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Epstein–Barr virus (EBV) is a paradigm for human tumor viruses: it is the first virus recognized to cause cancer in people; it causes both lymphomas and carcinomas; yet these tumors arise infrequently given that most people in the world are infected with the virus. EBV is maintained extrachromosomally in infected normal and tumor cells. Eighty-four percent of these viral plasmids replicate each S phase, are licensed, require a single viral protein for their synthesis, and can use two functionally distinct origins of DNA replication, oriP, and Raji ori. Eighty-eight percent of newly synthesized plasmids are segregated faithfully to the daughter cells. Infectious viral particles are not synthesized under these conditions of latent infection. This plasmid replication is consistent with survival of EBV’s host cells. Rare cells in an infected population either spontaneously or following exogenous induction support EBV’s lytic cycle, which is lethal for the cell. In this case, the viral DNA replicates 100-fold or more, uses a third kind of viral origin of DNA replication, oriLyt, and many viral proteins. Here we shall describe the three modes of EBV’s replication as a function of the viral origins used and the viral and cellular proteins that mediate the DNA synthesis from these origins focusing, where practical, on recent advances in our understanding. 相似文献
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Laurie Rey Julia M. Sidorova Nadine Puget Fran?ois Boudsocq Denis S. F. Biard Raymond J. Monnat Jr. Christophe Cazaux Jean-Sébastien Hoffmann 《Molecular and cellular biology》2009,29(12):3344-3354
Human DNA polymerase η (Pol η) modulates susceptibility to skin cancer by promoting translesion DNA synthesis (TLS) past sunlight-induced cyclobutane pyrimidine dimers. Despite its well-established role in TLS synthesis, the role of Pol η in maintaining genome stability in the absence of external DNA damage has not been well explored. We show here that short hairpin RNA-mediated depletion of Pol η from undamaged human cells affects cell cycle progression and the rate of cell proliferation and results in increased spontaneous chromosome breaks and common fragile site expression with the activation of ATM-mediated DNA damage checkpoint signaling. These phenotypes were also observed in association with modified replication factory dynamics during S phase. In contrast to that seen in Pol η-depleted cells, none of these cellular or karyotypic defects were observed in cells depleted for Pol ι, the closest relative of Pol η. Our results identify a new role for Pol η in maintaining genomic stability during unperturbed S phase and challenge the idea that the sole functional role of Pol η in human cells is in TLS DNA damage tolerance and/or repair pathways following exogenous DNA damage.Mutations in the POLH gene that encodes DNA polymerase η (Pol η) are responsible for the variant form of xeroderma pigmentosum (XP-V). XP-V is a rare autosomal recessive disorder characterized by extreme sensitivity to sunlight and a very high incidence of sunlight-induced skin cancer, as are the other forms of “classical” XP (17, 27). However, in contrast to the other nucleotide excision repair (NER)-defective XP complementation groups (XP-A to XP-G), XP-V cells have normal NER but cannot support translesion synthesis (TLS) past DNA-containing cyclobutane pyrimidine dimers (CPDs) (27). Purified Pol η, the TLS polymerase that is mutated in XP-V, is able to synthesize past this lesion with a high level of efficiency (28), and in a majority of cases it inserts the correct nucleotide, adenine, opposite the two thymines contained in the cyclobutane pyrimidine dimer ring (26).The ability to replicate efficiently past UV pyrimidine dimers has been the principal—or sole—function assigned thus far to Pol η. In the absence of Pol η, cells display an increased rate of UV-induced mutagenesis and carcinogenesis (23) that may reflect inefficient or error-prone synthesis by another polymerase. In mouse cells, this back-up polymerase may be Pol ι (12). Despite its ability to replicate past cyclobutane pyrimidine dimers, Pol η does not appear to be able to carry out TLS past the other major UV photoproduct, the pyrimidine (6-4) pyrimidone photoproduct [(6-4)PP] in vitro or in vivo. It can, however, replicate past a limited number of other types of DNA damage in vitro, albeit with a lower level of efficiency than past CPDs (21). Whether the bypass of these lesions is performed in vivo by Pol η is less clear. For example, XP-V cells are sensitive to cisplatin, suggesting that bypass of cisplatin lesions may depend on Pol η (1). Combined NER- and Pol η-mediated lesion bypass has also been suggested as the likely mechanism for repairing DNA interstrand cross-links formed by mitomycin C (46) and psoralen (32). In contrast, Pol η does not appear to play a role in replication past endogenous lesions such as 8-oxoguanine (3) or abasic sites (2).It has been difficult to visualize or identify sites of action of Pol η or any of the other TLS polymerases by immunofluorescence due to their low levels of expression. However, in cells that mildly overexpress Pol η, it has been possible to localize the polymerase to nuclear replication factories during S phase. This localization depends on several motifs located close to the C terminus of Pol η, including an NLS and a ubiquitin-binding zinc finger domain (7, 18). Localization of Pol η in replication factories may concentrate the polymerase near sites of replication to facilitate recruitment to carry out TLS. If cells cannot remove or synthesize through a lesion blocking the replication fork, then homology-dependent recombinational repair (HRR) may be used to restart the replication fork (11, 34). RAD51-mediated HRR has been shown to be important for the repair of DNA damage during replication in all organisms (20, 31, 42). Recent evidence has suggested that Pol η, in addition to its role in TLS, may participate in HRR. This has been suggested by analyses of gene conversion in chicken DT40 cells during immunoglobulin gene diversification (19), as well as by in vitro experiments showing that Pol η is capable of promoting extension of the invading strand in D-loop structures to facilitate RAD52-mediated second-end capture during recombination-mediated repair (29, 30). The functional importance of this observation is less clear. Recent evidence from yeast argues that the bulk of heteroduplex DNA strand extension during HRR is mediated by the preferential recruitment of a replicative DNA polymerase, Pol δ (25). Moreover, there is no obvious recombination deficit in XP-V patients or in XP-V cells beyond a modest elevation in the frequency of UV-induced sister chromatid exchanges (10).In order to better understand the functional roles and importance of Pol η in human cells, we used short hairpin RNAs (shRNAs) to selectively deplete Pol η from cells and then determined how the loss of Pol η affected cell cycle progression, DNA replication dynamics, and cell proliferation in otherwise unperturbed cells. These experiments revealed an unexpected role for Pol η in maintaining chromosomal stability and preventing common fragile site (CFS) breakage during unperturbed S phase. Our results thus broaden the functional role of Pol η in human cells to include the maintenance of genomic stability during unperturbed DNA replication in S phase. 相似文献
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Histone methylation is widely believed to contribute to epigenetic inheritance by persevering through DNA replication and subsequently templating methylation of daughter chromosome regions. However, a report in this issue (Petruk et?al.) suggests that chromatin association of the methytransferase complexes themselves persists through replication and re-establishes histone methylation. 相似文献
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3'' -> 5'' Exonucleases of DNA Polymerases ε and δ Correct Base Analog Induced DNA Replication Errors on opposite DNA Strands in Saccharomyces Cerevisiae 下载免费PDF全文
The base analog 6-N-hydroxylaminopurine (HAP) induces bidirectional GC -> AT and AT -> GC transitions that are enhanced in DNA polymerase ε and δ 3' -> 5' exonuclease-deficient yeast mutants, pol2-4 and pol3-01, respectively. We have constructed a set of isogenic strains to determine whether the DNA polymerases δ and ε contribute equally to proofreading of replication errors provoked by HAP during leading and lagging strand DNA synthesis. Site-specific GC -> AT and AT -> GC transitions in a Pol(+), pol2-4 or pol3-01 genetic background were scored as reversions of ura3 missense alleles. At each site, reversion was increased in only one proofreading-deficient mutant, either pol2-4 or pol3-01, depending on the DNA strand in which HAP incorporation presumably occurred. Measurement of the HAP-induced reversion frequency of the ura3 alleles placed into chromosome III near to the defined active replication origin ARS306 in two orientations indicated that DNA polymerases ε and δ correct HAP-induced DNA replication errors on opposite DNA strands. 相似文献
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Giovanni Maga Barbara van Loon Emmanuele Crespan Giuseppe Villani Ulrich H��bscher 《The Journal of biological chemistry》2009,284(21):14267-14275
Abasic (AP) sites are very frequent and dangerous DNA lesions. Their
ability to block the advancement of a replication fork has been always viewed
as a consequence of their inhibitory effect on the DNA synthetic activity of
replicative DNA polymerases (DNA pols). Here we show that AP sites can also
affect the strand displacement activity of the lagging strand DNA pol δ,
thus preventing proper Okazaki fragment maturation. This block can be overcome
through a polymerase switch, involving the combined physical and functional
interaction of DNA pol β and Flap endonuclease 1. Our data identify a
previously unnoticed deleterious effect of the AP site lesion on normal cell
metabolism and suggest the existence of a novel repair pathway that might be
important in preventing replication fork stalling.Loss of purine and pyrimidine bases is a significant source of DNA damage
in prokaryotic and eukaryotic organisms. Abasic (apurinic and apyrimidinic)
lesions occur spontaneously in DNA; in eukaryotes it has been estimated that
about 104 depurination and 102 depyrimidation events
occur per genome per day. An equally important source of abasic DNA lesions
results from the action of DNA glycosylases, such as uracil glycosylase, which
excises uracil arising primarily from spontaneous deamination of cytosines
(1). Although most AP sites are
removed by the base excision repair
(BER)5 pathway, a
small fraction of lesions persists, and DNA with AP lesions presents a strong
block to DNA synthesis by replicative DNA polymerases (DNA pols)
(2,
3). Several studies have been
performed to address the effects of AP sites on the template DNA strand on the
synthetic activity of a variety of DNA pols. The major replicative enzyme of
eukaryotic cells, DNA pol δ, was shown to be able to bypass an AP
lesion, but only in the presence of the auxiliary factor proliferating cell
nuclear antigen (PCNA) and at a very reduced catalytic efficiency if compared
with an undamaged DNA template
(4). On the other hand, the
family X DNA pols β and λ were shown to bypass an AP site but in a
very mutagenic way (5). Recent
genetic evidence in Saccharomyces cerevisiae cells showed that DNA
pol δ is the enzyme replicating the lagging strand
(6). According to the current
model for Okazaki fragment synthesis
(7–9),
the action of DNA pol δ is not only critical for the extension of the
newly synthesized Okazaki fragment but also for the displacement of an RNA/DNA
segment of about 30 nucleotides on the pre-existing downstream Okazaki
fragment to create an intermediate Flap structure that is the target for the
subsequent action of the Dna2 endonuclease and the Flap endonuclease 1
(Fen-1). This process has the advantage of removing the entire RNA/DNA hybrid
fragment synthesized by the DNA pol α/primase, potentially containing
nucleotide misincorporations caused by the lack of a proofreading exonuclease
activity of DNA pol α/primase. This results in a more accurate copy
synthesized by DNA pol δ. The intrinsic strand displacement activity of
DNA pol δ, in conjunction with Fen-1, PCNA, and replication protein A
(RP-A), has been also proposed to be essential for the S phase-specific long
patch BER pathway (10,
11). Although it is clear that
an AP site on the template strand is a strong block for DNA pol
δ-dependent synthesis on single-stranded DNA, the functional
consequences of such a lesion on the ability of DNA pol δ to carry on
strand displacement synthesis have never been investigated so far. Given the
high frequency of spontaneous hydrolysis and/or cytidine deamination events,
any detrimental effect of an AP site on the strand displacement activity of
DNA pol δ might have important consequences both for lagging strand DNA
synthesis and for long patch BER. In this work, we addressed this issue by
constructing a series of synthetic gapped DNA templates with a single AP site
at different positions with respect to the downstream primer to be displaced
by DNA pol δ (see Fig.
1A). We show that an AP site immediately upstream of a
single- to double-strand DNA junction constitutes a strong block to the strand
displacement activity of DNA pol δ, even in the presence of RP-A and
PCNA. Such a block could be resolved only through a “polymerase
switch” involving the concerted physical and functional interaction of
DNA pol β and Fen-1. The closely related DNA pol λ could only
partially substitute for DNA pol β. Based on our data, we propose that
stalling of a replication fork by an AP site not only is a consequence of its
ability to inhibit nucleotide incorporation by the replicative DNA pols but
can also stem from its effects on strand displacement during Okazaki fragment
maturation. In summary, our data suggest the existence of a novel repair
pathway that might be important in preventing replication fork stalling and
identify a previously unnoticed deleterious effect of the AP site lesion on
normal cell metabolism.Open in a separate windowFIGURE 1.An abasic site immediately upstream of a double-stranded DNA region
inhibits the strand displacement activity of DNA polymerase δ. The
reactions were performed as described under “Experimental
Procedures.” A, schematic representation of the various DNA
templates used. The size of the resulting gaps is indicated in nt. The
position of the AP site on the 100-mer template strand is indicated relative
to the 3′ end. Base pairs in the vicinity of the lesion are indicated by
dashes. The size of the gaps (35–38 nt) is consistent with the
size of ssDNA covered by a single RP-A molecule, which has to be released
during Okazaki fragment synthesis when the DNA pol is approaching the
5′-end of the downstream fragment. When the AP site is covered by the
downstream terminator oligonucleotide (Gap-3 and Gap-1 templates) the
nucleotide placed on the opposite strand is C to mimic the situation generated
by spontaneous loss of a guanine or excision of an oxidized guanine, whereas
when the AP site is covered by the primer (nicked AP template), the nucleotide
placed on the opposite strand is A to mimic the most frequent incorporation
event occurring opposite an AP site. B, human PCNA was titrated in
the presence of 15 nm (lanes 2–4 and
10–12) or 30 nm (lanes 6–8 and
14–16) recombinant human four subunit DNA pol δ, on a
linear control (lanes 1–8) or a 38-nt gap control (lanes
9–16) template. Lanes 1, 5, 9, and 13, control
reactions in the absence of PCNA. C, human PCNA was titrated in the
presence of 60 nm DNA pol δ, on a linear AP (lanes
2–4) or 38-nt gap AP (lanes 6–9) template. Lanes
1 and 5, control reactions in the absence of PCNA. 相似文献
11.
Impeded DNA replication or a deficiency of its control may critically threaten the genetic information of cells, possibly resulting in genome alterations, such as gross chromosomal translocations, microsatellite instabilities, or increased rates of homologous recombination (HR). We examined an Arabidopsis thaliana line derived from a forward genetic screen, which exhibits an elevated frequency of somatic HR. These HR events originate from replication stress in endoreduplicating cells caused by reduced expression of the gene coding for the catalytic subunit of the DNA polymerase δ (POLδ1). The analysis of recombination types induced by diverse alleles of polδ1 and by replication inhibitors allows the conclusion that two not mutually exclusive mechanisms lead to the generation of recombinogenic breaks at replication forks. In plants with weak polδ1 alleles, we observe genome instabilities predominantly at sites with inverted repeats, suggesting the formation and processing of aberrant secondary DNA structures as a result of the accumulation of unreplicated DNA. Stalled and collapsed replication forks account for the more drastic enhancement of HR in plants with strong polδ1 mutant alleles. Our data suggest that efficient progression of DNA replication, foremost on the lagging strand, relies on the physiological level of the polymerase δ complex and that even a minor disturbance of the replication process critically threatens genomic integrity of Arabidopsis cells. 相似文献
12.
A key set of reactions for the initiation of new DNA strands during herpes
simplex virus-1 replication consists of the primase-catalyzed synthesis of
short RNA primers followed by polymerase-catalyzed DNA synthesis
(i.e. primase-coupled polymerase activity). Herpes primase
(UL5-UL52-UL8) synthesizes products from 2 to ∼13 nucleotides long.
However, the herpes polymerase (UL30 or UL30-UL42) only elongates those at
least 8 nucleotides long. Surprisingly, coupled activity was remarkably
inefficient, even considering only those primers at least 8 nucleotides long,
and herpes polymerase typically elongated <2% of the primase-synthesized
primers. Of those primers elongated, only 4–26% of the primers were
passed directly from the primase to the polymerase (UL30-UL42) without
dissociating into solution. Comparing RNA primer-templates and DNA
primer-templates of identical sequence showed that herpes polymerase greatly
preferred to elongate the DNA primer by 650–26,000-fold, thus accounting
for the extremely low efficiency with which herpes polymerase elongated
primase-synthesized primers. Curiously, one of the DNA polymerases of the host
cell, polymerase α (p70-p180 or p49-p58-p70-p180 complex), extended
herpes primase-synthesized RNA primers much more efficiently than the viral
polymerase, raising the possibility that the viral polymerase may not be the
only one involved in herpes DNA replication.Herpes simplex virus 1
(HSV-1)2 encodes seven
proteins essential for replicating its double-stranded DNA genome; five of
these encode the heterotrimeric helicase-primase (UL5-UL52-UL8 gene products)
and the heterodimeric polymerase (UL30-UL42 gene products)
(1,
2). The helicase-primase
unwinds the DNA at the replication fork and generates single-stranded DNA for
both leading and lagging strand synthesis. Primase synthesizes short RNA
primers on the lagging strand that the polymerase presumably elongates using
dNTPs (i.e. primase-coupled polymerase activity). These two protein
complexes are thought to replicate the viral genome on both the leading and
lagging strands (1,
2).Previous studies have focused on the helicase-primase and polymerase
separately. The helicase-primase contains three subunits, UL5, UL52, and UL8
in a 1:1:1 ratio
(3–5).
The UL5 subunit has helicase-like motifs and the UL52 subunit has primase-like
motifs, yet the minimal active complex that demonstrates either helicase or
primase activities contains both UL5 and UL52
(6,
7). Although the UL8 subunit
has no known catalytic activity, several functions have been proposed,
including enhancing helicase and primase activities, enhancing primer
synthesis on ICP8 (the HSV-1 single-stranded binding protein)-coated DNA
strands, and facilitating formation of the replisome
(8–12).
Although primase will synthesize short
(2–3
nucleotides long) primers on a variety of template sequences, synthesis of
longer primers up to 13 nucleotides long requires the template sequence,
3′-deoxyguanidine-pyrimidine-pyrimidine-5′
(13). Primase initiates
synthesis at the first pyrimidine via the polymerization of two purine NTPs
(13). Even after initiation at
this sequence, however, the vast majority of products are only 2–3
nucleotides long (13,
14).The herpes polymerase consists of the UL30 subunit, which has polymerase
and 3′ → 5′ exonuclease activities
(1,
2), and the UL42 subunit, which
serves as a processivity factor
(15–17).
Unlike most processivity factors that encircle the DNA, the UL42 protein binds
double-stranded DNA and thus directly tethers the polymerase to the DNA
(18). Using pre-existing DNA
primer-templates as the substrate, the heterodimeric polymerase (UL30-UL42)
incorporates dNTPs at a rate of 150 s–1, a rate much faster
than primer synthesis (for primers >7 nucleotides long, 0.0002–0.01
s–1) (19,
20).We examined primase-coupled polymerase activity by the herpes primase and
polymerase complexes. Although herpes primase synthesizes RNA primers
2–13 nucleotides long, the polymerase only effectively elongates those
at least 8 nucleotides long. Surprisingly, the polymerase elongated only a
small fraction of the primase-synthesized primers (<1–2%), likely
because of the polymerase elongating RNA primer-templates much less
efficiently than DNA primer-templates. In contrast, human DNA polymerase
α (pol α) elongated the herpes primase-synthesized primers very
efficiently. The biological significance of these data is discussed. 相似文献
13.
14.
15.
Cell death can be divided into the anti-inflammatory process of apoptosis and the
pro-inflammatory process of necrosis. Necrosis, as apoptosis, is a regulated form of cell
death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP)
1/3 are major mediators. We previously showed that absence or inhibition of PARP-1
protects mice from nephritis, however only the male mice. We therefore hypothesized that
there is an inherent difference in the cell death program between the sexes. We show here
that in an immune-mediated nephritis model, female mice show increased apoptosis compared
to male mice. Treatment of the male mice with estrogens induced apoptosis to levels
similar to that in female mice and inhibited necrosis. Although PARP-1 was activated in
both male and female mice, PARP-1 inhibition reduced necrosis only in the male mice. We
also show that deletion of RIP-3 did not have a sex bias. We demonstrate here that male
and female mice are prone to different types of cell death. Our data also suggest that
estrogens and PARP-1 are two of the mediators of the sex-bias in cell death. We therefore
propose that targeting cell death based on sex will lead to tailored and better treatments
for each gender. 相似文献
16.
Michael W. Schmitt Ranga N. Venkatesan Marie-Jeanne Pillaire Jean-Sébastien Hoffmann Julia M. Sidorova Lawrence A. Loeb 《The Journal of biological chemistry》2010,285(42):32264-32272
DNA polymerase δ (pol δ) is one of the two main replicative polymerases in eukaryotes; it synthesizes the lagging DNA strand and also functions in DNA repair. In previous work, we demonstrated that heterozygous expression of the pol δ L604G variant in mice results in normal life span and no apparent phenotype, whereas a different substitution at the same position, L604K, is associated with shortened life span and accelerated carcinogenesis. Here, we report in vitro analysis of the homologous mutations at position Leu-606 in human pol δ. Four-subunit human pol δ variants that harbor or lack 3′ → 5′-exonucleolytic proofreading activity were purified from Escherichia coli. The pol δ L606G and L606K holoenzymes retain catalytic activity and processivity similar to that of wild type pol δ. pol δ L606G is highly error prone, incorporating single noncomplementary nucleotides at a high frequency during DNA synthesis, whereas pol δ L606K is extremely accurate, with a higher fidelity of single nucleotide incorporation by the active site than that of wild type pol δ. However, pol δ L606K is impaired in the bypass of DNA adducts, and the homologous variant in mouse embryonic fibroblasts results in a decreased rate of replication fork progression in vivo. These results indicate that different substitutions at a single active site residue in a eukaryotic polymerase can either increase or decrease the accuracy of synthesis relative to wild type and suggest that enhanced fidelity of base selection by a polymerase active site can result in impaired lesion bypass and delayed replication fork progression. 相似文献
17.
DNA polymerase η (pol η) synthesizes across from damaged DNA templates in order to prevent deleterious consequences like replication fork collapse and double-strand breaks. This process, termed translesion synthesis (TLS), is an overall positive for the cell, as cells deficient in pol η display higher mutation rates. This outcome occurs despite the fact that the in vitro fidelity of bypass by pol η alone is moderate to low, depending on the lesion being copied. One possible means of increasing the fidelity of pol η is interaction with replication accessory proteins present at the replication fork. We have previously utilized a bacteriophage based screening system to measure the fidelity of bypass using purified proteins. Here we report on the fidelity effects of a single stranded binding protein, replication protein A (RPA), when copying the oxidative lesion 7,8-dihydro-8-oxo-guanine(8-oxoG) and the UV-induced cis-syn thymine-thymine cyclobutane pyrimidine dimer (T-T CPD). We observed no change in fidelity dependent on RPA when copying these damaged templates. This result is consistent in multiple position contexts. We previously identified single amino acid substitution mutants of pol η that have specific effects on fidelity when copying both damaged and undamaged templates. In order to confirm our results, we examined the Q38A and Y52E mutants in the same full-length construct. We again observed no difference when RPA was added to the bypass reaction, with the mutant forms of pol η displaying similar fidelity regardless of RPA status. We do, however, observe some slight effects when copying undamaged DNA, similar to those we have described previously. Our results indicate that RPA by itself does not affect pol η dependent lesion bypass fidelity when copying either 8-oxoG or T-T CPD lesions. 相似文献
18.
19.
20.
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