may underpin the diverse roles of HELB in replication and recombination., Work in the laboratory of M.S.D. was supported by an Elizabeth Blackwell Early Career Fellowship from the University of Bristol (to O.J.W.) and Wellcome Trust Investigator Grant 100401/Z/12/Z (to M.S.D.). Work in the laboratory of E.A. was supported by NIH Grants GM130746 (to E.A.) and GM133967 (to E.A.). F.M.-H. acknowledges support from the European Research Council under European Union Horizon 2020 Research and Innovation Program Grant Agreement 681299. Work in the laboratory of F.M.-H. was also supported by Spanish Ministry of Science and Innovation Grants BFU2017-83794-P (AEI/FEDER, UE; to F.M.-H.) and PID2020-112998GB-100 (AEI/10.13039/501100011033; to F.M.-H.) and Comunidad de Madrid Grants Tec4-Bio–S2018/NMT-4443 (to F.M.-H.) and NanoBioCancer–Y2018/BIO-4747 (to F.M.-H.).

Human HELB is a poorly-characterised helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single molecule approaches to characterise the biochemical activities of HELB protein with a particular focus on its interactions with RPA and RPA-ssDNA filaments. HELB is a monomeric protein which binds tightly to ssDNA with a site size of ~20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5′-to-3′ direction accompanied by the formation of DNA loops and with an efficiency of 1 ATP per base. HELB also displays classical helicase activity but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from single-stranded DNA.

DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5' to 3' ssDNA translocase and 5' to 3' helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general., Wellcome Trust [100401/Z/12/Z to M.D.]; EuropeanResearch Council [681299 to F.M.-H.]; Spanish Min-istry of Economy and Competitiveness [BFU2017-83794-PAEI/FEDER, UE to F.M.-H.]; Comunidad de MadridTec4Bio [S2018/NMT-4443 to F.M.-H.]; NanoBioCancer[Y2018/BIO-4747 to F.M.-H.]. Funding for open accesscharge: Wellcome Trust [100401/Z/12/Z].

Promutagenic

A stapled peptide targets CtIP tetramers to inhibit DNA repair and exert synthetic lethality in BRCA1-mutant cancer cells., Cancer cells display high levels of DNA damage and replication stress, vulnerabilities that could be exploited by drugs targeting DNA repair proteins. Human CtIP promotes homology-mediated repair of DNA double-strand breaks (DSBs) and protects stalled replication forks from nucleolytic degradation, thus representing an attractive candidate for targeted cancer therapy. Here, we establish a peptide mimetic of the CtIP tetramerization motif that inhibits CtIP activity. The hydrocarbon-stapled peptide encompassing amino acid residues 18 to 28 of CtIP (SP18–28) stably binds to CtIP tetramers in vitro and facilitates their aggregation into higher-order structures. Efficient intracellular uptake of SP18–28 abrogates CtIP localization to damaged chromatin, impairs DSB repair, and triggers extensive fork degradation. Moreover, prolonged SP18–28 treatment causes hypersensitivity to DNA-damaging agents and selectively reduces the viability of BRCA1-mutated cancer cell lines. Together, our data provide a basis for the future development of CtIP-targeting compounds with the potential to treat patients with cancer.

DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterisedGeobacillus stearothermophilusBad, a novel bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5’ to 3’ ssDNA translocase and 5’ to 3’ helicase activity. Single molecule analysis reveals that Bad is a highly processive DNA motor capable of moving along DNA for distances of more than 4 kbp at a rate of ∼200 base pairs per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments also show that the enzyme loops DNA while translocating, which is an emerging feature of highly processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.

Toxin–antitoxin (TA) systems are present in many bacteria and play important roles in bacterial growth, physiology, and pathogenicity. Those that are best studied are the type II TA systems, in which both toxins and antitoxins are proteins. The HicAB system is one of the prototypic TA systems, found in many bacterial species. Complex interactions between the protein toxin (HicA), the protein antitoxin (HicB), and the DNA upstream of the encoding genes regulate the activity of this system, but few structural details are available about how HicA destabilizes the HicB–DNA complex. Here, we determined the X-ray structures of HicB and the HicAB complex to 1.8 and 2.5 Å resolution, respectively, and characterized their DNA interactions. This revealed that HicB forms a tetramer and HicA and HicB form a heterooctameric complex that involves structural reorganization of the C-terminal (DNA-binding) region of HicB. Our observations indicated that HicA has a profound impact on binding of HicB to DNA sequences upstream of hicAB in a stoichiometric-dependent way. At low ratios of HicA:HicB, there was no effect on DNA binding, but at higher ratios, the affinity for DNA declined cooperatively, driving dissociation of the HicA:HicB:DNA complex. These results reveal the structural mechanisms by which HicA de-represses the HicB–DNA complex.

SummaryCtIP is involved in the resection of double-stranded DNA breaks during the S and G2 phases of the cell cycle for repair by homologous recombination. Acting in concert with the MRN complex, it plays a particularly important role in handling complex DNA end structures by localised nucleolytic processing of DNA termini in preparation for longer range resection. Here we show that human CtIP is a tetrameric protein adopting a dumbbell architecture in which DNA binding domains are connected by long coiled-coils. The protein complex binds two short DNA duplexes with high affinity and bridges DNA molecules in trans. DNA binding is potentiated by dephosphorylation and is not specific for DNA end structures per se. However, the affinity for linear DNA molecules is increased if the DNA terminates with more complex structures including forked ssDNA overhangs and model nucleoprotein conjugates. This work provides a biochemical and structural basis for the function of CtIP at complex DNA breaks.

N-Nitrosation of glycine and its derivatives generates potent alkylating agents that can lead to the formation ofO6-carboxymethylguanine (O6-CMG) in DNA.O6-CMG has been identified in DNA derived from human colon tissue and its occurrence has been linked to diets high in red and processed meats, implying an association with the induction of colorectal cancer. By analogy toO6-methylguanine,O6-CMG is expected to be mutagenic, inducing G-to-A mutations that may be the molecular basis of increased cancer risk. Previously, the crystal structure of the DNA dodecamer d(CGCG[O6-CMG]ATTCGCG) has been reported, in whichO6-CMG forms a Watson–Crick-type pair with thymine similar to the canonical A:T pair. In order to further investigate the versatility ofO6-CMG in base-pair formation, the structure of the DNA dodecamer d(CGC[O6-CMG]AATTTGCG) containingO6-CMG at a different position has been determined by X-ray crystallography using four crystal forms obtained under conditions containing different solvent ions (Sr2+, Ba2+, Mg2+, K+or Na+) with and without Hoechst 33258. The most striking finding is that the pairing modes ofO6-CMG with T are quite different from those previously reported. In the present dodecamer, the T bases are displaced (wobbled) into the major groove to form a hydrogen bond between the thymine N3N—H and the carboxyl group ofO6-CMG. In addition, a water molecule is bridged through two hydrogen bonds between the thymine O2atom and the 2-amino group ofO6-CMG to stabilize the pairing. These interaction modes commonly occur in the four crystal forms, regardless of the differences in crystallization conditions. The previous and the present results show thatO6-CMG can form a base pair with T in two alternative modes: the Watson–Crick type and a high-wobble type, the nature of which may depend on the DNA-sequence context.

Alkyltransferase-like (ATL) proteins in Schizosaccharomyces pombe (Atl1) and Thermus thermophilus (TTHA1564) protect against the adverse effects of DNA alkylation damage by flagging O 6 -alkylguanine lesions for nucleotide excision repair (NER). We show that both ATL proteins bind with high affinity to oligodeoxyribonucleotides containing O 6 -alkylguanines differing in size, polarity, and charge of the alkyl group. However, Atl1 shows a greater ability than TTHA1564 to distinguish between O 6 -alkylguanine and guanine and in an unprecedented mechanism uses Arg69 to probe the electrostatic potential surface of O 6 -alkylguanine, as determined using molecular mechanics calculations. An unexpected consequence of this feature is the recognition of 2,6-diaminopurine and 2-aminopurine, as confirmed in crystal structures of respective Atl1-DNA complexes. O 6 -Alkylguanine and guanine discrimination is diminished for Atl1 R69A and R69F mutants, and S. pombe R69A and R69F mutants are more sensitive toward alkylating agent toxicity, revealing the key role of Arg69 in identifying O 6 -alkylguanines critical for NER recognition.

Nucleotide excision repair (NER) has long been known to remove DNA lesions induced by chemical carcinogens, and the molecular mechanism has been partially elucidated. Here we demonstrate that in Schizosaccharomyces pombe a DNA recognition protein, alkyltransferase-like 1 (Atl1), can play a pivotal role in selecting a specific NER pathway, depending on the nature of the DNA modification. The relative ease of dissociation of Atl1 from DNA containing small O(6)-alkylguanines allows accurate completion of global genome repair (GGR), whereas strong Atl1 binding to bulky O(6)-alkylguanines blocks GGR, stalls the transcription machinery, and diverts the damage to transcription-coupled repair. Our findings redraw the initial stages of the NER process in those organisms that express an alkyltransferase-like gene and raise the question of whether or not O(6)-alkylguanine lesions that are poor substrates for the alkyltransferase proteins in higher eukaryotes might, by analogy, signal such lesions for repair by NER.

We show that DNA containing a conformationally-locked anti analogue of O(6)-alkylguanine is a poor substrate for human O(6)-methylguanine-DNA methyltransferase (MGMT) and the alkyltransferase-like protein, Atl1. This highlights the requirement for the syn conformation and rationalises why certain O(6)-alkylguanines are poor MGMT substrates.

Previously, we reported the crystal structure of DNA d(CGCGXATTCGCG), which revealed that X (O6-carboxymethyl-G) at the 5th position forms a Watson-Crick (WC) type pair with T (Fig. 1a) similar to the canonical A:T pair [1]. In order to investigate the versatility of X in the base-pair formation, another DNA d(CGCXAATTTGCG) containing X at the 4th position has been X-ray analyzed using four different crystals. In the four crystals, the T bases are always largely wobbled into the major groove side to form a hydrogen bond between N3 of T and the carboxyl group of X. In addition, a water molecule bridges between the two bases through two hydrogen bonds to stabilize the pair formation (Fig.1b). This high-wobble (hW) pair is quite different from the previously reported one [1]. Now we succeeded to reveal the two types of pairing modes of X:T pair, WC type and hW type. Since the four crystals were obtained in different conditions, the large displacement of T cannot be ascribed to the interactions with solvent cations. Another possibility is Hoechst33258 used for crystallization. But, as the crystals were obtained in both the presence and the absence of Hoechst33258, this is not the case. The remaining possibility would be the location of the modified base, because the sequence of the used DNA contains an AATT tract at the center and two CGCG tracts at the both ends. The AATT tract is well known to be more flexible (ω*≍ -160) than CGCG tract (ω*≍ -8.70). We found that the WC type X:T pairing requires a large ω angle (-180) between the bases. When X is located at the 5th position (in the AATT tract), the WC type is allowed to occur, while at the 4th position (in the CGCG tract) the WC type is difficult to occur. Here it could be concluded that X form a pair with T in the mode of WC if it is in the flexible tract, while in the mode of hW if it is in the rigid tract, because T base is out of the base stacking. * propeller twist angle between the two bases.

gastrointesti-nal cancer allows thymine residue to be flexible between Watson-Crick type pairing and large wobbling. Fang Zhang, Masaru Tsunoda, Oliver Wilkinson, Christoper L. Millington, David M. Williams and Akio Takenaka Grad. Sch. Sci. & Eng., Fclty. Pharm., Iwaki-Meisei Univ., Iwaki 970-8044, Japan, and Ctr. Chem. Biol., Krebs Inst., Univ. Sheffield, Sheffield S3 7HK, UK E-mail: atakenak@iwakimu.ac.jp