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Multiple Functions of Nuclear DNA Helicase Ⅱ (RNA Helicase A) in Nucleic Acid Metabolism 总被引:6,自引:0,他引:6
Secondary structures of nucleic acids play an importantrole in regulating their transactions as carriers of thegenetic information, including DNA replication, trans-cription, RNA processing, RNA transport, and translation.Resolving double-stranded (ds) DNA or RNA is usually anenergy-dependent process that can be accomplished byproteins termed DNA or RNA helicases, which are presentin all prokaryotic and eukaryotic organisms. Earlier attemptsto find mammalian helicases led to the detect… 相似文献
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Chanmi Lee Ivan Liachko Roxane Bouten Zvi Kelman Bik K. Tye 《Molecular and cellular biology》2010,30(2):423-435
Functional coordination between DNA replication helicases and DNA polymerases at replication forks, achieved through physical linkages, has been demonstrated in prokaryotes but not in eukaryotes. In Saccharomyces cerevisiae, we showed that mutations that compromise the activity of the MCM helicase enhance the physical stability of DNA polymerase α in the absence of their presumed linker, Mcm10. Mcm10 is an essential DNA replication protein implicated in the stable assembly of the replisome by virtue of its interaction with the MCM2-7 helicase and Polα. Dominant mcm2 suppressors of mcm10 mutants restore viability by restoring the stability of Polα without restoring the stability of Mcm10, in a Mec1-dependent manner. In this process, the single-stranded DNA accumulation observed in the mcm10 mutant is suppressed. The activities of key checkpoint regulators known to be important for replication fork stabilization contribute to the efficiency of suppression. These results suggest that Mcm10 plays two important roles as a linker of the MCM helicase and Polα at the elongating replication fork—first, to coordinate the activities of these two molecular motors, and second, to ensure their physical stability and the integrity of the replication fork.The key players of the replication machinery are the DNA polymerases that synthesize the leading and lagging daughter strands and the replicative helicase that unwinds the parental strands ahead of the polymerases. Coordination between the helicase and the polymerases is critical during replication. Uncoupling of these two molecular machines, especially during lagging strand synthesis, may result in an unrestrained helicase and the exposure of extensive single-stranded DNA (ssDNA), as observed in checkpoint mutants treated with hydroxyurea (HU) (37). Although there is no direct evidence, the implication is that the replicative helicase would be moving at a faster pace than would the DNA polymerase if synchrony were destroyed. In Escherichia coli, the replicative helicase (DnaB) and the primase (DnaG) are coupled by direct contact to form a tight complex (3). In T7, processivity of the gp5 polymerase in lagging strand synthesis requires coupling to the gp4 helicase (16). Recent studies of the budding yeast Saccharomyces cerevisiae suggest that Mrc1 may couple DNA polymerase ɛ and the MCM helicase on the leading strand as well as activate the checkpoint response under replication stress (1, 22, 28). A candidate for coupling DNA polymerase α primase and the MCM helicase on the lagging strand is Mcm10, because Mcm10 interacts with subunits of the Mcm2-7 helicase (26, 29) as well as Polα (14, 33) and the stability of Polα requires Mcm10 in both budding yeast and human cells (8, 33). Mcm10 is an essential protein known to be involved in various aspects of the replication process. It is required during both initiation and elongation steps of DNA replication and interacts with a wide range of replication factors, such as ORC (17, 23, 29), MCM helicase, DNA polymerases ɛ and δ (23), Cdc45 (34), and Polα (33). Therefore, Mcm10 is important for the overall stability of the elongation complex, but its essential function remains unknown.Accumulating evidence suggests that the major function of many checkpoint proteins is the stabilization of the replication machinery at the fork (9, 22, 39), in addition to regulation of the temporal and spatial firing of origins and prevention of premature mitosis (31, 35, 39). The main signal that leads to checkpoint activation is believed to be the exposure of RPA-coated ssDNA (42). In Xenopus, ssDNA exposure has been shown to be mediated by a functional uncoupling between the polymerase and the helicase (7), and it has been shown that the level of checkpoint activation depended on the extent of ssDNA accumulation. This observation suggests that uncoupling of the polymerase and the helicase activity would result in ssDNA accumulation that in turn would activate the checkpoint pathway to stabilize the fork.In our study, we carried out a random and a gene-targeted mutagenesis screen to identify mutations that suppress the conditional lethality of mcm10 caused by the lability of Mcm10 in budding yeast (27). We found suppressor mutations in MCM2, which encodes one of the six distinct subunits of the MCM helicase. These mcm2 mutations correct the fork defects of mcm10, particularly that which leads to Polα instability. The altered helicase activity and activation of the checkpoint pathway of the mcm2 mutants appeared to be required for viability of mcm10 mcm2. We showed that uncoupling the MCM helicase and DNA polymerase α by destabilizing Mcm10 leads to accumulation of ssDNA, which is suppressed by reducing the MCM helicase activity. Our findings suggest that the physical coupling of Polα and the helicase by Mcm10 may be replaced by an alternative stabilization mechanism that involves slowing down the helicase and activating the checkpoint proteins. 相似文献
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Hao Huang Kun Zhao Diana R. Arnett Ellen Fanning 《The Journal of biological chemistry》2010,285(43):33475-33484
Replication of simian virus 40 (SV40) DNA, a model for eukaryotic chromosomal replication, can be reconstituted in vitro using the viral helicase (large tumor antigen, or Tag) and purified human proteins. Tag interacts physically with two cellular proteins, replication protein A and DNA polymerase α-primase (pol-prim), constituting the viral primosome. Like the well characterized primosomes of phages T7 and T4, this trio of proteins coordinates parental DNA unwinding with primer synthesis to initiate the leading strand at the viral origin and each Okazaki fragment on the lagging strand template. We recently determined the structure of a previously unrecognized pol-prim domain (p68N) that docks on Tag, identified the p68N surface that contacts Tag, and demonstrated its vital role in primosome function. Here, we identify the p68N-docking site on Tag by using structure-guided mutagenesis of the Tag helicase surface. A charge reverse substitution in Tag disrupted both p68N-binding and primosome activity but did not affect docking with other pol-prim subunits. Unexpectedly, the substitution also disrupted Tag ATPase and helicase activity, suggesting a potential link between p68N docking and ATPase activity. To assess this possibility, we examined the primosome activity of Tag with a single residue substitution in the Walker B motif. Although this substitution abolished ATPase and helicase activity as expected, it did not reduce pol-prim docking on Tag or primosome activity on single-stranded DNA, indicating that Tag ATPase is dispensable for primosome activity in vitro. 相似文献
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The Vibrio cholerae bacterium is the agent of cholera. The capacity to produce the cholera toxin, which is responsible for the deadly diarrhea associated with cholera epidemics, is encoded in the genome of a filamentous phage, CTXφ. Rolling-circle replication (RCR) is central to the life cycle of CTXφ because amplification of the phage genome permits its efficient integration into the genome and its packaging into new viral particles. A single phage-encoded HUH endonuclease initiates RCR of the proto-typical filamentous phages of enterobacteriaceae by introducing a nick at a specific position of the double stranded DNA form of the phage genome. The rest of the process is driven by host factors that are either essential or crucial for the replication of the host genome, such as the Rep SF1 helicase. In contrast, we show here that the histone-like HU protein of V. cholerae is necessary for the introduction of a nick by the HUH endonuclease of CTXφ. We further show that CTXφ RCR depends on a SF1 helicase normally implicated in DNA repair, UvrD, rather than Rep. In addition to CTXφ, we show that VGJφ, a representative member of a second family of vibrio integrative filamentous phages, requires UvrD and HU for RCR while TLCφ, a satellite phage, depends on Rep and is independent from HU. 相似文献
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Hepatitis E virus (HEV) is a causative agent of acute hepatitis, and it is the sole member of the genus Hepevirus in the family Hepeviridae. The open reading frame 1 (ORF1) protein of HEV encodes nonstructural polyprotein with putative domains for methyltransferase, cysteine protease, helicase and RNA-dependent RNA polymerase. It is not yet known whether ORF1 functions as a single protein with multiple domains or is processed to form separate functional units. On the basis of amino acid conserved motifs, HEV helicase has been grouped into helicase superfamily 1 (SF-1). In order to examine the RNA helicase activity of the NTPase/helicase domain of HEV, the region (amino acids 960 to 1204) was cloned and expressed as histidine-tagged protein in Escherichia coli (HEV Hel) and purified. HEV Hel exhibited NTPase and RNA unwinding activities. Enzyme hydrolyzed all rNTPs efficiently, dATP and dCTP with moderate efficiency, while it showed less hydrolysis of dGTP and dTTP. Enzyme showed unwinding of only RNA duplexes with 5′ overhangs showing 5′-to-3′ polarity. We also expressed and purified two HEV Hel mutants. Helicase mutant I, with substitution in the nucleotide-binding motif I (GKS to GAS), showed 30% ATPase activity. Helicase mutant II, with substitutions in the Mg2+ binding motif II (DEAP to AAAP), showed 50% ATPase activity. Both mutants completely lost ability to unwind RNA duplexes with 5′ overhangs. These findings represent the first report demonstrating NTPase/RNA helicase activity of the helicase domain of HEV ORF1.Viruses with single-strand positive-sense RNA genomes represent the largest class of viruses, which includes numerous pathogens of humans, plants, and animals. In these viruses, RNA replication occurs through negative-strand RNA intermediate, which may also act as the template for synthesis of subgenomic RNAs in some viruses. During replication, various nonstructural proteins remain associated with the viral polymerase in a small compartmentalized replisome. Most of the other accessory proteins are obtained from the cellular machinery.Helicase seems to be essential for RNA replication by many positive-sense RNA viruses (19). Many positive-strand RNA viruses encode their own RNA helicases and besides RNA-dependent RNA polymerase, helicase is the most conserved viral sequence in these viruses. It has been shown by direct mutagenesis studies in poliovirus (26, 39), alphaviruses (31), brome mosaic virus (2, 41), nidoviruses (40), and flaviviruses (15) that helicase functions are essential for viral replication. In addition, it may be involved in RNA translocation, genome packaging, protection of RNA at the replication center, modulating RNA-protein interactions, etc.Helicases are classified into six superfamilies, SF-1 to SF-6 (11, 35), and can be classified further into subfamilies, A (3′→5′) or B (5′→3′) depending on their unwinding directionality. Classic helicases (exhibiting both NTPase and unwinding activities) are referred to as subtype α, while translocases (with no unwinding activity) are referred to as subtype β (35). SF-1 and SF-2 constitute largest of these superfamilies with seven signature motifs (I, Ia, II, III, IV, V, and VI), which form core of the enzyme. Although these motifs are not comparable between SF-1 and SF-2, universal features of core domains include (i) conserved residues involved in binding and hydrolysis of the NTP and (ii) an arginine finger that plays a key role in energy coupling.Hepatitis E virus (HEV) is a nonenveloped virus in the genus Hepevirus of the family Hepeviridae. Hepatitis E is an important public health disease in many developing countries and is also endemic in some industrialized countries (8). Infection by HEV has a known association with increased mortality during pregnancy (22, 23). HEV has a positive-sense RNA genome of ∼7.2 kb, consisting of a 5′ noncoding region (5′NCR) of 27 to 35 nucleotides (nt), followed by three open reading frames (ORFs)—ORF1, ORF2, and ORF3—and a 3′NCR of 65 to 74 nt, ending with a poly(A) tail of variable length (37). The 5′ end has m7G cap (18). ORF1 is known to encode for the viral nonstructural polyprotein with a proposed molecular mass of ∼186 kDa (3). Based on protein sequence homology, the ORF1 polyprotein is proposed to contain four putative domains indicative of methyltransferase, papain-like cysteine protease, RNA helicase (Hel), and RNA-dependent RNA polymerase (RdRp) (24). ORF2 encodes the major structural protein (capsid protein), which has N-terminal signal peptide and three glycosylation sites and is translocated across the endoplasmic reticulum (ER). ORF2 protein associates with the 5′ end of the viral RNA, suggesting its regulatory role in the virus replication (36, 37, 44, 45). ORF3 encodes a protein which gets phosphorylated by the cellular mitogen activated protein kinase and is associated with cellular membranes and cytoskeleton fractions (43).HEV belongs to an “alpha-like” supergroup of positive-sense single-stranded RNA (+ssRNA) viruses with conserved motifs of replication-related proteins in the ORF1, with typical signature sequences homologous with the other members of the family (11, 12, 13). ORF1 of HEV encodes additional domains such as the Y domain, papainlike protease, “proline-rich hinge,” and the X domain. Methyltransferase (25), RdRp (1), and X domain (binding to poly-ADP-ribose) (9) in ORF1 have been characterized, whereas the functions of the other domains are yet to be identified. Intracellularly expressed RdRp localizes itself in the ER membranes (30), suggesting that HEV replicates probably in ER in the cytosolic compartment of the cells. It is still unknown whether ORF1 polyprotein undergoes cleavages to form separate functional units of the replication machinery or functions as a single protein with multiple functional domains.The putative RNA helicase of HEV contains all of the seven conserved segments typical of the SF-1 helicase (12, 13). Putative SF-1 helicases are extremely widespread among +ssRNA viruses. Based on sequence comparisons, such helicases have been identified in a variety of plant virus families, as well as in animal viruses such as alphavirus, rubivirus, hepatitis E virus, and coronavirus (11). When compared to other +ssRNA viral helicases belonging to SF-1, HEV helicase showed the highest overall similarity with the helicase of beet necrotic yellow vein virus, a plant furovirus. HEV helicase was speculated to have N-terminal NTPase and C-terminal RNA-binding domains (24). A major obstacle in studying HEV replication has been lack of cell culture system. We report here experimental verification of the helicase activity of the recombinant helicase domain protein of HEV. 相似文献
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Christopher Capp Jianhong Wu Tao-shih Hsieh 《The Journal of biological chemistry》2009,284(45):30845-30852
Members of the RecQ family of proteins are highly conserved DNA helicases that have important functions in the maintenance of genomic stability. Deficiencies in RecQ4 have been linked to human diseases including Rothmund-Thomson, RAPADILINO, and Baller-Gerold syndromes, all of which are characterized by developmental defects, tumor propensity, and genetic instability. However, there are conflicting results shown in the literature regarding the DNA helicase activity of RecQ4. We report here the expression of Drosophila melanogaster RecQ4 with a baculoviral vector and its purification to near homogeneity. The purified protein has a DNA-dependent ATPase activity and is a 3′-5′ DNA helicase dependent on hydrolysis of ATP. The presence of 5′-adenylyl-β,γ-imidodiphosphate (AMPPNP), a nonhydrolyzable ATP analog, promotes stable complex formation between RecQ4 and single-stranded DNA. Drosophila RecQ4 can also anneal complementary single strands; this activity was reduced in the presence of AMPPNP, possibly because of the stable protein-DNA complex formed under such conditions. A point mutation of the highly conserved lysine residue in the helicase domain, although retaining the wild type level of annealing activity, inactivated ATPase and helicase activities and eliminated stable complex formation. These results suggest that the helicase domain alone is responsible for the DNA unwinding action of the Drosophila enzyme. We generated a null recq4 mutant that is homozygous lethal, which we used to test the genetic function of the helicase-dead mutant in flies. Complementation tests showed that the helicase-dead mutant recq4 transgenes are incapable of rescuing the null mutation, demonstrating that the helicase activity has an essential biological function. 相似文献
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Saumitri Bhattacharyya Jeremy Keirsey Beatriz Russell Juraj Kavecansky Kate Lillard-Wetherell Kambiz Tahmaseb John J. Turchi Joanna Groden 《The Journal of biological chemistry》2009,284(22):14966-14977
The BLM helicase associates with the telomere structural proteins TRF1 and
TRF2 in immortalized cells using the alternative
lengthening of telomere (ALT) pathways. This work
focuses on identifying protein partners of BLM in cells using ALT. Mass
spectrometry and immunoprecipitation techniques have identified three proteins
that bind directly to BLM and TRF2 in ALT cells: telomerase-associated protein
1 (TEP1), heat shock protein 90 (HSP90), and topoisomerase IIα
(TOPOIIα). BLM predominantly co-localizes with these proteins in foci
actively synthesizing DNA during late S and G2/M phases of the cell
cycle when ALT is thought to occur. Immunoprecipitation studies also indicate
that only HSP90 and TOPOIIα are components of a specific complex
containing BLM, TRF1, and TRF2 but that this complex does not include TEP1.
TEP1, TOPOIIα, and HSP90 interact directly with BLM in vitro
and modulate its helicase activity on telomere-like DNA substrates but not on
non-telomeric substrates. Initial studies suggest that knockdown of
BLM in ALT cells reduces average telomere length but does not do so
in cells using telomerase.Bloom syndrome
(BS)4 is a genetic
disease caused by mutation of both copies of the human BLM gene. It
is characterized by sun sensitivity, small stature, immunodeficiency, male
infertility, and an increased susceptibility to cancer of all sites and types.
The high incidence of spontaneous chromosome breakage and other unique
chromosomal anomalies in cells from BS patients indicate an increase in
homologous recombination in somatic cells
(1). Another notable feature of
non-immortalized and immortalized cells from BS individuals is the presence of
telomeric associations (TAs) between homologous chromosomes
(2). Work from our group and
others have suggested a role for BLM in recombination-mediated mechanisms of
telomere elongation or ALT (alternative lengthening of telomeres), processes
that maintain/elongate telomeres in the absence of telomerase
(3–5).
However, the exact mechanism by which BLM contributes to telomere stability is
unknown.Several proteins interact with and regulate BLM helicase activity,
including two telomere-specific proteins, TRF1 and TRF2
(6,
7). Although TRF2 stimulates
BLM unwinding of telomeric and non-telomeric 3′-overhang substrates,
TRF1 inhibits BLM unwinding of telomeric substrates. TRF2-mediated stimulation
of BLM helicase activity on a telomeric substrate is observed when TRF2 is
present in excess or with equimolar amount of TRF1 but not when TRF1 is
present in molar excess. Both proteins associate with BLM specifically in ALT
cells in vivo, suggesting their involvement in the ALT pathways. In
addition to TRF1 and TRF2, the telomere single-strand DNA-binding protein POT1
strongly stimulates BLM helicase activity on long telomeric forked duplexes
and D-loop structures (8).
Other proteins also play an important role in telomere maintenance in
telomerase-negative cells, including RAD50, NBS1, and MRE11, which co-localize
with TRF1 and TRF2 in specialized ALT-associated promyelocytic leukemia (PML)
nuclear bodies (APBs)
(9–11).
Thus, we hypothesize that BLM complex formation may be essential for the ALT
mechanism, and its modification may occur dynamically during the specific
nucleic acid transactions required to protect the telomere in cells using the
ALT pathways.This study has identified previously unknown protein partners of BLM and
TRF2 in ALT cells using double immunoprecipitation and mass spectrometry (MS).
These include telomerase-associated protein 1 (TEP1), heat shock protein 90
(HSP90), and topoisomerase IIα (TOPOIIα). These proteins associate
with BLM and TRF2 in cells using ALT but not in cells using telomerase and
directly interact with BLM in vitro. This complex of proteins
localizes to sites of new DNA synthesis in vivo in ALT cells,
suggesting a role in telomere maintenance. We also identified HSP90 and
TOPOIIα in another ALT-specific complex consisting of BLM, TRF1, and
TRF2 but not TEP1. In vitro analyses demonstrate that HSP90 inhibits
BLM helicase activity using both telomeric and non-telomeric substrates,
whereas TEP1 and TOPOIIα initially slow the kinetics of BLM unwinding
only using telomeric substrates. These findings suggest the presence of
dynamic BLM-associated ALT complexes that include previously unidentified
interacting proteins. The function of TEP1 in the BLM·TRF2 complex
remains unclear, although its previously described interaction with the RNA
subunit of telomerase (12)
suggests an interesting hypothesis of cross-talk between mechanisms of
telomere elongation. 相似文献
11.
Hsiu-Fang Fan 《Biophysical journal》2009,96(5):1875-1883
Escherichia coli RecBCD helicase unwinds blunt-end duplex DNA to repair damaged DNA molecules in the homologous recombination pathway. Previous single-molecule experiments showed that RecBCD recognizes an 8 nt DNA sequence, χ, and lowers its unwinding rate afterward under saturating ATP condition. We have developed a single-molecule force-tethered particle motion (FTPM) method, which is modified from the conventional TPM method, and applied it to study RecBCD motion in detail. In the FTPM experiment, a stretching force is applied to the DNA-bead complex that suppresses the bead's Brownian motion, resulting in an improved spatial resolution at long DNA substrates. Based on the equipartition theorem, the mean-square displacement of the bead's Brownian motion measured by FTPM correlates linearly to DNA extension length with a predicted slope, circumventing the difficulties of conventional TPM experiments, such as nonlinearity and low resolution of long DNA substrates. The FTPM method offers the best resolution in the presence of only a small stretching force (0.20 pN). We used the FTPM method to investigate RecBCD helicase motion along 4.1 kb long χ-containing duplex DNA molecules, and observed that the translocation rate of RecBCD changes after the χ sequence under limited ATP concentrations. This suggests that χ recognition by RecBCD does not require saturating ATP conditions, contrary to what was previously reported. 相似文献
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Nalini Dhingra Irina Bruck Skye Smith Boting Ning Daniel L. Kaplan 《The Journal of biological chemistry》2015,290(12):7586-7601
Dpb11 is required for the initiation of DNA replication in budding yeast. Dpb11 binds to S-phase cyclin-dependent kinase-phosphorylated Sld2 and Sld3 to form a ternary complex during S phase. The replication fork helicase in eukaryotes is composed of Cdc45, Mcm2-7, and GINS. We show here, using purified proteins from budding yeast, that Dpb11 alone binds to Mcm2-7 and that Dpb11 also competes with GINS for binding to Mcm2-7. Furthermore, Dpb11 binds directly to single-stranded DNA (ssDNA), and ssDNA inhibits the Dpb11 interaction with Mcm2-7. We also found that Dpb11 can recruit Cdc45 to Mcm2-7. We identified a mutant of the BRCT4 motif of Dpb11 that remains bound to Mcm2-7 in the presence of ssDNA (dpb11-m1,m2,m3,m5), and this mutant exhibits a DNA replication defect when expressed in budding yeast cells. Expression of this mutant results in increased interaction between Dpb11 and Mcm2-7 during S phase, impaired GINS interaction with Mcm2-7 during S phase, and decreased replication protein A (RPA) interaction with origin DNA during S phase. We propose a model in which Dpb11 first recruits Cdc45 to Mcm2-7. Dpb11, although bound to Cdc45·Mcm2-7, can block the interaction between GINS and Mcm2-7. Upon extrusion of ssDNA from the central channel of Mcm2-7, Dpb11 dissociates from Mcm2-7, and Dpb11 binds to ssDNA, thereby allowing GINS to bind to Cdc45·Mcm2-7. Finally, we propose that Dpb11 functions with Sld2 and Sld3 to help control the assembly of the replication fork helicase. 相似文献
14.
Hao Huang Brian E. Weiner Haijiang Zhang Brian E. Fuller Yue Gao Brian M. Wile Kun Zhao Diana R. Arnett Walter J. Chazin Ellen Fanning 《The Journal of biological chemistry》2010,285(22):17112-17122
DNA polymerase α-primase (pol-prim) plays a central role in DNA replication in higher eukaryotes, initiating synthesis on both leading and lagging strand single-stranded DNA templates. Pol-prim consists of a primase heterodimer that synthesizes RNA primers, a DNA polymerase that extends them, and a fourth subunit, p68 (also termed B-subunit), that is thought to regulate the complex. Although significant knowledge about single-subunit primases of prokaryotes has accumulated, the functions and regulation of pol-prim remain poorly understood. In the SV40 replication model, the p68 subunit is required for primosome activity and binds directly to the hexameric viral helicase T antigen, suggesting a functional link between T antigen-p68 interaction and primosome activity. To explore this link, we first mapped the interacting regions of the two proteins and discovered a previously unrecognized N-terminal globular domain of p68 (p68N) that physically interacts with the T antigen helicase domain. NMR spectroscopy was used to determine the solution structure of p68N and map its interface with the T antigen helicase domain. Structure-guided mutagenesis of p68 residues in the interface diminished T antigen-p68 interaction, confirming the interaction site. SV40 primosome activity of corresponding pol-prim mutants decreased in proportion to the reduction in p68N-T antigen affinity, confirming that p68-T antigen interaction is vital for primosome function. A model is presented for how this interaction regulates SV40 primosome activity, and the implications of our findings are discussed in regard to the molecular mechanisms of eukaryotic DNA replication initiation. 相似文献
15.
Zhou B Arnett DR Yu X Brewster A Sowd GA Xie CL Vila S Gai D Fanning E Chen XS 《The Journal of biological chemistry》2012,287(32):26854-26866
DNA polymerase α-primase (Pol-prim) plays an essential role in eukaryotic DNA replication, initiating synthesis of the leading strand and of each Okazaki fragment on the lagging strand. Pol-prim is composed of a primase heterodimer that synthesizes an RNA primer, a DNA polymerase subunit that extends the primer, and a regulatory B-subunit (p68) without apparent enzymatic activity. Pol-prim is thought to interact with eukaryotic replicative helicases, forming a dynamic multiprotein assembly that displays primosome activity. At least three subunits of Pol-prim interact physically with the hexameric replicative helicase SV40 large T antigen, constituting a simple primosome that is active in vitro. However, structural understanding of these interactions and their role in viral chromatin replication in vivo remains incomplete. Here, we report the detailed large T antigen-p68 interface, as revealed in a co-crystal structure and validated by site-directed mutagenesis, and we demonstrate its functional importance in activating the SV40 primosome in cell-free reactions with purified Pol-prim, as well as in monkey cells in vivo. 相似文献
16.
ATPase Mechanism of the 5′-3′ DNA Helicase,RecD2: EVIDENCE FOR A PRE-HYDROLYSIS CONFORMATION CHANGE*
Christopher P. Toseland Martin R. Webb 《The Journal of biological chemistry》2013,288(35):25183-25193
The superfamily 1 helicase, RecD2, is a monomeric, bacterial enzyme with a role in DNA repair, but with 5′-3′ activity unlike most enzymes from this superfamily. Rate constants were determined for steps within the ATPase cycle of RecD2 in the presence of ssDNA. The fluorescent ATP analog, mantATP (2′(3′)-O-(N-methylanthraniloyl)ATP), was used throughout to provide a complete set of rate constants and determine the mechanism of the cycle for a single nucleotide species. Fluorescence stopped-flow measurements were used to determine rate constants for adenosine nucleotide binding and release, quenched-flow measurements were used for the hydrolytic cleavage step, and the fluorescent phosphate biosensor was used for phosphate release kinetics. Some rate constants could also be measured using the natural substrate, ATP, and these suggested a similar mechanism to that obtained with mantATP. The data show that a rearrangement linked to Mg2+ coordination, which occurs before the hydrolysis step, is rate-limiting in the cycle and that this step is greatly accelerated by bound DNA. This is also shown here for the PcrA 3′-5′ helicase and so may be a general mechanism governing superfamily 1 helicases. The mechanism accounts for the tight coupling between translocation and ATPase activity. 相似文献
17.
Ajit K. Satapathy Donald J. Crampton Benjamin B. Beauchamp Charles C. Richardson 《The Journal of biological chemistry》2009,284(21):14286-14295
The multifunctional protein encoded by gene 4 of bacteriophage T7 (gp4)
provides both helicase and primase activity at the replication fork. T7 DNA
helicase preferentially utilizes dTTP to unwind duplex DNA in vitro
but also hydrolyzes other nucleotides, some of which do not support helicase
activity. Very little is known regarding the architecture of the nucleotide
binding site in determining nucleotide specificity. Crystal structures of the
T7 helicase domain with bound dATP or dTTP identified Arg-363 and Arg-504 as
potential determinants of the specificity for dATP and dTTP. Arg-363 is in
close proximity to the sugar of the bound dATP, whereas Arg-504 makes a
hydrogen bridge with the base of bound dTTP. T7 helicase has a serine at
position 319, whereas bacterial helicases that use rATP have a threonine in
the comparable position. Therefore, in the present study we have examined the
role of these residues (Arg-363, Arg-504, and Ser-319) in determining
nucleotide specificity. Our results show that Arg-363 is responsible for dATP,
dCTP, and dGTP hydrolysis, whereas Arg-504 and Ser-319 confer dTTP
specificity. Helicase-R504A hydrolyzes dCTP far better than wild-type
helicase, and the hydrolysis of dCTP fuels unwinding of DNA. Substitution of
threonine for serine 319 reduces the rate of hydrolysis of dTTP without
affecting the rate of dATP hydrolysis. We propose that different nucleotides
bind to the nucleotide binding site of T7 helicase by an induced fit
mechanism. We also present evidence that T7 helicase uses the energy derived
from the hydrolysis of dATP in addition to dTTP for mediating DNA
unwinding.Helicases are molecular machines that translocate unidirectionally along
single-stranded nucleic acids using the energy derived from nucleotide
hydrolysis
(1–3).
The gene 4 protein encoded by bacteriophage T7 consists of a helicase domain
and a primase domain, located in the C-terminal and N-terminal halves of the
protein, respectively (4). The
T7 helicase functions as a hexamer and has been used as a model to study
ring-shaped replicative helicases. In the presence of dTTP, T7 helicase binds
to single-stranded DNA
(ssDNA)3 as a hexamer
and translocates 5′ to 3′ along the DNA strand using the energy of
hydrolysis of dTTP
(5–7).
T7 helicase hydrolyzes a variety of ribo and deoxyribonucleotides; however,
dTTP hydrolysis is optimally coupled to DNA unwinding
(5).Most hexameric helicases use rATP to fuel translocation and unwind DNA
(3). T7 helicase does hydrolyze
rATP but with a 20-fold higher Km as compared with dTTP
(5,
8). It has been suggested that
T7 helicase actually uses rATP in vivo where the concentration of
rATP is 20-fold that of dTTP in the Escherichia coli cell
(8). However, hydrolysis of
rATP, even at optimal concentrations, is poorly coupled to translocation and
unwinding of DNA (9). Other
ribonucleotides (rCTP, rGTP, and rUTP) are either not hydrolyzed or the poor
hydrolysis observed is not coupled to DNA unwinding
(8). Furthermore, Patel et
al. (10) found that the
form of T7 helicase found in vivo, an equimolar mixture of the
full-length gp4 and a truncated form lacking the zinc binding domain of the
primase, prefers dTTP and dATP. Therefore, in the present study we have
restricted our examination of nucleotides to the deoxyribonucleotides.The nucleotide binding site of the replicative DNA helicases, such as T7
gene 4 protein, bind nucleotides at the subunit interface
(Fig. 1) located between two
RecA-like subdomains that bind ATP
(11,
12). The location of the
nucleotide binding site at the subunit interface provides multiple
interactions of residues with the bound NTP. A number of cis- and
trans-acting amino acids stabilize the bound nucleotide in the
nucleotide binding site and also provide for communication between subunits
(13–15).
Earlier reports revealed that the arginine finger (Arg-522) in T7 helicase is
positioned to interact with the γ-phosphate of the bound nucleotide in
the adjacent subunit (12,
16). However, His-465
(phosphate sensor), Glu-343 (catalytic base), and Asp-424 (Walker motif B)
interacts with the γ-phosphate of the bound nucleotide in the same
subunit (12,
17,
18). The arginine finger and
the phosphate sensor have been proposed to couple NTP hydrolysis to DNA
unwinding. Substitution of Glu-343, the catalytic base, eliminates dTTP
hydrolysis (19), and
substitution of Asp-424 with Asn leads to a severe reduction in dTTP
hydrolysis (20). The conserved
Lys-318 in Walker motif A interacts with the β-phosphate of the bound
nucleotide and plays an important role in dTTP hydrolysis
(21).Open in a separate windowFIGURE 1.Crystal structure of T7 helicase. A, crystal structure of
the hexameric helicase C-terminal domain of gp4
(17). The structure reveals a
ring-shaped molecule with a central core through which ssDNA passes. The
inset shows the interface between two subunits of the helicase with
adenosine 5′-{β,γ-imidol}-triphosphate in the nucleotide
binding site. B, the nucleotide binding site of a monomer of the gp4
with the crucial amino acid residues reported earlier and in the present study
is shown in sticks. The crystal structures of the T7 gene 4 helicase
domain (12) with bound dTTP
(C) and dATP (D). The structures shown are the nucleotide
binding site of T7 helicase as viewed in Pymol by analyzing the PDB files 1cr1
and 1cr2 (12). Arg-504 and
Tyr-535 sandwiches the base of the bound dNTP. Additionally, Arg-504 forms a
hydrogen bridge with dTTP. Arg-363 interacts specifically with the 3-OH group
of bound dATP. AMPPNP, adenosine
5′-(β,γ-imino)triphosphate.Considering the wealth of information on the above residues that are
involved in the hydrolysis of dTTP and the coupling of hydrolysis to
unwinding, it is intriguing that little information is available on nucleotide
specificity. Several crystal structures of T7 helicase in complex with a
nucleotide triphosphate are available. However, most of structures were
crystallized with a non-hydrolyzable analogue of dTTP or the nucleotide was
diffused into the crystal. The crystal structure of the T7 helicase domain
bound with dTTP or dATP was reported by Sawaya et al.
(12). These structures
assisted us in identifying two basic residues (Arg-363 and Arg-504) in close
proximity to the sugar and base of the bound nucleotide whose orientation
suggested that these residues could be involved in nucleotide selection.
Arg-504 together with Tyr-535 sandwich the base of the bound nucleotide at the
subunit interface of the hexameric helicase
(Fig. 1). Arg-504 and Tyr-535
are structurally well conserved in various helicases
(12). However, Arg-504 could
make a hydrogen bridge with the OH group of thymidine, thus suggesting a role
in dTTP specificity. On the other hand, Arg-363 is in close proximity
(∼3.4 Å) to the sugar 3′-OH of bound dATP, whereas in the
dTTP-bound structure this residue is displaced by 7.12 Å
(Fig. 1) from the equivalent
position. Consequently Arg-363 could play a role in dATP binding. The crystal
structures do not provide any information on different interaction of residues
with the phosphates of dATP and dTTP. However, alignment of the residues in
the P-loops of different hexameric helicases reveals that the serine adjacent
to the invariant lysine at position 319 (Ser-319) is conserved in
bacteriophages, whereas bacterial helicases have a conserved threonine in the
equivalent position (supplemental Fig. 1). Bacterial helicases use rATP in the
DNA unwinding reactions. whereas T7 helicase preferentially uses dTTP, and
bacteriophage T4 gene 41 uses rGTP or rATP
(22).Although considerable information is available on the role of residues in
nucleotide binding and dTTP hydrolysis, very little is known on the
determinants of nucleotide specificity. In the present study we made an
attempt to address the role of a few selected residues (Arg-363, Arg-504, and
Ser-319) in determining nucleotide specificity, especially dTTP and dATP, both
of which are hydrolyzed and mediate DNA unwinding. We show that under
physiological conditions T7 helicase uses the energy derived from the
hydrolysis of dATP in addition to dTTP for mediating DNA unwinding. 相似文献
18.
19.
Homologous recombination is involved in the repair of DNA damage and collapsed replication fork, and is critical for the maintenance of genomic stability. Its process involves a network of proteins with different enzymatic activities. Human DNA helicase B (HDHB) is a robust 5′-3′ DNA helicase which accumulates on chromatin in cells exposed to DNA damage. HDHB facilitates cellular recovery from replication stress, but its role in DNA damage response remains unclear. Here we report that HDHB silencing results in reduced sister chromatid exchange, impaired homologous recombination repair, and delayed RPA late-stage foci formation induced by ionizing radiation. Ectopically expressed HDHB colocalizes with Rad51, Rad52, RPA, and ssDNA. In vitro, HDHB stimulates Rad51-mediated heteroduplex extension in 5′-3′ direction. A helicase-defective mutant HDHB failed to promote this reaction. Our studies implicate HDHB promotes homologous recombination in vivo and stimulates 5′-3′ heteroduplex extension during Rad51-mediated strand exchange in vitro. 相似文献
20.
Sabrina Kobbe Oliver Trapp Alexander Knoll Anja Manuss Holger Puchta 《Plant physiology》2015,169(4):2718-2729
DNA polymerase zeta catalytic subunit REV3 is known to play an important role in the repair of DNA damage induced by cross-linking and methylating agents. Here, we demonstrate that in Arabidopsis (Arabidopsis thaliana), the basic polymerase activity of REV3 is essential for resistance protection against these different types of damaging agents. Interestingly, its processivity is mainly required for resistance to interstrand and intrastrand cross-linking agents, but not alkylating agents. To better define the role of REV3 in relation to other key factors involved in DNA repair, we perform epistasis analysis and show that REV3-mediated resistance to DNA-damaging agents is independent of the replication damage checkpoint kinase ataxia telangiectasia-mutated and rad3-related homolog. REV3 cooperates with the endonuclease MMS and UV-sensitive protein81 in response to interstrand cross links and alkylated bases, whereas it acts independently of the ATP-dependent DNA helicase RECQ4A. Taken together, our data show that four DNA intrastrand cross-link subpathways exist in Arabidopsis, defined by ATP-dependent DNA Helicase RECQ4A, MMS and UV-sensitive protein81, REV3, and the ATPase Radiation Sensitive Protein 5A.The DNA of all living organisms is constantly exposed to damaging factors, and therefore a number of DNA damage repair and bypass mechanisms have evolved. DNA lesions that interfere with the replication machinery constitute a particular challenge for cells (Schröpfer et al., 2014a); if not repaired in a timely manner, such damage can result in the stalling or collapse of replication forks, which in turn can lead to cell death. Furthermore, one-sided double-strand breaks (DSBs) can occur when the replication fork encounters a single-strand break. Lesions within one DNA strand, such as alkylations or DNA intrastrand cross links, can be bypassed by postreplicative repair (PRR), a process that is best understood in yeast (Saccharomyces cerevisiae). This mechanism does not lead to repair of the lesion but prevents fatal long-lasting stalling of the replication fork. PRR can be divided into two branches: the error-prone pathway and the error-free pathway (for review, see Goodman and Woodgate, 2013; Haynes et al., 2015; Jansen et al., 2015). It is known from yeast that both branches of PRR are controlled by the Radiation sensitivity protein6 (Rad6) and Mms-Ubc13 E3 ubiquitin-conjugating enzyme complexes, which ubiquitinate the replicative processivity factor Proliferating Cellular Nuclear Antigen1. The monoubiquitination of PCNA at Lys-164 by Rad6-Rad18 initiates the error-prone pathway, whereas polyubiquitination additionally requires Mms2, Ubc13, and Rad5, and triggers the error-free PRR branch (Hoege et al., 2002; Moldovan et al., 2007; Lee and Myung, 2008). There are two possible competing models postulated for the error-free bypass of lesions at the replication fork, both of which depend on template-switch mechanisms; if the lesion concerns only one of the two sister strands, the undamaged strand can be used as the template for bypassing the lesion. One of the two models features the so-called overshoot synthesis, whereby the newly synthesized strand on the undamaged parental strand is elongated further than the strand blocked by the lesion. Regression of the replication fork then leads to the formation of a special type of four-way junction called a chicken-foot structure. This regression mechanism is thought to be accomplished by helicases, such as the RecQ helicase Bloom Syndrome Protein (BLM) in humans (Croteau et al., 2014). AtRECQ4A is the respective BLM homolog in Arabidopsis (Arabidopsis thaliana), and this enzyme has the ability to regress replication forks in vitro (Hartung et al., 2007, 2008; Schröpfer et al., 2014b). The second error-free subpathway entails invasion of the newly synthesized strand on the blocked sister chromatid into the complementary newly replicated strand on the other sister chromatid. Such a step forms a displacement loop-like structure in which synthesis over the damaged region can occur. In yeast, both error-free pathways are dependent on the multifunctional protein Rad5, which is known to recruit PRR factors and also exhibits helicase activity itself (Blastyák et al., 2007). We previously identified AtRAD5A as a functional Arabidopsis homolog of Rad5 (Chen et al., 2008). Interestingly, AtRAD5A is required for efficient repair by homologous recombination via the synthesis-dependent strand-annealing mechanism, a pathway that in some steps is related to the invasion model of PRR (Mannuss et al., 2010).The error-prone pathway is based on the function of translesion synthesis (TLS) polymerases, which promote replication through DNA lesions (Prakash et al., 2005). In a mechanism termed polymerase switch, the replicative polymerase is exchanged by such a TLS polymerase at a damaged site. After incorporation of a nucleotide opposite the damaged base by the TLS polymerase, a second polymerase switch exchanges the TLS polymerase for the replicative polymerase so that replication can proceed (Prakash and Prakash, 2002; Lehmann et al., 2007). TLS polymerases possess no 5′-3′-exonuclease activity, and therefore act in a potentially mutagenic manner. Nevertheless, depending on the damage incurred and the TLS polymerase used, damage bypass can be error free (Haracska et al., 2000; McCulloch et al., 2004).Polymerases can be divided into at least six families based on their amino acid sequences and crystal structures: A, B, C, D, X, and Y. All of them share the common structure analogous to a right hand grasping DNA with palm, finger, and thumb domains (Steitz, 1999). The amino acid sequences of the finger and thumb domains of different polymerase families are highly variable, whereas the palm domains share high similarity. The palm domain forms the largest part of the polymerase active site and contains highly conserved Asp residues that have been postulated to be involved in the catalytic activity of the enzyme (Joyce and Steitz, 1995; Steitz, 1999).Most TLS polymerases belong to the Y family of polymerases, a class of specially structured enzymes that catalyze replication over damaged templates (Ohmori et al., 2001; Sale et al., 2012). Although some polymerases of the A, B, or X family can also exhibit TLS activity, this is often not their primary function (Prakash et al., 2005). DNA Polymerase Zeta (POLζ) is a B family polymerase and consists of a DNA Polymerase Zeta subunit REV3-REV7 heterodimer, in which REV3 is the catalytic subunit with its accessory subunit and processivity factor REV7 (Nelson et al., 1996). Recent studies in yeast and human cells have shown that POLζ contains two additional subunits, Pol31 and Pol32 in yeast, orthologs to human POLD2 and POLD3, which are known to be accessory subunits of the replicative polymerase POLδ (Johnson et al., 2012; Lee et al., 2014). REV3 contains three regions that are highly conserved between organisms: an N-terminal region, a REV7 binding domain, and a B family-type polymerase domain. The polymerase domain carries the six common conserved regions, I to VI (IV-II-VI-III-I-V), of which I is the most and VI the least conserved region. The A (II), B (III), and C (I) motifs, located within regions I, II, and III (Wong et al., 1988), form the active site of the enzyme, and each harbors an essential Asp residue that coordinates two catalytic metal ions. Deficiency of REV3 in mice is embryo lethal (Bemark et al., 2000; Esposito et al., 2000), and vertebrate cells depleted in REV3 show hypersensitivity to various DNA-damaging agents, including UV and ionizing irradiation, cisplatin, MMS, and mitomycin C (MMC; Sonoda et al., 2003; Sharma and Canman, 2012). In Arabidopsis, rev3 mutants exhibit no obvious phenotype under standard growth conditions, but are hypersensitive to UV-B and gamma irradiation, MMC, MMS, and cisplatin (Sakamoto et al., 2003).Our previous work demonstrated the existence of several different pathways in Arabidopsis involved in repairing the DNA damage induced by cross-linking and methylating agents. These independent pathways are defined by the ATPase RAD5A, the helicase RECQ4A, and MMS and UV-sensitive protein81 (MUS81; Mannuss et al., 2010). The structure-specific endonuclease MUS81 together with its noncatalytic subunit (Mms4 in yeast, Eme1 in Schizosaccharomyces pombe, and MMS4 or Crossover Junction Endonuclease (EME1) in humans and plants) functions in the rescue of stalled replication forks. The enzyme is able to cleave the stalled fork at the lesion site, which leads to a one-sided DSB that is repaired by homologous recombination (HR) to restore the stalled fork (Hanada et al., 2006). We previously showed that mus81 transfer DNA (T-DNA) insertion lines in Arabidopsis are highly sensitive to treatment with MMS, cisplatin, hydroxyurea, ionizing irradiation, and MMC. We also found that AtMUS81 can form a complex with its heterologous binding partners AtEME1A or AtEME1B that is able to process intricate DNA structures, such as nicked Holliday junctions, which might also form at stalled replication forks (Hartung et al., 2006; Geuting et al., 2009).In the current study, we address whether fully functional polymerase activity is required for the repair of DNA damage induced by alkylating and cross-link-inducing agents. Moreover, we sought to clarify whether REV3 cooperates with other key factors identified in Arabidopsis in the repair of these different types of damage. Indeed, it has already been shown that AtREV3 and AtRAD5A do not cooperate in the repair of such DNA damage, confirming independent pathways of error-prone and error-free PRR in plants (Wang et al., 2011). However, as plants, animals, and yeast differ in their DNA cross-link repair machinery (e.g. Mannuss et al., 2010; Knoll et al., 2012; Dangel et al., 2014; Herrmann et al., 2015), it is of particular importance to define the role of REV3 in relation to MUS81 and RECQ4A. 相似文献