End Of ssDNA Research Articles

Biochemical Characterization of Bacteriophage T4 Mre11-Rad50 Complex

The Mre11-Rad50 complex (MR) from bacteriophage T4 (gp46/47) is involved in the processing of DNA double-strand breaks. Here, we describe the activities of the T4 MR complex and its modulation by proteins involved in homologous recombination. T4 Mre11 is a Rad50- and Mn(2+)-dependent dsDNA exonuclease and ssDNA endonuclease. ATP hydrolysis is required for the removal of multiple nucleotides via dsDNA exonuclease activity but not for the removal of the first nucleotide or for ssDNA endonuclease activity, indicating ATP hydrolysis is only required for repetitive nucleotide removal. By itself, Rad50 is a relatively inefficient ATPase, but the presence of Mre11 and dsDNA increases ATP hydrolysis by 20-fold. The ATP hydrolysis reaction exhibits positive cooperativity with Hill coefficients ranging from 1.4 for Rad50 alone to 2.4 for the Rad50-Mre11-DNA complex. Kinetic assays suggest that approximately four nucleotides are removed per ATP hydrolyzed. Directionality assays indicate that the prevailing activity is a 3' to 5' dsDNA exonuclease, which is incompatible with the proposed role of MR in the production of 3' ssDNA ends. Interestingly, we found that in the presence of a recombination mediator protein (UvsY) and ssDNA-binding protein (gp32), Mre11 is capable of using Mg(2+) as a cofactor for its nuclease activity. Additionally, the Mg(2+)-dependent nuclease activity, activated by UvsY and gp32, results in the formation of endonuclease reaction products. These results suggest that gp32 and UvsY may alter divalent cation preference and facilitate the formation of a 3' ssDNA overhang, which is a necessary intermediate for recombination-mediated double-strand break repair.

Break-induced ATR and Ddb1–Cul4Cdt2 ubiquitin ligase-dependent nucleotide synthesis promotes homologous recombination repair in fission yeast

Nucleotide synthesis is a universal response to DNA damage, but how this response facilitates DNA repair and cell survival is unclear. Here we establish a role for DNA damage-induced nucleotide synthesis in homologous recombination (HR) repair in fission yeast. Using a genetic screen, we found the Ddb1-Cul4(Cdt)² ubiquitin ligase complex and ribonucleotide reductase (RNR) to be required for HR repair of a DNA double-strand break (DSB). The Ddb1-Cul4(Cdt)² ubiquitin ligase complex is required for degradation of Spd1, an inhibitor of RNR in fission yeast. Accordingly, deleting spd1(+) suppressed the DNA damage sensitivity and the reduced HR efficiency associated with loss of ddb1(+) or cdt2(+). Furthermore, we demonstrate a role for nucleotide synthesis in postsynaptic gap filling of resected ssDNA ends during HR repair. Finally, we define a role for Rad3 (ATR) in nucleotide synthesis and HR through increasing Cdt2 nuclear levels in response to DNA damage. Our findings support a model in which break-induced Rad3 and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent Spd1 degradation and RNR activation promotes postsynaptic ssDNA gap filling during HR repair.

Visualizing RAD51-mediated joint molecules: implications for recombination mechanism and the effect of sequence heterology

The defining event in homologous recombination is the exchange of base-paired partners between a single-stranded (ss) DNA and a homologous duplex driven by recombinase proteins, such as human RAD51. To understand the mechanism of this essential genome maintenance event, we analyzed the structure of RAD51–DNA complexes representing strand exchange intermediates at nanometer resolution by scanning force microscopy. Joint molecules were formed between substrates with a defined ssDNA segment and homologous region on a double-stranded (ds) partner. We discovered and quantified several notable architectural features of RAD51 joint molecules. Each end of the RAD51-bound joints had a distinct structure. Using linear substrates, a 10-nt region of mispaired bases blocked extension of joint molecules in all examples observed, whereas 4 nt of heterology only partially blocked joint molecule extension. Joint molecules, including 10 nt of heterology, had paired DNA on either side of the heterologous substitution, indicating that pairing could initiate from the free 3′end of ssDNA or from a region adjacent to the ss–ds junction. RAD51 filaments covering joint ss–dsDNA regions were more stable to disassembly than filaments covering dsDNA. We discuss how distinct structural features of RAD51-bound DNA joints can play important roles as recognition sites for proteins that facilitate and control strand exchange.

Label-free and sequence-specific DNA detection down to a picomolar level with carbon nanotubes as support for probe DNA

This study describes a simple and label-free electrochemical impedance spectroscopic (EIS) method for sequence-specific detection of DNA by using single-walled carbon nanotubes (SWNTs) as the support for probe DNA. SWNTs are confined onto gold electrodes with mixed self-assembly monolayers of thioethanol and cysteamine. Single-stranded DNA (ssDNA) probe is anchored onto the SWNT support through covalent binding between carboxyl groups at the nanotubes and amino groups at 5′ ends of ssDNA. Hybridization of target DNA with the anchored probe DNA greatly increases the interfacial electron-transfer resistance ( R et) at the double-stranded DNA (dsDNA)-modified electrodes for the redox couple of Fe(CN) 6 3−/4−, which could be used for label-free and sequence-specific DNA detection. EIS results demonstrate that the utilization of SWNTs as the support for probe DNA substantially increases the surface loading of probe DNA onto electrode surface and thus remarkably lowers the detection limit for target DNA. Under the conditions employed here, R et is linear with the concentration of target DNA within a concentration range from 1 to 10 pM with a detection limit down to 0.8 pM ( S/ N = 3). This study may offer a novel and label-free electrochemical approach to sensitive sequence-specific DNA detection.

A Model for Oligomeric Regulation of APOBEC3G Cytosine Deaminase-dependent Restriction of HIV

APOBEC3G (A3G) restricts HIV-1 infection by catalyzing processive C --> U deaminations on single-stranded DNA (ssDNA) with marked 3' --> 5' deamination polarity. Here we show that A3G exists in oligomeric states whose composition is dictated primarily by interactions with DNA, with salt playing an important, yet secondary, role. Directional deaminations correlate with the presence of dimers, tetramers, and larger oligomers observed by atomic force microscopy, and random deaminations appear to correlate mainly with monomers. The presence of a 30-nt weakly deaminated "dead" zone located at the 3'-ssDNA end implies the presence of a preferred asymmetric direction for A3G catalysis. Single turnover reaction rates reveal a salt-dependent inhibition of C deamination toward the 3'-ssDNA region, offering a molecular basis underlying A3G deamination polarity. Presteady state analysis demonstrates rapid diffusion-limited A3G-ssDNA binding, a slower salt-dependent conformational change, possibly indicative of DNA wrapping, and long (5-15 min) protein-DNA complex lifetimes. We suggest that diverse A3G oligomerization modes contribute to the human immunodeficiency virus, type 1, proviral DNA mutational bias.

A novel one-step method to incorporate ss DNA into bilayer lipid membranes supported on an agar electrode

A one-step method has been developed for incorporating ss DNA into a bilayer lipid membrane (BLM) on agar electrode, which displayed good responses to complementary ss DNA. The 5′-terminal phosphated end of ss DNA formed a phosphormidate bond with the amino group of N, N-diethylamidobenzene (NDAB) in the BLM forming solution. The method is easy to perform and has good reproducibility. Fourier transform infrared spectroscopy (FTIR) showed that the ss DNA was incorporated into the BLM.

Dynamic structures of Bacillus subtilis RecN–DNA complexes

Genetic and cytological evidences suggest that Bacillus subtilis RecN acts prior to and after end-processing of DNA double-strand ends via homologous recombination, appears to participate in the assembly of a DNA repair centre and interacts with incoming single-stranded (ss) DNA during natural transformation. We have determined the architecture of RecN–ssDNA complexes by atomic force microscopy (AFM). ATP induces changes in the architecture of the RecN–ssDNA complexes and stimulates inter-complex assembly, thereby increasing the local concentration of DNA ends. The large CII and CIII complexes formed are insensitive to SsbA (counterpart of Escherichia coli SSB or eukaryotic RPA protein) addition, but RecA induces dislodging of RecN from the overhangs of duplex DNA molecules. Reciprocally, in the presence of RecN, RecA does not form large RecA–DNA networks. Based on these results, we hypothesize that in the presence of ATP, RecN tethers the 3′-ssDNA ends, and facilitates the access of RecA to the high local concentration of DNA ends. Then, the resulting RecA nucleoprotein filaments, on different ssDNA segments, might promote the simultaneous genome-wide homology search.

Extensive ssDNA end formation at DNA double-strand breaks in non-homologous end-joining deficient cells during the S phase

BackgroundEfficient and correct repair of DNA damage, especially DNA double-strand breaks, is critical for cellular survival. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer. Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks in mammalian cells. The ability of other repair pathways, such as homologous recombination, to compensate for loss of NHEJ and the ways in which contributions of different pathways are regulated are far from fully understood.ResultsIn this report we demonstrate that long single-stranded DNA (ssDNA) ends are formed at radiation-induced DNA double-strand breaks in NHEJ deficient cells. At repair times ≥ 1 h, processing of unrejoined DNA double-strand breaks generated extensive ssDNA at the DNA ends in cells lacking the NHEJ protein complexes DNA-dependent protein kinase (DNA-PK) or DNA Ligase IV/XRCC4. The ssDNA formation was cell cycle dependent, since no ssDNA ends were observed in G1-synchronized NHEJ deficient cells. Furthermore, in wild type cells irradiated in the presence of DNA-PKcs (catalytic subunit of DNA-PK) inhibitors, or in DNA-PKcs deficient cells complemented with DNA-PKcs mutated in six autophosphorylation sites (ABCDE), no ssDNA was formed. The ssDNA generation also greatly influences DNA double-strand break quantification by pulsed-field gel electrophoresis, resulting in overestimation of the DNA double-strand break repair capability in NHEJ deficient cells when standard protocols for preparing naked DNA (i. e., lysis at 50°C) are used.ConclusionWe provide evidence that DNA Ligase IV/XRCC4 recruitment by DNA-PK to DNA double-strand breaks prevents the formation of long ssDNA ends at double-strand breaks during the S phase, indicating that NHEJ components may downregulate an alternative repair process where ssDNA ends are required.

Structure-Based Model of the Stepping Motor of PcrA Helicase

DNA helicases are ubiquitous molecular motors involved in cellular DNA metabolism. They move along single-stranded DNA (ssDNA) and separate duplex DNA into its component strands, utilizing the free energy from ATP hydrolysis. The PcrA helicase from Bacillus stearothermophilus translocates as a monomer progressively from the 3′ end to the 5′ end of ssDNA and is one of the smallest motor proteins structurally known in full atomic detail. Using high-resolution crystal structures of the PcrA-DNA complex, we performed nanosecond molecular dynamics simulations and derived potential energy profiles governing individual domain movement of the PcrA helicase along ssDNA. Based on these profiles, the millisecond translocation of the helicase along ssDNA was described through Langevin dynamics. The calculations support a domain stepping mechanism of PcrA helicase, in which, during one ATP hydrolysis cycle, the pulling together and pushing apart of domains 2A and 1A are synchronized with alternating mobilities of the individual domains in such a fashion that PcrA moves unidirectionally along ssDNA. By combining short timescale (nanoseconds) molecular dynamics and long timescale (milliseconds) stochastic-dynamics descriptions, our study suggests a structure-based mechanism of the ATP-powered unidirectional movement of PcrA helicase.

BRCA1 Regulates RAD51 Function in Response to DNA Damage and Suppresses Spontaneous Sister Chromatid Replication Slippage: Implications for Sister Chromatid Cohesion, Genome Stability, and Carcinogenesis

The breast/ovarian cancer susceptibility proteins BRCA1 and BRCA2 maintain genome stability, at least in part, through a functional role in DNA damage repair. They both colocalize with RAD51 at sites of DNA damage/replication and activate RAD51-mediated homologous recombination repair of DNA double-strand breaks (DSB). Whereas BRCA2 interacts directly with and regulates RAD51, the role of BRCA1 in this process is unclear. However, BRCA1 may regulate RAD51 in response to DNA damage or through its ability to interact with and regulate MRE11/RAD50/NBS1 (MRN) during the processing of DSBs into single-strand DNA (ssDNA) ends, prerequisite substrates for RAD51, or both. To test these hypotheses, we measured the effect of BRCA1 on the competition between RAD51-mediated homologous recombination (gene conversion and crossover) versus RAD51-independent homologous recombination [single-strand annealing (SSA)] for ssDNA at a site-specific chromosomal DSB within a DNA repeat, a substrate for both homologous recombination pathways. Expression of wild-type BRCA1 in BRCA1-deficient human recombination reporter cell lines promoted both gene conversion and SSA but greatly enhanced gene conversion. In addition, BRCA1 also suppressed both spontaneous gene conversion and deletion events, which can arise from either crossover or sister chromatid replication slippage (SCRS), a RAD51-independent process. BRCA1 does not seem to block crossover. From these results, we conclude that (a) BRCA1 regulates RAD51 function in response to the type of DNA damage and (b) BRCA1 suppresses SCRS, suggesting a role for this protein in sister chromatid cohesion/alignment. Loss of such control in response to estrogen-induced DNA damage after BRCA1 inactivation may be a key initial event that triggers genome instability and carcinogenesis.

A Sisyphean helicase

Some helicases continually bang their heads against the wall, if a new report from Sua Myong, Taekjip Ha (University of Illinois, Urbana-Champaign, IL), and colleagues is any indication. The Rep helicase, the group finds, repeatedly motors along a track of DNA, hits an obstacle, and returns to square one. Figure Collisions with duplex DNA close Rep's 2B domain and trigger its return to the starting point. Not all helicases can unwind DNA, but they can all motor along it. While studying how Rep motors in a 5′ direction on single-stranded (ss) DNA, Ha's group noticed that a duplex DNA obstacle did not knock Rep off its template. Their FRET analysis instead suggested that the helicase returned to its original binding site and tried again. Unlike the 5′ motoring, the return step was almost instantaneous. “At first it seemed like Rep was doing some sort of quantum tunneling,” says Ha, who is a physicist by training. But more FRET studies cleared up the situation—Rep, it seemed, was transiently bound both to DNA near the obstacle and to its initial 3′ binding site, creating a ssDNA loop. Closing of a regulatory region of Rep called domain 2B coincided with the sudden 3′ end capture. As Rep approached the obstacle, 2B was increasingly likely to be in a closed conformation. Ha guesses that collisions with the duplex—which would be more frequent as Rep gets closer—might push 2B into its closed position. This closed conformation might then trigger high-affinity binding to 3′ ends of ssDNA—a form of DNA that is, says Ha, “very flexible, like spaghetti.” In addition to free 3′ ends, Rep also has a high affinity for the three-way junctions at stalled replication forks. The group found that Rep shuttled repeatedly between a fork structure and an Okazaki fragment (the equivalent of the duplex obstacle). The ssDNA at stalled replication forks is a target for RecA binding, which promotes recombination. But Rep prevented RecA filament formation. The findings might thus explain why Rep mutations lead to increased recombination in bacteria. If so, perhaps the clearing, not the unwinding, of DNA is this helicase's main duty. Reference: Myong, S., et al. 2005. Nature. 437:1321–1325. [PubMed]

Bacillus subtilis RecN binds and protects 3'-single-stranded DNA extensions in the presence of ATP

Bacillus subtilis RecN appears to be an early detector of breaks in double-stranded DNA. In vivo, RecN forms discrete nucleoid-associated structures and in vitro exhibits Mg2+-dependent single-stranded (ss) DNA binding and ssDNA-dependent ATPase activities. In the presence of ATP or ADP, RecN assembles to form large networks with ssDNA molecules (designated complexes CII and CIII) that involve ATP binding and requires a 3′-OH at the end of ssDNA molecule. Addition of dATP–RecA complexes dissociates RecN from these networks, but this is not observed following addition of an ssDNA binding protein. Apparently, ATP modulates the RecN–ssDNA complex for binding to ssDNA extensions and, in vivo, RecN–ATP bound to 3′-ssDNA might sequester ssDNA ends within complexes that protect the ssDNA while the RecA accessory proteins recruit RecA. With the association of RecA to ssDNA, RecN would dissociate from the DNA end facilitating the subsequent steps in DNA repair.

Kinetic Studies of DNA Cleavage Reactions Catalyzed by an ATP-Dependent Deoxyribonuclease on a 27-MHz Quartz-Crystal Microbalance

Catalytic DNA cleavage reactions by an ATP-dependent deoxyribonuclease (DNase) from Micrococcus luteus were monitored directly with a DNA-immobilized 27-MHz quartz-crystal microbalance (QCM). The 27-MHz QCM is a very sensitive mass-measuring device in aqueous solution, as the frequency decreases linearly with increasing mass on the electrode at a nanogram level. Three steps in ATP-dependent DNA hydrolysis reactions, including (1) binding of DNase to the end of double-stranded DNA (dsDNA) on the QCM electrode (mass increase), (2) degradation of one strand of dsDNA in the 3' --> 5' direction depending on ATP (mass decrease), and (3) release of the enzyme from the nonhydrolyzed 5'-free-ssDNA (mass decrease), could be monitored stepwise from the time dependencies of QCM frequency changes. Kinetic parameters for each step were obtained as follows. The binding constant (K(a)) of DNase to the dsDNA was determined as (28 +/- 2) x 10(6) M(-)(1) (k(on) = (8.0 +/- 0.3) x 10(3) M (-)(1) s(-)(1) and k(off) = (0.29 +/-0.01) x 10(-)(3) s(-)(1)), and it decreased to (0.79 +/- 0.16) x 10(6) M(-)(1) (k'(on) = (2.3 +/- 0.2) x 10(3) M (-)(1) s(-)(1) and k'(off) = (2.9 +/- 0.1) x 10(-)(3) s(-)(1)) for the completely nonhydrolyzed 5'-free ssDNA. This is the reason the DNase bound to the dsDNA substrate can easily release from the nonhydrolyzed 5'-free-ssDNA after the complete hydrolysis of the 3' --> 5' direction of the complementary ssDNA. K(a) values depended on the DNA structures on the QCM, and the order of these values was as follows: the dsDNA having a 4-base-mismatched base-pair end (3) > the dsDNA having a 5' 15-base overhanging end (2) > the dsDNA having a blunt end (1) > the ssDNA having a 3'-free end (4) >> the ssDNA having a 5'-free end (5). Thus, DNase hardly recognized the free 5' end of ssDNA. Michaelis-Menten parameters (K(m) for ATP and k(cat)) of the hydrolysis process also could be obtained, and the order of k(cat)/K(m) was as follows: the dsDNA having a blunt end (1) approximately the dsDNA having a 4-base-mismatched base-pair end (3) > the ssDNA having a free 3' end (4) >> the ssDNA having a free 5' end (5). Thus, DNase could not recognize and not hydrolyze the free 5' end of ssDNA. The DNA hydrolysis reaction could be driven by dATP and GTP (purine base) as well as ATP, whereas the cleavage efficiency was very low driven with UTP, CTP (pyrimidine base), ADP, and AMP.

DNA Elongation by the Human DNA Polymerase λ Polymerase and Terminal Transferase Activities Are Differentially Coordinated by Proliferating Cell Nuclear Antigen and Replication Protein A

DNA polymerase lambda contains template-dependent (DNA polymerase) and template-independent (terminal transferase) activities. In this study we enzymologically characterized the terminal transferase activity of polymerase lambda (pol lambda-tdt). Pol lambda-tdt activity was strongly influenced by the nature of the 3'-terminal sequence of the DNA substrate, and it required a single-stranded (ss) DNA 3'-overhang of about 9-12 nucleotides for optimal activity. The strong preference observed for pyrimidine versus purine nucleotide incorporation was found to be due, at least partially, to a steric block imposed by the residue Tyr-505 in the active site of pol lambda. Pol lambda-tdt was found to be able to elongate a 3'-ssDNA end by two alternative mechanisms: first, a template-independent one resulting in addition of 1 or 2 nucleotides, and second, a template-dependent one where a homopolymeric tract as short as 3 nucleotides at the 3'-end could be used as a template to direct DNA polymerization by a looping back mechanism. Furthermore repetitive cycles of DNA synthesis resulted in the expansion of such a short homopolymeric terminal sequence. Most importantly we found that the proliferating cell nuclear antigen was able to selectively block the looping back mechanism while stimulating the single terminal nucleotide addition. Finally replication protein A completely suppressed the transferase activity of pol lambda while stimulating the polymerase activity, suggesting that proliferating cell nuclear antigen and replication protein A can coordinate the polymerase and the terminal transferase activities of pol lambda.

Alteration of Nucleic Acid Structure and Stability Modulates the Efficiency of Minus-Strand Transfer Mediated by the HIV-1 Nucleocapsid Protein

During human immunodeficiency virus type 1 minus-strand transfer, the nucleocapsid protein (NC) facilitates annealing of the complementary repeat regions at the 3'-ends of acceptor RNA and minus-strand strong-stop DNA ((-) SSDNA). In addition, NC destabilizes the highly structured complementary trans-activation response element (TAR) stem-loop (TAR DNA) at the 3'-end of (-) SSDNA and inhibits TAR-induced self-priming, a dead-end reaction that competes with minus-strand transfer. To investigate the relationship between nucleic acid secondary structure and NC function, a series of truncated (-) SSDNA and acceptor RNA constructs were used to assay minus-strand transfer and self-priming in vitro. The results were correlated with extensive enzymatic probing and mFold analysis. As the length of (-) SSDNA was decreased, self-priming increased and was highest when the DNA contained little more than TAR DNA, even if NC and acceptor were both present; in contrast, truncations within TAR DNA led to a striking reduction or elimination of self-priming. However, destabilization of TAR DNA was not sufficient for successful strand transfer: the stability of acceptor RNA was also crucial, and little or no strand transfer occurred if the RNA was highly stable. Significantly, NC may not be required for in vitro strand transfer if (-) SSDNA and acceptor RNA are small, relatively unstructured molecules with low thermodynamic stabilities. Collectively, these findings demonstrate that for efficient NC-mediated minus-strand transfer, a delicate thermodynamic balance between the RNA and DNA reactants must be maintained.

Specific inhibition of the E.coli RecBCD enzyme by Chi sequences in single-stranded oligodeoxyribonucleotides.

RecBCD is an ATP-dependent helicase and exonuclease which generates 3' single-stranded DNA (ssDNA) ends used by RecA for homologous recombination. The exonuclease activity is altered when RecBCD encounters a Chi sequence (5'-GCTGGTGG-3') in double-stranded DNA (ds DNA), an event critical to the generation of the 3'-ssDNA. This study tests the effect of ssDNA oligonucleotides having a Chi sequence (Ch+) or a single base change that abolishes the Chi sequence (Chi(o)), on the enzymatic activities of RecBCD. Our results show that a 14 and a 20mer with Chi+ in the center of the molecule inhibit the exonuclease and helicase activities of RecBCD to a greater extent than the corresponding Chi(o) oligonucleotides. Oligonucleotides with the Chi sequence at one end, or the Chi sequence alone in an 8mer, failed to show Chi-specific inhibition of RecBCD. Thus, Chi recognition requires that Chi be flanked by DNA at either end. Further experiments indicated that the oligonucleotides inhibit RecBCD from binding to its dsDNA substrate. These results suggest that a specific site for Chi recognition exists on RecBCD, which binds Chi with greater affinity than a non-Chi sequence and is probably adjacent to non-specific DNA binding sites.

Helicase and Nuclease Activities of Hyperthermophile Pyrococcus horikoshii Dna2 Inhibited by Substrates with RNA Segments at 5′-End

Dna2 protein plays an important role in Okazaki fragment maturation on the lagging strand and also participates in DNA repair in Eukarya. Herein, we report the first biochemical characterization of a Dna2 homologue from Archaea, the hyperthermophile Pyrococcus horikoshii (Dna2Pho). Dna2Pho has both a RecB-like nuclease motif and seven conserved helicase motifs similar to Dna2 from Saccharomyces cerevisiae. Dna2Pho has single-stranded (ss) DNA-stimulated ATPase activity, DNA helicase activity (5' to 3' direction) requiring ATP, and nuclease activity, which prefers free 5'-ends of ssDNA as substrate. These activities depend on MgCl(2) concentrations. Dna2Pho requires a higher concentration of MgCl(2) for the nuclease than helicase activity. Both the helicase and nuclease activities of Dna2Pho were inhibited by substrates with RNA segments at the 5'-end of flap DNA, whereas the nuclease activity of Dna2 from S. cerevisiae was reported to be stimulated by RNA segments in the 5'-tail (Bae, S.-H., and Seo, Y. S. (2000) J. Biol. Chem. 38022-38031).

DNA-dependent Protein Kinase Catalytic Subunit: STRUCTURAL REQUIREMENTS FOR KINASE ACTIVATION BY DNA ENDS

DNA-dependent protein kinase (DNA-PK) is a DNA end-activated protein kinase composed of a catalytic subunit, DNA-PKcs, and a DNA binding subunit, Ku, that is involved in repair of DNA double-stranded breaks (DSBs). We have previously shown that DNA-PKcs interacts with single-stranded DNA (ssDNA) ends with a separate ssDNA binding site to be activated for its kinase activity. Here, the properties of the ssDNA binding site were examined by using DNA fragments with modified ssDNA extensions. DNA fragments with a wide range of ssDNA modifictations activated DNA-PKcs, indicating a relaxed specificity for the chemical structure of terminal nucleotides of a DSB. Methyl substitution of the phosphate backbone impaired kinase activation but not binding, indicating that interaction with the DNA backbone was involved in kinase activation. Experiments with RNA and RNA/DNA hybrid fragments suggested that the discrimination between RNA and DNA ends resides in the double-stranded DNA binding function of DNA-PKcs. DNA fragments exposing only one ssDNA end activated DNA-PKcs poorly, suggesting that DNA-PKcs distinguishes between DSBs and ssDNA breaks by simultaneous interaction with two ssDNA ends. These properties potentially explain how DNA-PKcs can be specifically activated by DSBs but still recognize the diverse chemical structures exposed when DSBs are introduced by ionizing radiation.

The RecOR proteins modulate RecA protein function at 5' ends of single-stranded DNA.

The Escherichia coli RecF, RecO and RecR pro teins have previously been implicated in bacterial recombinational DNA repair at DNA gaps. The RecOR-facilitated binding of RecA protein to single-stranded DNA (ssDNA) that is bound by single-stranded DNA-binding protein (SSB) is much faster if the ssDNA is linear, suggesting that a DNA end (rather than a gap) facilitates binding. In addition, the RecOR complex facilitates RecA protein-mediated D-loop formation at the 5' ends of linear ssDNAs. RecR protein remains associated with the RecA filament and its continued presence is required to prevent filament disassembly. RecF protein competes with RecO protein for RecR protein association and its addition destabilizes RecAOR filaments. An enhanced function of the RecO and RecR proteins can thus be seen in vitro at the 5' ends of linear ssDNA that is not as evident in DNA gaps. This function is countered by the RecF/RecO competition for association with the RecR protein.

Direct measurement of single-stranded DNA translocation by PcrA helicase using the fluorescent base analogue 2-aminopurine.

Use of the fluorescent base analogue 2-aminopurine has provided a direct demonstration of the translocation of PcrA helicase toward the 5'-end of single-stranded DNA. Single 2-aminopurine bases are introduced into otherwise standard oligonucleotides and produce a fluorescence signal when PcrA reaches their position. We demonstrate that random binding of PcrA to ssDNA is followed by translocation in an ATP-dependent manner toward the 5'-terminus at 80 bases per second at 20 degrees C. The data also provide information on the kinetics of ssDNA binding to the helicase and of the protein dissociation from the 5'-end of ssDNA. A full kinetic model is presented for ATP-dependent DNA translocation by PcrA helicase.