Fork-like Structures Research Articles

Dual function of Zika virus NS2B-NS3 protease.

Zika virus (ZIKV) serine protease, indispensable for viral polyprotein processing and replication, is composed of the membrane-anchored NS2B polypeptide and the N-terminal domain of the NS3 polypeptide (NS3pro). The C-terminal domain of the NS3 polypeptide (NS3hel) is necessary for helicase activity and contains an ATP-binding site. We discovered that ZIKV NS2B-NS3pro binds single-stranded RNA with a Kd of ~0.3 μM, suggesting a novel function. We tested various structural modifications of NS2B-NS3pro and observed that constructs stabilized in the recently discovered "super-open" conformation do not bind RNA. Likewise, stabilizing NS2B-NS3pro in the "closed" (proteolytically active) conformation using substrate inhibitors abolished RNA binding. We posit that RNA binding occurs when ZIKV NS2B-NS3pro adopts the "open" conformation, which we modeled using highly homologous dengue NS2B-NS3pro crystallized in the open conformation. We identified two positively charged fork-like structures present only in the open conformation of NS3pro. These forks are conserved across Flaviviridae family and could be aligned with the positively charged grove on NS3hel, providing a contiguous binding surface for the negative RNA strand exiting helicase. We propose a "reverse inchworm" model for a tightly intertwined NS2B-NS3 helicase-protease machinery, which suggests that NS2B-NS3pro cycles between open and super-open conformations to bind and release RNA enabling long-range NS3hel processivity. The transition to the closed conformation, likely induced by the substrate, enables the classical protease activity of NS2B-NS3pro.

Distribution of virulence genes and biofilm characterization of human isolates of Streptococcus agalactiae: A pilot study

This study included 21 newly isolated clinical samples of Streptococcus agalactiae (Group B Streptococcus) screened in patients (six male, fifteen female) from various states of India with different infections (urinary tract infections, blood, pus and eye infections). All isolates were identified as Group B Streptococcus (GBS) using hemolytic properties, serogrouping and MALDI-TOF-MS analysis. Six virulence genes, cfb (100%), cylE (90.4%), lmp (85.7%), bca (71.4%), rib (38%) and bac (4.7%) were detected via polymerase chain reaction (PCR). Distribution studies of these six genes revealed five isolates containing five virulence genes (23.8%), followed by ten isolates containing four virulence genes (47.6%). The twenty GBS isolates selected on the glass surface included non-biofilm producers (n = 6, 30%), weak (n = 11, 55%) and moderate biofilm producers (n = 3, 15%). On the polystyrene surface, weak (n = 4, 20%), moderate (n = 2, 10%) and strong (n = 14, 70%) biofilm producers were detected. Live-dead cell staining revealed that more viable cells accumulated in the S. ag 7420 isolate than in the AH1 isolate. Scanning electron microscope (SEM) biofilm analysis showed S. ag AH1 cells appeared as chain-like structures, whereas the S. ag 7420 isolate biofilm cells appeared as fork-like structures on the glass surface. Biofilm elements were analyzed using Energy Dispersive X-Ray Analysis (EDAX) for both isolates and 13 elements with different orders of composition were found. Thus, virulence gene detection, distribution and biofilm formation by these new clinical isolates suggested the virulent nature of these pathogens, which might cause different levels of disease severity in humans.

Mind the replication gap.

Unlike bacteria, mammalian cells need to complete DNA replication before segregating their chromosomes for the maintenance of genome integrity. Thus, cells have evolved efficient pathways to restore stalled and/or collapsed replication forks during S-phase, and when necessary, also to delay cell cycle progression to ensure replication completion. However, strong evidence shows that cells can proceed to mitosis with incompletely replicated DNA when under mild replication stress (RS) conditions. Consequently, the incompletely replicated genomic gaps form, predominantly at common fragile site regions, where the converging fork-like DNA structures accumulate. These branched structures pose a severe threat to the faithful disjunction of chromosomes as they physically interlink the partially duplicated sister chromatids. In this review, we provide an overview discussing how cells respond and deal with the under-replicated DNA structures that escape from the S/G2 surveillance system. We also focus on recent research of a mitotic break-induced replication pathway (also known as mitotic DNA repair synthesis), which has been proposed to operate during prophase in an attempt to finish DNA synthesis at the under-replicated genomic regions. Finally, we discuss recent data on how mild RS may cause chromosome instability and mutations that accelerate cancer genome evolution.

A priA Mutant Expressed in Two Pieces Has Almost Full Activity in Escherichia coli K-12.

The ability to restart broken DNA replication forks is essential across all domains of life. In Escherichia coli, the priA, priB, priC, and dnaT genes encode the replication restart proteins (RRPs) to accomplish this task. PriA plays a critical role in replication restart such that its absence reveals a dramatic phenotype: poor growth, high basal levels of SOS expression, poorly partitioned nucleoids (Par-), UV sensitivity, and recombination deficiency (Rec-). PriA has 733 amino acids, and its structure is composed of six domains that enable it to bind to DNA replication fork-like structures, remodel the strands of DNA, interact with SSB (single-stranded DNA binding protein), PriB, and DnaT, and display ATPase, helicase, and translocase activities. We have characterized a new priA mutation called priA316::cat It is a composite mutation involving an insertion that truncates the protein within the winged-helix domain (at the 154th codon) and an ACG (Thr)-to-ATG (Met) mutation that allows reinitiation of translation at the 157th codon such that PriA is expressed in two pieces. priA316::cat phenotypes are like those of the wild type for growth, recombination, and UV resistance, revealing only a slightly increased level of SOS expression and defects in nucleoid partitioning in the mutant. Both parts of PriA are required for activity, and the N-terminal fragment can be optimized to yield wild-type activity. A deletion of the lon protease suppresses priA316::cat phenotypes. We hypothesize the two parts of PriA form a complex that supplies most of the PriA activity needed in the cell.IMPORTANCE PriA is a highly conserved multifunctional protein that plays a crucial role in the essential process of replication restart. Here we characterize an insertion mutation of priA with an intragenic suppressor such that it is now made in two parts. These two pieces split the winged-helix domain to separate the N-terminal 3' DNA-binding domain from the C-terminal domain of PriA. It is hypothesized that the two pieces form a complex that is capable of almost wild type priA function. The composite mutation leads to a moderate level of SOS expression and defects in partitioning of the chromosomes. Full function is restored by deletion of lon, suggesting that stability of this complex may be a reason for the partial phenotypes seen.

Irradiation of zinc single crystal with 500 keV singly-charged carbon ions: surface morphology, structure, hardness, and chemical modifications

Cylindrical specimens of (1 0 4) oriented zinc single crystal (diameter = 6 mm and length = 5 mm) were irradiated with 500 keV C+1 ions with the help of a Pelletron accelerator. Six specimens were irradiated in an ultra-high vacuum (~10‒8 Torr) with different ion doses, namely 3.94 × 1014, 3.24 × 1015, 5.33 × 1015, 7.52 × 1015, 1.06 × 1016, and 1.30 × 1016 ions cm−2. A field emission scanning electron microscope (FESEM) was utilized for the morphological study of the irradiated specimens. Formation of nano- and sub-micron size rods, clusters, flower- and fork-like structures, etc, was observed. Surface roughness of the irradiated specimens showed an increasing trend with the ions dose. Energy dispersive x-ray spectroscopy (EDX) helped to determine chemical modifications in the specimens. It was found that carbon content varied in the range 22.86–31.20 wt.% and that oxygen content was almost constant, with an average value of 10.16 wt.%. The balance content was zinc. Structural parameters, i.e. crystallite size and lattice strain, were determined by Williamson–Hall analysis using x-ray diffraction (XRD) patterns of the irradiated specimens. Both crystallite size and lattice strain showed a decreasing trend with the increasing ions dose. A good linear relationship between crystallite size and lattice strain was observed. Surface hardness depicted a decreasing trend with the ions dose and followed an inverse Hall–Petch relation. FTIR spectra of the specimens revealed that absorption bands gradually diminish as the dose of singly-charged carbon ions is increased from 3.94 × 1014 ions cm−1 to 1.30 × 1016 ions cm−1. This indicates progressive deterioration of chemical bonds with the increase in ion dose.

Replication Restart in Bacteria

In bacteria, replication forks assembled at a replication origin travel to the terminus, often a few megabases away. They may encounter obstacles that trigger replisome disassembly, rendering replication restart from abandoned forks crucial for cell viability. During the past 25 years, the genes that encode replication restart proteins have been identified and genetically characterized. In parallel, the enzymes were purified and analyzed in vitro, where they can catalyze replication initiation in a sequence-independent manner from fork-like DNA structures. This work also revealed a close link between replication and homologous recombination, as replication restart from recombination intermediates is an essential step of DNA double-strand break repair in bacteria and, conversely, arrested replication forks can be acted upon by recombination proteins and converted into various recombination substrates. In this review, we summarize this intense period of research that led to the characterization of the ubiquitous replication restart protein PriA and its partners, to the definition of several replication restart pathways in vivo, and to the description of tight links between replication and homologous recombination, responsible for the importance of replication restart in the maintenance of genome stability.

RECQL5 plays co-operative and complementary roles with WRN syndrome helicase.

Humans have five RecQ helicases, whereas simpler organisms have only one. Little is known about whether and how these RecQ helicases co-operate and/or complement each other in response to cellular stress. Here we show that RECQL5 associates longer at laser-induced DNA double-strand breaks in the absence of Werner syndrome (WRN) protein, and that it interacts physically and functionally with WRN both in vivo and in vitro. RECQL5 co-operates with WRN on synthetic stalled replication fork-like structures and stimulates its helicase activity on DNA fork duplexes. Both RECQL5 and WRN re-localize from the nucleolus into the nucleus after replicative stress and significantly associate with each other during S-phase. Further, we show that RECQL5 is essential for cell survival in the absence of WRN. Loss of both RECQL5 and WRN severely compromises DNA replication, accumulates genomic instability and ultimately leads to cell death. Collectively, our results indicate that RECQL5 plays both co-operative and complementary roles with WRN. This is an early demonstration of a significant functional interplay and a novel synthetic lethal interaction among the human RecQ helicases.

On Adhesive and Buckling Instabilities in the Mechanics of Carbon Nanotubes Bundles

Many recently synthesized materials feature aligned arrays or bundles of carbon nanotubes (CNTs) whose mechanical properties are partially determined by the van der Waals interactions between adjacent tubes. Of particular interest in this paper are instances where the resulting interaction between a pair of CNTs often produces a forklike structure. The mechanical properties of this structure are noticeably different from those for isolated individual CNTs. In particular, while one anticipates buckling phenomena in the forked structure, an adhesion instability may also be present. New criteria for buckling and adhesion instabilities in forklike structures are presented in this paper. The criteria are illuminated with a bifurcation analyses of the response of the forklike structure to applied compressive and shear loadings.

Resonance effects and quantum beats in attosecond transient absorption of helium

We present theoretical simulations of the attosecond transient absorption of singly-excited states of helium atoms in the presence of a dressing near-infrared or infrared laser. In particular, we aim to address several unresolved questions in the transient absorption of helium and to resolve the remaining discrepancies between theory and experiment. We initially focus on the forklike structures in the Autler–Townes splitting of the 1s2p state and the effects of resonant coupling to the 1s2s and 1s3s states. We find that the delay-dependent features of the Autler–Townes doublet depend strongly on both the laser frequency detuning from resonance and on the laser pulse duration, and explain the lack of such structures in current experimental data. Next, we identify the interference mechanism which causes the half-cycle oscillations in the absorption spectrum below the excited state manifold. Finally, we observe for the first time the presence of quantum beating in the simulated transient absorption spectrogram, and discuss the conditions under which such wavepacket dynamics could be observed experimentally.

Hypersensitivity of mouse NEIL1-knockdown cells to hydrogen peroxide during S phase

Oxidative base damage occurs spontaneously due to reactive oxygen species generated as byproducts of respiration and other pathological processes in mammalian cells. Many oxidized bases are mutagenic and/or toxic, and most are repaired through the base excision repair pathway. Human endonuclease VIII-like protein 1 (hNEIL1) is thought to play an important role during the S phase of the cell cycle by removing oxidized bases in DNA replication fork-like (bubble) structures, and the protein level of hNEIL1 is increased in S phase. Compared with hNEIL1, there is relatively little information on the properties of the mouse ortholog mNEIL1. Since mouse cell nuclei lack endonuclease III-like protein (NTH) activity, in contrast to human cell nuclei, mNEIL1 is a major DNA glycosylase for repair of oxidized pyrimidines in mouse nuclei. In this study, we made mNEIL1-knockdown cells using an shRNA expression vector and examined the cell cycle-related variation in hydrogen peroxide (H2O2) sensitivity. Hypersensitivity to H2O2 caused by mNEIL1 knockdown was more significant in S phase than in G1 phase, suggesting that mNEIL1 has an important role during S phase, similarly to hNEIL1.

RECQL5 plays co-operative and complementary roles with WRN syndrome helicase

Humans have five RecQ helicases, whereas simpler organisms have only one. Little is known about whether and how these RecQ helicases co-operate and/or complement each other in response to cellular stress. Here we show that RECQL5 associates longer at laser-induced DNA double-strand breaks in the absence of Werner syndrome (WRN) protein, and that it interacts physically and functionally with WRN both in vivo and in vitro. RECQL5 co-operates with WRN on synthetic stalled replication fork-like structures and stimulates its helicase activity on DNA fork duplexes. Both RECQL5 and WRN re-localize from the nucleolus into the nucleus after replicative stress and significantly associate with each other during S-phase. Further, we show that RECQL5 is essential for cell survival in the absence of WRN. Loss of both RECQL5 and WRN severely compromises DNA replication, accumulates genomic instability and ultimately leads to cell death. Collectively, our results indicate that RECQL5 plays both co-operative and complementary roles with WRN. This is an early demonstration of a significant functional interplay and a novel synthetic lethal interaction among the human RecQ helicases.

The Human RecQ Helicases, BLM and RECQ1, Display Distinct DNA Substrate Specificities

RecQ helicases maintain chromosome stability by resolving a number of highly specific DNA structures that would otherwise impede the correct transmission of genetic information. Previous studies have shown that two human RecQ helicases, BLM and WRN, have very similar substrate specificities and preferentially unwind noncanonical DNA structures, such as synthetic Holliday junctions and G-quadruplex DNA. Here, we extend this analysis of BLM to include new substrates and have compared the substrate specificity of BLM with that of another human RecQ helicase, RECQ1. Our findings show that RECQ1 has a distinct substrate specificity compared with BLM. In particular, RECQ1 cannot unwind G-quadruplexes or RNA-DNA hybrid structures, even in the presence of the single-stranded binding protein, human replication protein A, that stimulates its DNA helicase activity. Moreover, RECQ1 cannot substitute for BLM in the regression of a model replication fork and is very inefficient in displacing plasmid D-loops lacking a 3'-tail. Conversely, RECQ1, but not BLM, is able to resolve immobile Holliday junction structures lacking an homologous core, even in the absence of human replication protein A. Mutagenesis studies show that the N-terminal region (residues 1-56) of RECQ1 is necessary both for protein oligomerization and for this Holliday junction disruption activity. These results suggest that the N-terminal domain or the higher order oligomer formation promoted by the N terminus is essential for the ability of RECQ1 to disrupt Holliday junctions. Collectively, our findings highlight several differences between the substrate specificities of RECQ1 and BLM (and by inference WRN) and suggest that these enzymes play nonoverlapping functions in cells.

Saccharomyces cerevisiae Mus81-Mms4 is a catalytic, DNA structure-selective endonuclease.

The DNA structure-selective endonuclease Mus81-Mms4/Eme1 is a context-specific recombination factor that supports DNA replication, but is not essential for DSB repair in Saccharomyces cerevisiae. We overexpressed Mus81-Mms4 in S. cerevisiae, purified the heterodimer to apparent homogeneity, and performed a classical enzymological characterization. Kinetic analysis (kcat, KM) demonstrated that Mus81-Mms4 is catalytically active and identified three substrate classes in vitro. Class I substrates reflect low KM (3–7 nM) and high kcat (∼1 min−1) and include the nicked Holliday junction, 3′-flapped and replication fork-like structures. Class II substrates share low KM (1–6 nM) but low kcat (≤0.3 min−1) relative to Class I substrates and include the D-loop and partial Holliday junction. The splayed Y junction defines a class III substrate having high KM (∼30 nM) and low kcat (0.26 min−1). Holliday junctions assembled from oligonucleotides with or without a branch migratable core were negligibly cut in vitro. We found that Mus81 and Mms4 are phosphorylated constitutively and in the presence of the genotoxin MMS. The endogenous complex purified in either modification state is negligibly active on Holliday junctions. Hence, Holliday junction incision activity in vitro cannot be attributed to the Mus81-Mms4 heterodimer in isolation.

A Mutant Allele ofMRE11Found in Mismatch Repair-deficient Tumor Cells Suppresses the Cellular Response to DNA Replication Fork Stress in a Dominant Negative Manner

The interaction of ataxia-telangiectasia mutated (ATM) and the Mre11/Rad50/Nbs1 (MRN) complex is critical for the response of cells to DNA double-strand breaks; however, little is known of the role of these proteins in response to DNA replication stress. Here, we report a mutant allele of MRE11 found in a colon cancer cell line that sensitizes cells to agents causing replication fork stress. The mutant Mre11 weakly interacts with Rad50 relative to wild type and shows little affinity for Nbs1. The mutant protein lacks 3'-5' exonuclease activity as a result of loss of part of the conserved nuclease domain; however, it retains binding affinity for single-stranded DNA (ssDNA), double-stranded DNA with a 3' single-strand overhang, and fork-like structures containing ssDNA regions. In cells, the mutant protein shows a time- and dose-dependent accumulation in chromatin after thymidine treatment that corresponds with increased recruitment and hyperphosphorylation of replication protein A. ATM autophosphorylation, Mre11 foci, and thymidine-induced homologous recombination are suppressed in cells expressing the mutant allele. Together, our results suggest that the mutant Mre11 suppresses the cellular response to replication stress by binding to ssDNA regions at disrupted forks and impeding replication restart in a dominant negative manner.

Slx4 becomes phosphorylated after DNA damage in a Mec1/Tel1-dependent manner and is required for repair of DNA alkylation damage

Members of the RecQ family of DNA helicases, mutated in several syndromes associated with cancer predisposition, are key regulators of genome stability. The Saccharomyces cerevisiae SLX4 gene is required for cell viability in the absence of Sgs1, the only yeast RecQ helicase. SLX4 encodes one subunit of the heterodimeric Slx1-Slx4 endonuclease, although its cellular function is not clear. Slx1-Slx4 was reported to preferentially cleave replication fork-like structures in vitro, and cells lacking SLX4 are hypersensitive to DNA alkylation damage. Here we report that Slx4 becomes phosphorylated in cells exposed to a wide range of genotoxins. Even though it has been proposed that the role of Slx4 is restricted to S-phase, Slx4 phosphorylation is observed in cells arrested in G1 or G2 phases of the cell cycle, but not during an unperturbed cell cycle. Slx4 phosphorylation is completely abolished in cells lacking the Mec1 and Tel1 protein kinases, critical regulators of genome stability, but is barely affected in the absence of both Rad53 and Chk1 kinases. Finally we show that, whereas both Slx1 and Slx4 are dispensable for activation of cell-cycle checkpoints, Slx4, but not Slx1, is required for repair of DNA alkylation damage in both aynchronously growing cells and in G2-phase-arrested cells. These results reveal Slx4 as a new target of the Mec1/Tel1 kinases, with a crucial role in DNA repair that is not restricted to the processing of stalled replisomes.

Ape1 Abasic Endonuclease Activity is Regulated by Magnesium and Potassium Concentrations and is Robust on Alternative DNA Structures

Abasic lesions are common mutagenic or cytotoxic DNA damages. Ape1 is the major human apurinic/apyrimidinic (AP) endonuclease and initiates repair of abasic sites by catalyzing strand cleavage at the lesion. I show here that Ape1 single-stranded (ss) AP site incision activity prefers 0.5 mM or 2 mM MgCl(2) and low concentrations (< or =50 mM) of KCl, whereas its double-stranded (ds) activity favors 10 mM MgCl(2) and 50 mM KCl or 2 mM MgCl(2) and 200 mM KCl. Both activities favor a pH between 7.0 and 7.5, suggesting a common catalytic mechanism. In conditions designed to mimic the intracellular environment (pH 7.2; 100 mM KCl; 1 mM MgCl(2)), Ape1 ssAP site incision activity is either about fivefold more active or approximately 20-fold less efficient than its ds activity, depending on the oligonucleotide employed. Secondary structure predictions suggest a role for the DNA conformational state in determining the effectiveness of Ape1. Ape1 complex stability in the presence of EDTA (non-incising conditions) is significantly weaker for ssDNA than dsDNA, regardless of the AP substrate. Duplexes where the AP site is positioned opposite the 3' terminus of a complementary primer strand are incised with an efficiency similar (less than twofold difference) to that of the ssAP substrate alone. Moreover, Ape1 cleaved AP sites in fork-like and bubble DNA structures with an efficiency that is identical or up to sevenfold higher than ssAP-DNA. The findings here suggest that Ape1 ssAP and dsAP endonuclease activities are regulated by sequence context and the relative concentrations of certain chemical elements in vivo, and that Ape1 incision activity occurs on complex replication, recombination, and/or transcription DNA intermediates.

Modulation of enzymatic activities of Escherichia coli DnaB helicase by single-stranded DNA-binding proteins.

The modulation of enzymatic activities of Escherichia coli DnaB helicase by homologous and heterologous single-stranded DNA-binding proteins (SSBs) and its DNA substrates were analyzed. Although DnaB helicase can unwind a variety of DNA substrates possessing different fork-like structures, the rate of DNA unwinding was significantly diminished with substrates lacking a 3' fork. A 5 nt fork appeared to be adequate to attain the maximum rate of DNA unwinding. Efficient helicase action of DnaB requires the participation of SSBs. Studies involving heterologous SSBs demonstrated that they can stimulate the helicase activity of DnaB protein under certain conditions. However, this stimulation occurs in a manner distinctly different from that observed with cognate E.coli SSB. The E.coli SSB was found to stimulate the helicase activity over a wide range of SSB concentrations and was unique in its strong inhibition of single-stranded DNA-dependent ATPase activity when uncoupled from the DNA helicase activity. In the presence of a helicase substrate, the ATPase activity of DnaB helicase remained uninhibited. Thus, E.coli SSB appears to coordinate and couple the ATPase activity to the DNA helicase activity by suppressing unproductive ATP hydrolysis by DnaB helicase.

Translesion Replication by DNA Polymerase β Is Modulated by Sequence Context and Stimulated by Fork-like Flap Structures in DNA

Mutations in the human genome are clustered in hot-spot regions, suggesting that some sequences are more prone to accumulate mutations than others. These regions are therefore more likely to lead to the development of cancer. Several pathways leading to the creation of mutations may be influenced by the DNA sequence, including sensitivity to DNA damaging agents, and repair mechanisms. We have analyzed sequence context effects on translesion replication, the error-prone repair of single-stranded DNA regions carrying lesions. By using synthetic oligonucleotides containing systematic variations of sequences flanking a synthetic abasic site, we show that translesion replication by the repair polymerase DNA polymerase beta is stimulated to a moderate extent by low stacking levels of the template nucleotides downstream of the lesion, combined with homopolymeric runs flanking the lesion both upstream and downstream. A strong stimulation of translesion replication by DNA polymerase beta was seen when fork-like flap structures were introduced into the DNA substrate downstream of the lesion. Unlike for gapped substrates, this stimulation was independent of the presence of a phosphate group at the 5' terminus of the flap. These results suggest that DNA polymerase beta may participate in cellular DNA transactions involving higher order structures. The significance of these results for in vivo translesion replication is discussed.

A chloroplast DNA helicase II from pea that prefers fork-like replication structures

A DNA helicase, called chloroplast DNA (ctDNA) helicase II, was purified to apparent homogeneity from pea (Pisum sativum). The enzyme contained intrinsic, single-stranded, DNA-dependent ATPase activity and an apparent molecular mass of 78 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The DNA helicase was markedly stimulated by DNA substrates with fork-like replication structures. A 5'-tailed fork was more active than the 3'-tailed fork, which itself was more active than substrates without a fork. The direction of unwinding was 3' to 5' along the bound strand, and it failed to unwind blunt-ended duplex DNA. DNA helicase activity required only ATP or dATP hydrolysis. The enzyme also required a divalent cation (Mg2+>Mn2+>Ca2+) for its unwinding activity and was inhibited at 200 mM KCl or NaCl. This enzyme could be involved in the replication of ctDNA. The DNA major groove-intercalating ligands nogalamycin and daunorubicin were inhibitory to unwinding (Ki approximately 0.85 &mgr;M and 2.2 &mgr;M, respectively) and ATPase (Ki approximately 1.3 &mgr;M and 3.0 &mgr;M, respectively) activities of pea ctDNA helicase II, whereas ellipticine, etoposide (VP-16), and camptothecin had no effect on the enzyme activity. These ligands may be useful in further studies of the mechanisms of chloroplast helicase activities.

A complex between replication factor A (SSB) and DNA helicase stimulates DNA synthesis of DNA polymerase α on double-stranded DNA

A helicase-like DNA unwinding activity was found in highly purified fractions of the calf thymus single-stranded DNA binding protein (ctSSB). also known as replication protein A (RP-A) or replication factor A (RF-A). This activity depended on the hydrolysis of ATP or dATP, and used CTP with a lower efficiency. ctSSB promoted the homologous DNA polymerase α to perform DNA synthesis on double-stranded templates containing replication fork-like structures. The rate and amount of DNA synthesis was found to be dependent on the concentration of ctSSB. At a 10-fold mass excess of ctSSB over double-stranded DNA, products of 200–600 nucleotides in length were obtained. This comprises or even exceeds the length of a eukaryotic Okazaki fragment. The ctSSB-associated DNA helicase activity is most likely a distinct protein rather than an inherent property of SSB, as inferred from titration experiments between SSB and DNA. The association of a helicase with SSB and the stimulatory action of this complex to the DNA polymerase α-catalyzed synthesis of double-stranded DNA suggests a cooperative function of the three enzymatic activities in the process of eukaryotic DNA replication.