From the
Nuclear DNA helicase II (NDH II) unwinds both DNA and RNA
(Zhang, S., and Grosse, F.(1994) Biochemistry 33,
3906-3912). Here, we report on the molecular cloning and sequence
determination of the complementary DNA (cDNA) coding for this DNA and
RNA helicase. The full-length cDNA sequence was derived from
overlapping clones that were detected by immunoscreening of a calf
thymus cDNA library in bacteriophage
Nucleic acid helicases unwind double-stranded DNA and RNA
coupled with the hydrolysis of a nucleoside 5`-triphosphate for energy
supply. Numerous processes of nucleic acid metabolism including DNA
replication, DNA repair, DNA recombination, transcription, RNA
splicing, and translation are aided by DNA and/or RNA helicases. These
enzymes play a key role in resolving secondary structures to expose
single-stranded templates for various DNA or RNA polymerases to copy
on, to facilitate the binding of sequence-specific regulatory proteins,
to reorganize the nucleoprotein complex, and to mediate sequential dis-
and reassembly of higher order nucleic acid structures for the
expression of their destined functions (for reviews, see Refs.
1-4). On the basis of computer-assisted amino acid comparisons, a
so called superfamily of helicases has been found that contains common
helicase domains, including a nucleotide-binding motif(5) . The
members of this protein family can be subdivided into superfamily I and
superfamily II according to their different sequence
signatures(5) . A DEAD/H motif found in domain II defines an
expanding group of DNA and RNA helicases from superfamily
II(6, 7, 8) , which include proteins with
important regulatory functions in several pathways of genome
expression.
We have recently purified and characterized two DNA
helicases from calf thymus nuclei, designated as nuclear DNA helicase I
(NDH I)
The 5`-terminal sequence of NDH II
cDNA was obtained by using a 5`-anchored PCR method(16) . 1
µg of calf thymus total RNA, prepared by the guanidinium
isothiocyanate/acidic phenol method(17) , was reverse
transcribed with avian myeloblastosis virus reverse transcriptase (Life
Technologies, Inc.) and the 21-mer antisense primer A1
(5`-GCCTTAGCATTTTCCAAGGTC-3`, see Fig. 1) that was complementary
to a region at 460 bp downstream of the 5`-end of clone 6. After
removal of excess primer by GlassMAX spin cartridge (Life Technologies,
Inc.), the first-strand cDNA was tailed with dGTP in a terminal
transferase-catalyzed reaction(18) . The 5`-anchored PCR product
was obtained by amplifications with two anchor primers, ANpolyC
(5`-GCATGCGCGCGGCCGCGGAGGCCCCCCCCCCCCCC-3`) and AN
(5`-GCATGCGCGCGGCCGCGGAGGCC-3`) at a ratio of 1:9, and the second
internal cDNA primer A2 (5`-GGGTCGACTTTCAAGGGTCGCTTGGACTT-3`) (Fig. 1) containing a SacII and a SalI
restriction site (underlined), respectively. Southern blot
hybridizations were performed to examine the successful synthesis of
first-round PCR products with the third internal primer A3
(5`-ATATCCTTCGACGCGAACCT-3`) that was 5`-labeled with
[
Most significantly, NDH II
contains a glycine-rich region of 121 amino acids at the very C
terminus (Fig. 3), comprising 16 imperfect amino acid repeats
arranged as GGG(G/D)(Y/V)(G/S/V/R)GG. The glycine-rich domain forms
five so called ``RGG'' boxes that are thought to be involved
in RNA binding(28, 29, 30) . Similar
glycine-rich arrangements have been found in many other proteins that
interact with nucleic acids, including ribonuclear particle core
proteins A1 (31) and U(28) , the chromatin assembly
factor nucleolin(32) , and the Epstein-Barr virus nuclear
antigen 1(30) . The multitude of glycine residues may allow to
form flexible loop structures; the regularly interspersed amino acids
tyrosine/phenylalanine and the positively charged amino acid arginine
could provide additional affinity to nucleic acids. Interestingly, the
glycine-rich sequence is not stringently conserved within the three Mle
proteins. For Drosophila Mle, the numbers of repeats vary
between different Mle wild type forms(13) . Although bovine NDH
II is strongly homologous to human RNA helicase A, it diverges
considerably at the glycine-rich C terminus(11) . Obviously,
some deletion mutations must have occurred, which led to different
reading frames for the C termini of NDH II and RNA helicase A.
Surprisingly, the C-terminal region of bovine NDH II is more similar to
the corresponding region of the Drosophila Mle protein than to
that of human RNA helicase II.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Jerard Hurwitz (Memorial Sloan-Kettering
Cancer Center, Sloan-Kettering Institute, New York) for the kind gift
of anti-human RNA helicase A antibodies. We also thank Torsten
Mummenbrauer (The Heinrich-Pette-Institut (HPI, Hamburg), which is
financially supported by the Freie und Hansestadt Hamburg and
Bundesministerium für Gesundheit) for help with the
computer-assisted sequence comparisons, and Wolfgang Deppert (HPI) for
critically reading the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
gt11. This cDNA was 4,528
bases in length, which corresponded well with a
4.5-4.7-kilobase-long mRNA as detected by Northern blot analysis.
The open reading frame of NDH II cDNA predicts a polypeptide of 1287
amino acids and a calculated molecular mass of 141,854 daltons. NDH II
is related to a group of nucleic acid helicases from the DEAD/H box
family II, with the signature motif DEIH in domain II. Two further
proteins of this family, i.e. human RNA helicase A and Drosophila Maleless (Mle) protein, were found to be highly
homologous to NDH II. With RNA helicase A, there was 91.5% identity and
95.5% similarity between the amino acid residues; with Mle protein, we
observed a 50% identity and an 85% similarity. Antibodies against human
RNA helicase A cross-reacted with NDH II, further supporting that NDH
II is the bovine homologue of human RNA helicase A. Immunofluorescence
studies revealed a mainly nuclear localization of NDH II. A role for
NDH II in nuclear DNA and RNA metabolism is suggested.
(
)and nuclear DNA helicase II (NDH II)
(9). Homogeneous NDH II displays a rather high molecular mass of around
130-140 kDa. While this enzyme was initially identified as DNA
helicase, it was subsequently shown to be associated with an
RNA-dependent NTPase and to be able to unwind double-stranded
RNA(10) . Here, we report on the sequence of the NDH II-encoding
cDNA. The derived amino acid sequence of bovine NDH II turned out to be
91.5% identical and 95.5% similar to that of human RNA helicase
A(11) , an enzyme previously identified as RNA helicase
only(12) . Furthermore, there was a 50% amino acid identity, and
a 85% similarity with the Maleless protein (Mle) of Drosophila(13) , suggesting that NDH II is the bovine
homologue of these two proteins. Combining data from sequence analysis
and biochemical properties, Mle proteins might be considered as DNA and
RNA helicases that are required for the regulation of both DNA and RNA
secondary structure in gene expression and perhaps also in DNA
replication.
Polyclonal Antibodies against NDH
II
400 µg of pure NDH II was injected subcutaneously
into a rabbit. After 2 weeks, a second injection was performed with 200
µg of protein, followed by two further injections of 200 µg
(each) after another 4 and 8 weeks. Rabbit antiserum was collected
7-10 days after each injection. Quantity and specificity of the
antibodies were examined by dot-blot assays. Serum harvested after the
fourth immunization (10 weeks after the first injection) was used for
library screening, Western blotting, and immunofluorescence studies.
Isolation and Sequencing of cDNA Clones Encoding NDH
II
An oligo(dT)-primed gt11 library, constructed from
calf thymus poly(A)-containing RNA, was purchased from Clontech. About
2
10
recombinant phages were screened with the
antibodies described above by following standard procedures(4) .
As many as 40 colonies producing immunopositive signals were identified
after the initial round of library screening. Thereof, 20 colonies were
selected for further purification by two successive rounds of antibody
screening. Insert sizes of positive clones were determined by PCR using
5`-GGTGGCGACGACTCCTGGAGCCCG-3` as forward primer and
5`-TTGACACCAGACCAACTGGTAATG-3` (flanking the EcoRI site of the
gt11 vector) as reverse primer(15) . Clone 6, containing
2168 bp, and clone 12, containing 1419 bp (Fig. 1), were selected
and subcloned into the EcoRI-digested vector M13 mp18.
Nucleotide sequences of the cDNA inserts on both strands from the M13
mp18 vector were determined by Sanger's dideoxy
method(14) . 7-Deaza-dGTP (Boehringer Mannheim) was used to
eliminate band compression effects.
Figure 1:
Strategy for cloning the cDNA of NDH
II. The relative positions of the cDNA subclones, PCR primers, and the
PCR probe are indicated. For more details, see
text.
The remaining immunopositive
clones were PCR-screened by using the 24-mer oligonucleotide 12-F-6 (Fig. 1) as forward primer and a reverse primer from gt11.
By using this method, clone 7 was found to extend most to the C
terminus. The 3.3-kb insert of clone 7 was subcloned into M13 mp18 and
sequenced (Fig. 1). From this a coding length containing 110
amino acids downstream of clone 6 cDNA was obtained. Because we did not
obtain full-length cDNA by antibody screening, we rescreened the cDNA
library with a PCR probe (300 bp in length) that was synthesized from a
downstream part of clone 7 (Fig. 1). About 3
10
phages were screened with the randomly labeled PCR probe. Nucleic
acid hybridization and detection of the clones were achieved by
standard procedures(14) . In total, 20 positive clones were
obtained by using this method. To identify the clones covering the
missing sequence at the C terminus of the reading frame, PCR
characterization of cDNA inserts was performed as described above,
using the insert-specific primer 7-F-2 (Fig. 1), combined with
one of the
gt11 vector forward and reverse primers, to account for
cases where cDNA inserts might have been oriented in both directions.
In this way, two clones, clone 4 (1450 bp) and clone 10 (2150 bp), both
of which provided the longest extensions over the 3` terminus of clone
7, were subcloned and sequenced.
-
P]ATP. Hybridized DNA bands were
identified on 1% agarose gels at lengths between 650 and 700 bp.
1-5 µl of agarose, containing DNA bands of this length, were
transferred to a PCR reaction mixture for the second round of
amplification, again with primers AN and A2. The second round PCR
products were sequentially digested by SacII and SalI
and were subsequently cloned into the plasmid vector Bluescript
SK+ II and transformed into the Escherichia coli strain
XL-Blue (Stratagene). Colonies carrying NDH II-5`-anchored inserts were
screened with the radiolabeled primer A3 (see above). Plasmid DNA was
prepared by alkaline lysis followed by ultracentrifugation through a
CsCl gradient(14) . Purified plasmid DNA was denatured by alkali
and sequenced in both directions with M13 universal primers and
sequence-specific primers.
Northern Blot Analysis
Enrichment of calf
thymus poly(A) RNA from total RNA was achieved by
using oligotex-dT mRNA spin columns obtained from Qiagen. 2.5 µg of
poly(A)
RNA was electrophoresed in a 1.2% agarose gel
containing 2.2 M formaldehyde and transferred onto a Nitran
membrane (Schleicher & Schuell). The membrane was baked at 80
°C under vacuum for 2 h. Prehybridization was performed by
incubating the membrane in a roller bottle with 5 ml of 5
SSC
(1
SSC contained 0.15 M NaCl, 0.015 M sodium
citrate), 2
Denhardt's solution (1
Denhardt's solution contained 0.02% Ficoll, 0.02%
polyvinylpyrrolidone, 0.02% bovine serum albumin), 0.1% SDS, and 100
µg of salmon sperm DNA per ml for 2 h at 65 °C. Hybridization
was performed by incubation for 16-18 h at 65 °C with an NDH
II-specific PCR probe. The probe, spanning clone 6 from nucleotides
1117 to 2012, was labeled to a specific radioactivity of
1
10
cpm/ml by using random primers and
[
-
P]dCTP. Following hybridization, the
membrane was washed twice for 15 min each at room temperature in 2
SSC and 0.1% SDS, followed by washing for 15 min at 65 °C
in 1
SSC and 0.1% SDS and 30 min at 65 °C in 0.1
SSC and 0.1% SDS. Autoradiography was performed at -80 °C for
3 weeks.
Western Blot Analysis
Calf thymus nuclear
extract was prepared as described(9) . 10 µl of nuclear
lysate at a concentration of 9.45 mg of protein/ml and 0.5 µg of
pure NDH II were electrophoresed through a 7.5% SDS-polyacrylamide gel (19) and transferred onto a Hybond-C extra membrane (Amersham
Corp.) by using a semi-dry electroblotter. The membrane was incubated
with rabbit polyclonal antibodies against NDH II and human RNA helicase
A (kindly donated by Dr. J. Hurwitz, Memorial Sloan-Kettering Cancer
Center, NY) at a dilution of 1 to 1000. The primary antigen-antibody
complexes were visualized by an enhanced chemiluminescent (ECL)
immunodetection procedure as recommended by the manufacturer
(Amersham).
Subcellular Localization of NDH II by
Immunofluorescence Microscopy
Monkey TC-7 kidney cells were
adhesively grown to subconfluence on coverslips within cell dishes. The
coverslips were removed and rinsed for several times with PBS (PBS
contained 10 mM sodium phosphate, pH 7.5, 140 mM NaCl, 3 mM KCl), air dried, and then fixed with acetone
for 10 min at room temperature. Following removal of residual acetone
by air drying, the cells were briefly washed with PBS. Acetone-fixed
cells were incubated for 1 h with anti-NDH II serum at room
temperature. The optimal dilution of antiserum was experimentally
determined as 1:700 in PBS. After several washes with PBS, the cells
were incubated for another 1 h at room temperature with biotinylated
anti-rabbit IgG antibody (Amersham), diluted 1:200 with PBS. The cells
were washed and subsequently stained with fluorescence dye-conjugated
avidin (Cy3-conjugated avidin; Dianova, Hamburg) that was diluted 1:400
in PBS. Finally, the cells were washed and prepared for microscopy by
adding a drop of mounting medium (10% glycerol, 5% polyvinyl alcohol in
PBS) and topping them with a thin coverslip. The cells were viewed with
a Zeiss fluorescence microscope. Antibody-depleted control serum was
prepared by incubating 1:700-diluted anti-NDH II serum with an excess
of purified NDH II.
Cloning and Sequencing NDH II-encoding
cDNA
The total cDNA sequence of NDH II was obtained by
immunoscreening of a gt11 cDNA library of calf thymus
poly(A)
mRNA; the very 5` and 3` termini of the cDNA
were determined by using PCR approaches. Combining the nucleotide
sequences from several overlapping clones resulted in a cDNA of 4528 bp
in length (Fig. 1). Between the 141-bp-long 5`-untranslated
region and the 523-bp-long 3`-untranslated region, NDH II-encoding cDNA
contains an open reading frame of 3864 bp. The open reading frame
starts with the initiation codon ATG at nucleotides 142-144 and
ends with the stop codon TAA at nucleotides 4003-4005. It encodes
a polypeptide of 1287 amino acids and a predicted molecular mass of
141,854 daltons. A purine-rich, putative ribosomal binding site
(5`-GAAGAAGA-3`) could be identified at the 5`-untranslated region
prior to the initiation codon at nucleotides 124-131; four
polyadenylation signals (5`-AATAAA-3`) were found downstream of the
stop codon at nucleotide positions 4042-4047, 4192-4197,
4290-4295, and 4457-4462.
NDH II Is the Bovine Homologue of Human RNA Helicase
A and Drosophila Maleless Protein
The predicted protein
sequence of NDH II was used for searching similar sequences in several
data bases, such as the SwissProt, the PIR, and the GenBank/EMBL data
base, by using the FASTA computer program. The NDH II protein sequence
displayed high scoring indexes with two proteins, namely human RNA
helicase A (11) and Drosophila Maleless protein
(Mle)(13) . With RNA helicase A there was a 91.5% identity and
95.5% similarity between the amino acid residues (Fig. 2A); with Mle protein we observed a 50% identity
and an 85% similarity (Fig. 2B). All three proteins
belong to superfamily II of DNA and RNA
helicases(5, 6, 7, 8) . Members of this
family contain six highly conserved amino acid domains and the
signature motif DEXH. Bovine NDH II contains the conserved
domains within 353 amino acids spanning from residue 411 to 764 (Fig. 3). Here, domain I contains the putative Walker-type
nucleotide binding site(20) , consisting of motif A as GCGKT and
motif B as FILDD (Fig. 3). In domain II, all three helicases show
the DEXH signature motif as DEIH (Fig. 3). In domain VI,
the motif QRKGRAGR is strongly conserved between NDH II, Mle, and RNA
helicases A (Fig. 3) as well as the vaccinia virus-encoded RNA
helicase nucleic acid-dependent phosphohydrolase II(21) . This
so-called HRIGRXXR region has been shown to be involved in RNA
binding and RNA-dependent ATP hydrolysis of the eukaryotic translation
initiation factor eIF-4A (22). Within the six conserved domains, NDH II
displays weak but still detectable similarities to three putative RNA
helicases from yeast, i.e. the splicing factors
PRP2(23) , PRP16(24) , and PRP22 (25).
Figure 2:
Protein
matrix comparisons between NDH II and RNA helicase A (A) and
between NDH II and Mle protein (B). The comparison was made by
using DNA Strider software (38) with a window of 23 and a stringency of
7 amino acids. The identity and similarity between NDH II and RNA
helicase A was 91.5 and 95.5%, respectively, and that between NDH II
and Mle protein was 50 and 85%, respectively. Dots in the lowerrightcorner of each plot represent
the 16 imperfect amino acid repeats
GGG(G/D)(Y/V)(G/S/V/R)GG.
Figure 3:
Schematic representation of NDH II. A, depicted are sequence motifs that have been also found in
other proteins. dsRBD I and dsRBD II represent double
strand RNA binding domains I and II, NTP represents the
Walker-type nucleotide binding domain, DEIH is the so called
DEAD/H box motif of helicase superfamily II, NLS stands for a
putative nuclear localization sequence, NA bdg. represents a
conserved nucleic acid binding domain, and G-rich mirrors the location
of the glycine-rich C-terminal repeats. Furthermore, the relative
positions of the six conserved domains common to helicases are
indicated by Romannumerals. B, direct amino
acid comparison of the six highly conserved helicase regions between
NDH II, RNA helicase A (HelA), and Mle protein. Only differences to the
NDH II sequence are indicated; dashes represent identical
amino acids. Underlined are the NTP binding domains A and B in
motif Ia, the DEIH box in motif II, and the conserved nucleic acid
binding domain in motif VI.
In addition to
the conserved domains, there are further non-canonical helicase motifs
present in NDH II. There is a (presumed) nuclear localization sequence,
PPPKDKKKD, that is highly conserved between helicase A and NDH II (Fig. 3), while in Mle a comparable lysine-rich motif is found
closer toward the N terminus. There are also two putative
double-stranded RNA binding domains, named dsRBD I and dsRBD
II(26) , at the N-terminal region of NDH II (Fig. 3).
Interestingly, the interferon -induced double-stranded
RNA-activated protein kinase DAI, which like NDH II has a strong
affinity to poly(rI
rC), bears similarity to NDH II regarding both
dsRBD domains(27) . Therefore, the dsRBD regions might
contribute to poly(rI
rC) binding.
NDH II Is Encoded by a 4.5-4.7-kb-long
mRNA
Both the occurrence of two protein bands in highly
purified preparations of NDH II (9) and the observation of
untranslatable sequences in several cDNA clones (see
``Experimental Procedures'') suggested that there might have
been alternative splicing events at the level of pre-mRNA. Therefore,
we analyzed thymus and several other tissues for the presence, the
amounts, and sizes of the corresponding mRNAs. Direct hybridization to
total RNA of crude cellular extracts gave no positive signals,
suggesting that there was only very little NDH II-encoding mRNA
present. To increase the signal to noise ratio, we utilized
poly(A) RNA from calf thymus as target for Northern
blot analysis and a 895-bp-long PCR product obtained from clone 6 as
hybridization probe. This probe detected an mRNA transcript migrating
at about 4.5-4.7 kb (Fig. 4), a position that was expected
from cDNA cloning. With another cDNA probe that contained the
glycine-rich region (587 bp from nucleotides 3732 to 4319), again only
the 4.5-4.7-kb-long mRNA was detected (data not shown). Visible
hybridization signals were only obtained at exposition times of 3 weeks
or longer and when more than 2.5 µg of poly(rA)
RNA was loaded onto the gel.
Figure 4:
Northern blot analysis of
poly(A)-containing RNA. Calf thymus poly(A)-containing RNA (2.5 µg)
was subjected to the Northern blot analysis, as described under
``Experimental Procedures.'' The size of the RNA for NDH II
was estimated to be about 4.5-4.7 kb in length based on its
migration to marker RNAs of known length (leftside).
Exposure time was 21 days.
NDH II Has a Molecular Mass of 140 kDa in
Vivo
In contrast to the low abundant mRNA, NDH II protein
could be isolated in milligram amounts from a kilogram of calf thymus.
Highly purified preparations of this protein displayed two polypeptides
with molecular masses of 130 and 100 kDa(9) . To determine the
native form of NDH II in living cells, we undertook a Western blot
analysis of calf thymus nuclear extracts with the same anti-NDH II
serum used for cDNA screening. Purified NDH II, consisting of p130 and
p100, served as control. In nuclear extracts, there was only one
protein band with a molecular mass of around 140 kDa (Fig. 5).
This molecular weight is in agreement with the molecular weight
predicted from the cDNA and strongly suggests that NDH II becomes
degraded to p130 and p100 during purification. A similar observation
has been made with hnRNP A1(31) : the earlier purified
``unwinding protein 1'' (UP1) (33) was subsequently
shown to be a degradation product of hnRNP A1 that arose by deletion of
the C-terminal glycine-rich domain(31, 34) .
Figure 5:
Western blot analysis of nuclear extracts
of calf thymus. Nuclear extract prepared from calf thymus (lanes1 and 3) and purified NDH II (lanes2 and 4) were subjected to the immunoblot
analysis using anti-NDH II antibodies (lanes1 and 2) or anti-RNA helicase A antibodies (lanes3 and 4) at a dilution of 1 to 1000 each. The primary
antigen-antibody complexes were visualized by an ECL immunodetection
procedure using the secondary anti-rabbit IgG antibody at a 1 to 5000
dilution.
Cross-reactivity of Bovine NDH II with Antibodies
against Human RNA Helicase A
Since there is a 91.5% amino
acid identity among bovine NDH II and human RNA helicase A, we expected
a high cross-reactivity with antibodies derived from protein of either
species. Antibodies elicited against human RNA helicase A were used to
probe the same amount of nuclear extract and purified NDH II on
one-half of the membrane shown in Fig. 5. With the same dilution
of antibodies, equal intensities of immunostaining were observed as
with the antibody directed against the bovine enzyme (Fig. 5).
The strong cross-reactivity confirmed the expected homology and,
moreover, provided strong evidence for the authenticity of the cDNA
clones.
Intracellular Localization of NDH II
To
obtain further information on the likely physiological role of NDH II,
we examined the intracellular localization of this enzyme by
immunofluorescence. For these studies, monkey TC-7 cells were fixed
with acetone and subsequently incubated with anti-NDH II antiserum and
a secondary, biotinylated anti-rabbit antibody. Incubation with
carboxymethylindocyanine-conjugated avidin and subsequent fluorescence
microscopy revealed a mainly nuclear localization for NDH II (Fig. 6). NDH II was found at both the nuclear periphery and the
nucleoplasm with widespread staining foci throughout the nuclear area.
Such a staining pattern does not allow an unambiguous assignment of NDH
II to specific locations at either peri- or interchromatin regions.
Nevertheless, nucleolar-like structures were not stained, which may
allow the conclusion that NDH II is probably not involved in ribosomal
RNA synthesis.
Figure 6:
Intracellular localization of NDH II.
Monkey TC-7 kidney cells were grown to subconfluence on coverslips.
After fixation with acetone, the cells were incubated with anti-NDH II
serum. Then, the cells were incubated with biotinylated anti-rabbit IgG
and subsequently stained with fluorescence dye-conjugated avidin. After
addition of mounting solution, the cells were viewed at a 400-fold
magnification (A and B) or a 1000-fold magnification (C and D) by using a Zeiss fluorescence microscope
and the fluorescein Cy3 excitation wavelength. The staining of the
cytosol and the perinuclear region was also visible with control serum (B and D).
Comparison of Bovine NDH II and Human RNA Helicase
A
The extensive sequence homology between bovine NDH II and
its human homologue RNA helicase A is reflected by very similar
biochemical properties, such as a comparable molecular weight, the same
directionality of unwinding, and the capability for using all four
dNTPs and rNTPs(10, 12) . RNA helicase A binds to DNA as
demonstrated by affinity chromatography on single-stranded DNA
cellulose; furthermore, its ATPase can be stimulated by poly(dT) and
poly(dI) (12). Despite this and in contrast to NDH II, RNA helicase A
is apparently unable to unwind DNA(12) . However, as we have
shown earlier, NDH II-catalyzed DNA unwinding is salt sensitive,
leading to a complete inhibition of the DNA unwinding activity in the
presence of 75 mM NaCl(9) . On the other hand, RNA
unwinding is barely affected by 75 mM salt(10) .
Therefore, we suspect that a DNA unwinding capability of RNA helicase A
has remained undetected because of too high salt conditions in the
corresponding assay.
Possible Physiological Role(s) of Mle
Proteins
The demonstration of a DNA helicase activity for
NDH II might provide a novel clue for the function of the highly
homologous and genetically characterized Mle protein from fruit flies.
Mle helicase is required for the equalization of the X-linked gene
dosage between the two sexes, probably by altering the chromatin
structure of the X chromosome to increase transcription from this
chromosome(35) . The 85% similarity between Mle and NDH II
speaks in favor of a conserved function for both proteins. However, sex
determination in mammals is completely different from that in Drosophila. In mammals, the only X chromosome of males
contributes as much as both X chromosomes of females, because one of
the female X chromosomes is randomly inactivated. Hence, we cannot
expect that NDH II performs a role comparable to that of Mle protein in Drosophila. But there is also evidence for Mle being involved
in non-sex-specific processes. For example, the Mle mutation nap affects in both sexes the expression
of the gene para, which encodes a sodium channel essential for
the membrane excitability in the initiation and propagation of the
action potential(36, 37) . NAP
contains an
amino acid substitution near the nucleotide binding motif of Mle, which
in turn may lead to an impaired helicase function and consequently an
inefficient expression of the sodium channel(37) . A still
hypothesized DNA unwinding function of Mle protein (analogous to that
of NDH II) might help to decondense the chromatin structure;
alternatively, DNA unwinding might increase RNA polymerase II-dependent
transcription by either enhancing strand opening during elongation or
facilitating the release of the transcript. Further experiments are
necessary to distinguish between these alternatives and to assign
physiological function(s) to this class of highly conserved helicases.
/EMBL Data Bank with accession number(s)
X82829.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.