Metabolism of Pentose Sugars in the Hyperthermophilic Archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius

We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous pathway. On further investigation, the catalytic promiscuity of the first enzyme in this pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, d-xylose and l-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this pathway for the catabolism of d-xylose and l-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.     

Structural Basis for the Inefficient Nucleotide Incorporation Opposite Cisplatin-DNA Lesion by Human DNA Polymerase β

Human DNA polymerase β (polβ) has been suggested to play a role in cisplatin resistance, especially in polβ-overexpressing cancer cells. Polβ has been shown to accurately albeit slowly bypass the cisplatin-1,2-d(GpG) (Pt-GG) intramolecular cross-link in vitro. Currently, the structural basis for the inefficient Pt-GG bypass mechanism of polβ is unknown. To gain structural insights into the mechanism, we determined two ternary structures of polβ incorporating dCTP opposite the templating Pt-GG lesion in the presence of the active site Mg²⁺ or Mn²⁺. The Mg²⁺-bound structure shows that the bulky Pt-GG adduct is accommodated in the polβ active site without any steric hindrance. In addition, both guanines of the Pt-GG lesion form Watson-Crick base pairing with the primer terminus dC and the incoming dCTP, providing the structural basis for the accurate bypass of the Pt-GG adduct by polβ. The Mn²⁺-bound structure shows that polβ adopts a catalytically suboptimal semiclosed conformation during the insertion of dCTP opposite the templating Pt-GG, explaining the inefficient replication across the Pt-GG lesion by polβ. Overall, our studies provide the first structural insights into the mechanism of the potential polβ-mediated cisplatin resistance.     

In vitro gap-directed translesion DNA synthesis of an abasic site involving human DNA polymerases epsilon, lambda, and beta

DNA polymerase (pol) ε is thought to be the leading strand replicase in eukaryotes, whereas pols λ and β are believed to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). In this study, we present in vitro evidence that human pols λ, β, and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε, likely by initiating the 3'OHs created at the lesion by the arrested pol ε. However, in the case of pols λ and β, this TLS requires the presence of a DNA gap downstream from the product synthesized by the pol ε, and the optimal gap for efficient TLS is different for the two polymerases. The presence of gaps did not affect the TLS capacity of human pol η. Characterization of the reaction products showed that pol β inserted dAMP opposite the AP site, whereas gap filling synthesis by pol λ resulted in single or double deletions opposite the lesion. The synthesis up to the AP site by pol ε and the subsequent TLS by pols λ and β are not influenced by human processivity factor proliferating cell nuclear antigen and human single-stranded DNA-binding protein replication protein A. The bypass capacity of pol λ at the AP site is greatly reduced when a truncated form of the enzyme, which has lost the BRCA1 C-terminal and proline-rich domains, is used. Collectively, our in vitro results support the existence of a mechanism of gap-directed TLS at an AP site involving a switch between the replicative pol ε and the repair pols λ and β.     

Studies on human DNA polymerase epsilon and GINS complex and their role in DNA replication

In eukaryotic cells, DNA replication is carried out by the coordinated action of three DNA polymerases (Pols), Pol α, δ, and ε. In this report, we describe the reconstitution of the human four-subunit Pol ε and characterization of its catalytic properties in comparison with Pol α and Pol δ. Human Pol ε holoenzyme is a monomeric complex containing stoichiometric subunit levels of p261/Pol 2, p59, p17, and p12. We show that the Pol ε p261 N-terminal catalytic domain is solely responsible for its ability to catalyze DNA synthesis. Importantly, human Pol (hPol) ε was found more processive than hPol δ in supporting proliferating cell nuclear antigen-dependent elongation of DNA chains, which is in keeping with proposed roles for hPol ε and hPol δ in the replication of leading and lagging strands, respectively. Furthermore, GINS, a component of the replicative helicase complex that is composed of Sld5, Psf1, Psf2, and Psf3, was shown to interact weakly with all three replicative DNA Pols (α, δ, and ε) and to markedly stimulate the activities of Pol α and Pol ε. In vivo studies indicated that siRNA-targeted depletion of hPol δ and/or hPol ε reduced cell cycle progression and the rate of fork progression. Under the conditions used, we noted that depletion of Pol ε had a more pronounced inhibitory effect on cellular DNA replication than depletion of Pol δ. We suggest that reduction in the level of Pol δ may be less deleterious because of its collision-and-release role in lagging strand synthesis.     

Enhancement of Human DNA Polymerase η Activity and Fidelity Is Dependent Upon a Bipartite Interaction with the Werner Syndrome Protein

We have investigated the interaction between human DNA polymerase η (hpol η) and the Werner syndrome protein (WRN). Functional assays revealed that the WRN exonuclease and RecQ C-terminal (RQC) domains are necessary for full stimulation of hpol η-catalyzed formation of correct base pairs. We find that WRN does not stimulate hpol η-catalyzed formation of mispairs. Moreover, the exonuclease activity of WRN prevents stable mispair formation by hpol η. These results are consistent with a proofreading activity for WRN during single-nucleotide additions. ATP hydrolysis by WRN appears to attenuate stimulation of hpol η. Pre-steady-state kinetic results show that k_pol is increased 4-fold by WRN. Finally, pulldown assays reveal a bipartite physical interaction between hpol η and WRN that is mediated by the exonuclease and RQC domains. Taken together, these results are consistent with alteration of the rate-limiting step in polymerase catalysis by direct protein-protein interactions between WRN and hpol η. In summary, WRN improves the efficiency and fidelity of hpol η to promote more effective replication of DNA.     

Human DNA polymerase beta mutations allowing efficient abasic site bypass

The DNA of every cell in the human body gets damaged more than 50,000 times a day. The most frequent damages are abasic sites. This kind of damage blocks proceeding DNA synthesis by several DNA polymerases that are involved in DNA replication and repair. The mechanistic basis for the incapability of these DNA polymerases to bypass abasic sites is not clarified. To gain insights into the mechanistic basis, we intended to identify amino acid residues that govern for the pausing of DNA polymerase β when incorporating a nucleotide opposite to abasic sites. Human DNA polymerase β was chosen because it is a well characterized DNA polymerase and serves as model enzyme for studies of DNA polymerase mechanisms. Moreover, it acts as the main gap-filling enzyme in base excision repair, and human tumor studies suggest a link between DNA polymerase β and cancer. In this study we employed high throughput screening of a library of more than 11,000 human DNA polymerase β variants. We identified two mutants that have increased ability to incorporate a nucleotide opposite to an abasic site. We found that the substitutions E232K and T233I promote incorporation opposite the lesion. In addition to this feature, the variants have an increased activity and a lower fidelity when processing nondamaged DNA. The mutations described in this work are located in well characterized regions but have not been reported before. A crystallographic structure of one of the mutants was obtained, providing structural insights.     

Contributions of the specialised DNA polymerases to replication of structured DNA

It is becoming increasingly clear that processive DNA replication is threatened not only by DNA damage but also by secondary structures that can form in the DNA template. Failure to resolve these structures promptly leads to both genetic instability, for instance DNA breaks and rearrangements, and to epigenetic instability, in which inaccurate propagation of the parental chromatin state leads to unscheduled changes in gene expression. Multiple overlapping mechanisms are needed to deal with the wide range of potential DNA structural challenges to replication. This review focuses on the emerging mechanisms by which specialised DNA polymerases, best known for their role in the replication of damaged DNA, contribute to the replication of undamaged but structured DNA, particularly G quadruplexes.     

Coupling dTTP hydrolysis with DNA unwinding by the DNA helicase of bacteriophage T7

The DNA helicase encoded by gene 4 of bacteriophage T7 assembles on single-stranded DNA as a hexamer of six identical subunits with the DNA passing through the center of the toroid. The helicase couples the hydrolysis of dTTP to unidirectional translocation on single-stranded DNA and the unwinding of duplex DNA. Phe(523), positioned in a β-hairpin loop at the subunit interface, plays a key role in coupling the hydrolysis of dTTP to DNA unwinding. Replacement of Phe(523) with alanine or valine abolishes the ability of the helicase to unwind DNA or allow T7 polymerase to mediate strand-displacement synthesis on duplex DNA. In vivo complementation studies reveal a requirement for a hydrophobic residue with long side chains at this position. In a crystal structure of T7 helicase, when a nucleotide is bound at a subunit interface, Phe(523) is buried within the interface. However, in the unbound state, it is more exposed on the outer surface of the helicase. This structural difference suggests that the β-hairpin bearing the Phe(523) may undergo a conformational change during nucleotide hydrolysis. We postulate that upon hydrolysis of dTTP, Phe(523) moves from within the subunit interface to a more exposed position where it contacts the displaced complementary strand and facilitates unwinding.     

Translation initiation complex formation in the crenarchaeon Sulfolobus solfataricus

The function of initiation factors in and the sequence of events during translation initiation have been intensively studied in Bacteria and Eukaryotes, whereas in Archaea knowledge on these functions/processes is limited. By employing chemical probing, we show that translation initiation factor aIF1 of the model crenarchaeon Sulfolobus solfataricus binds to the same area on the ribosome as the bacterial and eukaryal orthologs. Fluorescence energy transfer assays (FRET) showed that aIF1, like its eukaryotic and bacterial orthologs, has a fidelity function in translation initiation complex formation, and that both aIF1 and aIF1A exert a synergistic effect in stimulating ribosomal association of the Met-tRNAi^Met binding factor a/eIF2. However, as in Eukaryotes their effect on a/eIF2 binding appears to be indirect. Moreover, FRET was used to analyze for the first time the sequence of events toward translation initiation complex formation in an archaeal model system. These studies suggested that a/eIF2-GTP binds first to the ribosome and then recruits Met-tRNAi^Met, which appears to comply with the operational mode of bacterial IF2, and deviates from the shuttle function of the eukaryotic counterpart eIF2. Thus, despite the resemblance of eIF2 and a/eIF2, recruitment of initiator tRNA to the ribosome is mechanistically different in Pro- and Eukaryotes.     

Biochemical characterization of two Cdc6/ORC1-like proteins from the crenarchaeon Sulfolobus solfataricus

The biological role of archaeal proteins, homologous to the eukaryal replication initiation factors of cell division control (Cdc6) and origin recognition complex (ORC1), has not yet been clearly established. The hyperthermophilic crenarchaeon Sulfolobus solfataricus (referred to as Sso) possesses three Cdc6/ORC1-like factors, which are named Sso Cdc6-1, Cdc6-2 and Cdc6-3. This study is a report on the biochemical characterization of Sso Cdc6-1 and Cdc6-3. It has been found that either Sso Cdc6-1 or Cdc6-3 behave as monomers in solutions by gel filtration analyses. Both factors are able to bind to various single-stranded and double-stranded DNA ligands, but Sso Cdc6-3 shows a higher DNA-binding affinity. It has also been observed that either Sso Cdc6-1 or Cdc6-3 inhibit the DNA unwinding activity of the S. solfataricus homo-hexameric mini-chromosome maintenance (MCM)-like DNA helicase (Sso MCM); although they strongly stimulate the interaction of the Sso MCM with bubble-containing synthetic oligonucleotides. The study has also showed, with surface plasmon resonance measurements, that Sso Cdc6-2 physically interacts with either Sso Cdc6-1 or Sso Cdc6-3. These findings may provide important clues needed to understand the biological role that is played by each of these three Cdc6 factors during the DNA replication initiation process in the S. solfataricus cells.     

Human DNA helicase B (HDHB) binds to replication protein A and facilitates cellular recovery from replication stress

Maintenance of genomic stability in proliferating cells depends on a network of proteins that coordinate chromosomal replication with DNA damage responses. Human DNA helicase B (HELB or HDHB) has been implicated in chromosomal replication, but its role in this coordinated network remains undefined. Here we report that cellular exposure to UV irradiation, camptothecin, or hydroxyurea induces accumulation of HDHB on chromatin in a dose- and time-dependent manner, preferentially in S phase cells. Replication stress-induced recruitment of HDHB to chromatin is independent of checkpoint signaling but correlates with the level of replication protein A (RPA) recruited to chromatin. We show using purified proteins that HDHB physically interacts with the N-terminal domain of the RPA 70-kDa subunit (RPA70N). NMR spectroscopy and site-directed mutagenesis reveal that HDHB docks on the same RPA70N surface that recruits S phase checkpoint signaling proteins to chromatin. Consistent with this pattern of recruitment, cells depleted of HDHB display reduced recovery from replication stress.     

Formation of a stable RuvA protein double tetramer is required for efficient branch migration in vitro and for replication fork reversal in vivo

In bacteria, RuvABC is required for the resolution of Holliday junctions (HJ) made during homologous recombination. The RuvAB complex catalyzes HJ branch migration and replication fork reversal (RFR). During RFR, a stalled fork is reversed to form a HJ adjacent to a DNA double strand end, a reaction that requires RuvAB in certain Escherichia coli replication mutants. The exact structure of active RuvAB complexes remains elusive as it is still unknown whether one or two tetramers of RuvA support RuvB during branch migration and during RFR. We designed an E. coli RuvA mutant, RuvA2(KaP), specifically impaired for RuvA tetramer-tetramer interactions. As expected, the mutant protein is impaired for complex II (two tetramers) formation on HJs, although the binding efficiency of complex I (a single tetramer) is as wild type. We show that although RuvA complex II formation is required for efficient HJ branch migration in vitro, RuvA2(KaP) is fully active for homologous recombination in vivo. RuvA2(KaP) is also deficient at forming complex II on synthetic replication forks, and the binding affinity of RuvA2(KaP) for forks is decreased compared with wild type. Accordingly, RuvA2(KaP) is inefficient at processing forks in vitro and in vivo. These data indicate that RuvA2(KaP) is a separation-of-function mutant, capable of homologous recombination but impaired for RFR. RuvA2(KaP) is defective for stimulation of RuvB activity and stability of HJ·RuvA·RuvB tripartite complexes. This work demonstrates that the need for RuvA tetramer-tetramer interactions for full RuvAB activity in vitro causes specifically an RFR defect in vivo.     

Eukaryotic lagging strand DNA replication employs a multi-pathway mechanism that protects genome integrity

In eukaryotic nuclear DNA replication, one strand of DNA is synthesized continuously, but the other is made as Okazaki fragments that are later joined. Discontinuous synthesis is inherently more complex, and fragmented intermediates create risks for disruptions of genome integrity. Genetic analyses and biochemical reconstitutions indicate that several parallel pathways evolved to ensure that the fragments are made and joined with integrity. An RNA primer is removed from each fragment before joining by a process involving polymerase-dependent displacement into a single-stranded flap. Evidence in vitro suggests that, with most fragments, short flaps are displaced and efficiently cleaved. Some flaps can become long, but these are also removed to allow joining. Rarely, a flap can form structure, necessitating displacement of the entire fragment. There is now evidence that post-translational protein modification regulates the flow through the pathways to favor protection of genomic information in regions of actively transcribed chromatin.     

Translocation and Stability of Replicative DNA Helicases upon Encountering DNA-Protein Cross-links

DNA-protein cross-links (DPCs) are formed when cells are exposed to various DNA-damaging agents. Because DPCs are extremely large, steric hindrance conferred by DPCs is likely to affect many aspects of DNA transactions. In DNA replication, DPCs are first encountered by the replicative helicase that moves at the head of the replisome. However, little is known about how replicative helicases respond to covalently immobilized protein roadblocks. In the present study we elucidated the effect of DPCs on the DNA unwinding reaction of hexameric replicative helicases in vitro using defined DPC substrates. DPCs on the translocating strand but not on the nontranslocating strand impeded the progression of the helicases including the phage T7 gene 4 protein, simian virus 40 large T antigen, Escherichia coli DnaB protein, and human minichromosome maintenance Mcm467 subcomplex. The impediment varied with the size of the cross-linked proteins, with a threshold size for clearance of 5.0–14.1 kDa. These results indicate that the central channel of the dynamically translocating hexameric ring helicases can accommodate only small proteins and that all of the helicases tested use the steric exclusion mechanism to unwind duplex DNA. These results further suggest that DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. The helicases stalled by DPC had limited stability and dissociated from DNA with a half-life of 15–36 min. The implications of the results are discussed in relation to the distinct stabilities of replisomes that encounter tight but reversible DNA-protein complexes and irreversible DPC roadblocks.     

Roles of ChlR1 DNA helicase in replication recovery from DNA damage

The ChlR1 DNA helicase is mutated in Warsaw breakage syndrome characterized by developmental anomalies, chromosomal breakage, and sister chromatid cohesion defects. However, the mechanism by which ChlR1 preserves genomic integrity is largely unknown. Here, we describe the roles of ChlR1 in DNA replication recovery. We show that ChlR1 depletion renders human cells highly sensitive to cisplatin; an interstrand-crosslinking agent that causes stalled replication forks. ChlR1 depletion also causes accumulation of DNA damage in response to cisplatin, leading to a significant delay in resolution of DNA damage. We also report that ChlR1-depleted cells display defects in the repair of double-strand breaks induced by the I-PpoI endonuclease and bleomycin. Furthermore, we demonstrate that ChlR1-depeleted cells show significant delays in replication recovery after cisplatin treatment. Taken together, our results indicate that ChlR1 plays an important role in efficient DNA repair during DNA replication, which may facilitate efficient establishment of sister chromatid cohesion.     

Distinct complexes of DNA polymerase I (Klenow fragment) for base and sugar discrimination during nucleotide substrate selection

During each catalytic cycle, DNA polymerases select deoxyribonucleoside triphosphate (dNTP) substrates complementary to a templating base with high fidelity from a pool that includes noncomplementary dNTPs and both complementary and noncomplementary ribonucleoside triphosphates (rNTPs). The Klenow fragment of Escherichia coli DNA polymerase I (KF) achieves this through a series of conformational transitions that precede the chemical step of phosphodiester bond formation. Kinetic evidence from fluorescence and FRET experiments indicates that discrimination of the base and sugar moieties of the incoming nucleotide occurs in distinct, sequential steps during the selection pathway. Here we show that KF-DNA complexes formed with complementary rNTPs or with noncomplementary nucleotides can be distinguished on the basis of their properties when captured in an electric field atop the α-hemolysin nanopore. The average nanopore dwell time of KF-DNA complexes increased as a function of complementary rNTP concentration. The increase was less than that promoted by complementary dNTP, indicating that the rNTP complexes are more stable than KF-DNA binary complexes but less stable than KF-DNA-dNTP ternary complexes. KF-DNA-rNTP complexes could also be distinguished from KF-DNA-dNTP complexes on the basis of ionic current amplitude. In contrast to complementary rNTPs, noncomplementary dNTPs and rNTPs diminished the average nanopore dwell time of KF-DNA complexes in a concentration-dependent manner, suggesting that binding of a noncomplementary nucleotide keeps the KF-DNA complex in a less stable state. These results imply that nucleotide selection proceeds through a series of complexes of increasing stability in which substrates with the correct moiety promote the forward transitions.     

Association between the Herpes Simplex Virus-1 DNA Polymerase and Uracil DNA Glycosylase

Herpes simplex virus-1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery and other enzymes involved in DNA transactions. We recently reported that the HSV-1 DNA polymerase catalytic subunit (UL30) exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities. Moreover, UL30, in conjunction with the viral uracil DNA glycosylase (UL2), cellular apurinic/apyrimidinic endonuclease, and DNA ligase IIIα-XRCC1, performs uracil-initiated base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we show that the HSV-1 UL2 associates with the viral replisome. We identified UL2 as a protein that co-purifies with the DNA polymerase through numerous chromatographic steps, an interaction that was verified by co-immunoprecipitation and direct binding studies. The interaction between UL2 and the DNA polymerase is mediated through the UL30 subunit. Moreover, UL2 co-localizes with UL30 to nuclear viral prereplicative sites. The functional consequence of this interaction is that replication of uracil-containing templates stalls at positions −1 and −2 relative to the template uracil because of the fact that these are converted into non-instructional abasic sites. These findings support the existence of a viral repair complex that may be capable of replication-coupled base excision repair and further highlight the role of DNA repair in the maintenance of the HSV-1 genome.     

Interactions of a replication initiator with histone H1-like proteins remodel the condensed mitochondrial genome

Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomatids, consists of several thousand topologically interlocked DNA circles. Mitochondrial histone H1-like proteins were implicated in the condensation of kDNA into a nucleoid structure in the mitochondrial matrix. However, the mechanism that remodels kDNA, promoting its accessibility to the replication machinery, has not yet been described. Analyses, using yeast two hybrid system, co-immunoprecipitation, and protein-protein cross-linking, revealed specific protein-protein interactions between the kDNA replication initiator protein universal minicircle sequence-binding protein (UMSBP) and two mitochondrial histone H1-like proteins. Fluorescence and electron microscopy, as well as biochemical analyses, demonstrated that these protein-protein interactions result in the decondensation of kDNA. UMSBP-mediated decondensation rendered the kDNA network accessible to topological decatenation by topoisomerase II, yielding free kDNA minicircle monomers. Hence, UMSBP has the potential capacity to function in vivo in the activation of the prereplication release of minicircles from the network, a key step in kDNA replication, which precedes and enables its replication initiation. These observations demonstrate the prereplication remodeling of a condensed mitochondrial DNA, which is mediated via specific interactions of histone-like proteins with a replication initiator, rather than through their posttranslational covalent modifications.     

Direct observation of translocation in individual DNA polymerase complexes

Complexes of phi29 DNA polymerase and DNA fluctuate on the millisecond time scale between two ionic current amplitude states when captured atop the α-hemolysin nanopore in an applied field. The lower amplitude state is stabilized by complementary dNTP and thus corresponds to complexes in the post-translocation state. We have demonstrated that in the upper amplitude state, the DNA is displaced by a distance of one nucleotide from the post-translocation state. We propose that the upper amplitude state corresponds to complexes in the pre-translocation state. Force exerted on the template strand biases the complexes toward the pre-translocation state. Based on the results of voltage and dNTP titrations, we concluded through mathematical modeling that complementary dNTP binds only to the post-translocation state, and we estimated the binding affinity. The equilibrium between the two states is influenced by active site-proximal DNA sequences. Consistent with the assignment of the upper amplitude state as the pre-translocation state, a DNA substrate that favors the pre-translocation state in complexes on the nanopore is a superior substrate in bulk phase for pyrophosphorolysis. There is also a correlation between DNA sequences that bias complexes toward the pre-translocation state and the rate of exonucleolysis in bulk phase, suggesting that during DNA synthesis the pathway for transfer of the primer strand from the polymerase to exonuclease active site initiates in the pre-translocation state.