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J. Biol. Chem., Vol. 277, Issue 2, 1614-1618, January 11, 2002
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From the
Received for publication, September 13, 2001, and in revised form, November 5, 2001
We show that certain DNA sequences have the
ability to influence the positioning of RecA monomers in RecA-DNA
complexes. A tendency for RecA monomers to be phased was observed in
RecA protein complexes with several oligonucleotides containing a
recombinational hotspot sequence, the chi-site from Escherichia
coli. This influence was observed in both the 5' to 3' and 3' to
5' directions with respect to chi. A 5'-end phosphate group and
probably some other features in DNA also influence the phasing of RecA
monomers. We conclude that natural DNAs contain a number of features
that influence the positioning of RecA monomers. The ability of
specific DNA sequences to influence the positioning of RecA monomers
demonstrates some specificity in the binding of individual bases at
different sites within a RecA monomer and, most likely, reflects the
stereochemical non-equivalence of these sites. The possible
biological implications of the phasing of RecA monomers in presynaptic
DNA-protein cofilaments are discussed.
RecA protein is the central component of homologous recombination
in Escherichia coli. In the last few years a number of RecA protein analogues from different sources have been characterized. That
these proteins exhibit rather similar properties suggests that the
fundamental features of homologous recombination are common in
prokaryotes and eukaryotes (for review, see Refs. 1 and 2).
In vitro, the first step of RecA-promoted strand exchange
reaction is the formation of a RecA protein complex with
single-stranded DNA (presynaptic complex). RecA protein monomers bind
cooperatively, completely cover the DNA, and thus form a close-packed
DNA-protein co-filament. The most widely accepted binding stoichiometry
is that each RecA monomer interacts with 3 nucleotides of
single-stranded (ss)1 DNA (3,
4). Thus, the repeat unit of the presynaptic complex should correspond
to 3 nucleotides interacting with one RecA monomer.
Earlier we demonstrated that when RecA protein binding is initiated on
a fluorescent dye molecule attached to the 5'-end of an oligonucleotide
there is a modulation of the DMS reactivity of a guanine residue along
the oligonucleotide with a period of 3 bases (5). This result indicated
that there was a strictly ordered (phased) arrangement of RecA monomers
on the dye-tagged oligonucleotides. In the present study we have asked
the question of whether natural DNA contains some features that can
influence the positioning of RecA monomers.
Homologous recombination is considered to proceed mainly in a
sequence-independent manner. At the same time, however, it has been
demonstrated that there are some preferred DNA binding and pairing
sequences for RecA and the chi-site sequence is among these (6). The
chi-site is a recombination hot spot in E. coli (7) with the
sequence -GCTGGTGG (8) that contains two 3-base repeats that conform
with the RecA protein-DNA binding stoichiometry and might provide a
physical basis for the ordered arrangement of RecA monomers in RecA
protein complexes with chi-containing DNA. For these reasons the
chi-site sequence has been chosen in the present study as an example of
a DNA sequence that may influence the positioning of RecA monomers
along a DNA strand.
The present study provides evidence that DNA sequences can influence
the phasing of RecA monomers in presynaptic RecA-DNA complexes. That
is, that natural DNAs can introduce some order in the arrangement of
the RecA monomers in complexes with DNA participating in homologous
recombination. The possible biological implications of this phenomenon
are discussed.
Protein and Oligonucleotides--
RecA protein was purified by
the method of Cox et al. (18). Oligonucleotides were
synthesized by automated
Radioactive label was introduced on the 5'-end of an oligonucleotide
with the use of polynucleotide kinase (Promega) or on the 3'-end via
the attachment of an additional nucleotide ([ Complexes, Methylation, and Registration--
RecA protein
complexes with oligonucleotides were formed by incubating 15 µM single-stranded oligonucleotides and 6.5 µM protein in the presence of 0.25 mM ATP
The reaction products were analyzed by electrophoresis on 16%
denaturing polyacrylamide gels followed by visualization and quantitation in a PhosphorImager (Molecular Dynamics). The
electrophoretic profiles were normalized to the signal amplitudes for
unmodified oligonucleotides.
The approach used was described earlier (5) and is briefly
outlined here. The oligonucleotides used for the formation of complexes
with RecA protein contained a chi-site sequence at different locations
and a DNA sequence (referred to as a Whereas in ss-DNA·RecA complexes the guanine bases react with DMS
with the same efficiency as in free ss-DNA, the reactivity of guanine
toward DMS is increased in RecA-double-stranded (ds) DNA complexes
(5;9). Therefore, to apply the DNA chemical modification method, two
shorter oligonucleotides complementary to different regions in the
chi-site containing oligonucleotide were added after formation of
RecA·ss-oligonucleotide presynaptic complexes (Fig. 1B).
One of these oligonucleotide annealed to the control sequence, another
to the chi-site-containing region. This experimental design resulted in
the formation of RecA protein ds-DNA complexes in these regions and
allowed us to characterize the arrangement of RecA monomers in both of
these regions.
Position of Chi Influences the Phasing of RecA Monomers on
ss-DNA--
Fig. 2 presents the results
of DMS modification of RecA protein complexes with 5'-end-radiolabeled
oligonucleotides (Table I, series I). The
patterns of DMS modification in the control sequence region exhibit
periodic changes that depend on the position of chi along the
oligonucleotides.
A comparison of the modification efficiency of the two
neighboring G residues in the control sequences (G-2 and G-3) reveals changes that proceed as a result of the shift of the chi-site by one
nucleotide (compare lanes 1 and 2; 2 and 3; 3 and 4). That the pattern
reverts after a shift of the chi-site by 3 nucleotides can be gleaned
by comparing lanes 1 and 4. Thus, the
modification pattern shows a repeat with a period of 3 nucleotides.
Analysis of the relative reactivity of each of the other G residues in
the control sequence confirms the presence of a 3-nucleotide periodicity in the modification pattern. According to their
As expected there is not a dependence of the modification pattern on
the distance between the chi-site and the control sequence on naked
duplex DNA and in the absence of RecA the modification patterns of the
control sequence was the same for all the oligonucleotides (data not shown).
The results presented demonstrate that in RecA protein complexes with
these oligonucleotides RecA monomers tend to arrange in a phased manner
depending on the position of chi in the oligonucleotide. This
phenomenon is similar to the phasing of RecA on a fluorescent dye
molecule located at the 5'-end of an oligonucleotide described earlier
(5).
An additional experiment was carried out where only the complementary
oligonucleotide to the control sequence was added to the ss-DNA·RecA
complex (see scheme in Fig. 1B). The changes of the
modification pattern of the control sequence observed were the same as
when both complementary oligonucleotides were added (data not shown).
This result indicates that the phasing of RecA monomers occurs when
RecA binds to the chi sequence in ss-DNA.
Similar modification patterns were obtained for the case of RecA
complexes with 3'-labeled oligonucleotides with a dephosphorylated 5'-end (not shown).
Phasing of RecA Is Observed in Both Directions from the Origin of
Phasing--
The above example demonstrated the capability of the
chi-site to influence the phasing in the 5'- to 3'-end direction. To check that this influence expands in the opposite direction as well,
another set of radiolabeled oligonucleotides was used (Table I, series
II and III). These oligonucleotides contained chi in the central part
of the oligonucleotide and control sequences near both oligonucleotide
ends. As demonstrated in Fig. 3 the shift
of the position of chi along the oligonucleotide induces changes of the
modification patterns in both directions from chi (compare scans 2 and
3 in Fig. 3) and confirms the capability of chi to influence phasing in
both directions.
A Phosphorylated 5'-End Influences the Phasing of RecA
Monomers--
Fig. 3 shows that in the case of 3'-labeled
oligonucleotides with a dephosphorylated 5'-end the characteristic
changes in the modification pattern are to some extent greater than for
the case of oligonucleotides with phosphorylated radiolabeled 5'-ends. A possible explanation is that the 5'-end phosphate acts as another phasing origin for RecA in competition with chi.
An analysis of the modification patterns of the chi-site regions also
reveals changes in the modification of the G residues in those regions
that correlates with the position of the chi-site in the
oligonucleotides. In this case also, the modification pattern reverts
after a shift by 3 nucleotides (Fig. 3, compare profiles 1 and 4).
This last result also may be explained by the presence of some origin
of RecA phasing other than the chi-site and that the shift of the
chi-site relative to this origin induces changes in the chi-site
pattern of modification. To locate this origin of phasing, a series of
oligonucleotides with the same internal sequences but containing
additional (or removed) nucleotides near the 5'- or 3'-ends were used.
As demonstrated in Fig. 4, changing the
oligonucleotide length by adding (or removing) nucleotides at the
phosphorylated 5'-end of the oligonucleotide resulted in corresponding
changes both in the chi-site and the control sequence modification
patterns whereas the introduction of an additional nucleotide to the
3'-end had no effect (data not shown). This result demonstrates the
influence of a phosphorylated 5'-end on the phasing of RecA.
Therefore, the modification patterns of the control sequences exhibit a
dependence upon both the chi-site position and the changes of the
oligonucleotides lengths by adding (or removing) nucleotides from the
phosphorylated 5'-end. These results may be explained by the presence
in solution of a mixture of two kinds of complexes. In one the binding
of RecA monomers has been initiated at chi and generates periodic
changes in the modification pattern of the control sequence in the set
of oligonucleotides of the same length (Figs. 2 and 3). In another kind
of complexe the binding was initiated at the phosphorylated 5'-end of
the oligonucleotide (Fig. 4). These two modes of binding generate
changes in both the control sequence and the chi-site modification patterns.
The data presented demonstrate that a specific DNA sequence, in
the present case the chi-site from E. coli, is able to
influence the positioning of RecA monomers in recombinational
protein-DNA filaments. The phasing occurs when the RecA protein
interacts with ss-DNA; that is, when the presynaptic complex is formed
and can be observed in both the 5' and 3' directions from the origin of phasing.
If the RecA monomers have some preference to bind to a particular DNA
sequence the cooperative binding of RecA will ensure that the ensuing
closed-packed filament will tend to be phased with respect to this
sequence. Given that each RecA monomer contains three different sites
of binding for DNA nucleotides, the local environment of one of these
sites within a RecA monomer is either more hydrophobic (10) or alters
the accessibility of guanines such that G residues at one of these
sites is most easily modified. It is this differential reactivity of G
residues to DMS in the DNA in a RecA·DNA complex that allows us to
observe the phasing of RecA monomers (Fig.
5). That is, when a guanine is situated in one of these sites a stronger modification signal is observed (Fig.
5, A, B, and D) and when guanines
appear between these sites they exhibit a relatively low level of
modification (Fig. 5C). The modification pattern reverts
after the shift of the chi-site by 3 nucleotides (Fig. 5, A
and D) in agreement with the stoichiometry of RecA protein
binding to ss-DNA.
Influence of DNA Sequence on the Positioning of RecA Monomers in
RecA-DNA Cofilaments*
§ and
Institute of Molecular Genetics of the
Russian Academy of Sciences, Kurchatov sq., 123182 Moscow, Russia
and the ¶ Genetics and Biochemistry Branch, NIDDK, National
Institutes of Health, Bethesda, Maryland 20892-1810
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyanoethyl phosphoramidite DNA synthesis
on a 380B DNA synthesizer (Applied Biosystems). All oligonucleotides
were purified by PAGE with a standard protocol (19).
-32P]ATP)
with terminal deoxynucleotidyl transferase (Promega) in accordance with
the manufacturer's recommendations. The sequences of synthesized
oligonucleotides are listed in Table I.
S
in a buffer containing 20 mM of triethanolamine acetate
(TEA-Ac) (pH 7.5), 2 mM MgAc, 5% glycerol at 37 °C for
30 min. The complexes formed were chilled on ice, and DMS was added to
a final concentration of 0.4%. After 30 min at 0 °C the reaction
was stopped by the addition of mercaptoethanol and SDS (to 0.2%)
followed by ethanol precipitation. DNA cleavage reactions were
performed at 95 °C by two successive incubations: 10 min in 10 mM TEA-Ac (pH 7.5) and 10 min in 0.1 M NaOH.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
control sequence
) located in the same position within the oligonucleotide. An example of
the oligonucleotides and a general outline of the experimental approach
is presented in Fig. 1. The control
sequence contained four guanine bases (designated in Fig. 1A
by 1-4) conveniently modifiable with DMS.
View larger version (26K):
[in a new window]
Fig. 1.
Outline of the experimental procedure used to
study the phasing of RecA monomers on oligonucleotides containing a
chi-site. A, the sequences of oligonucleotides that
were used for the formation of presynaptic complexes with RecA protein.
Underlined are the regions containing the chi-site and
control sequences. G residues in the control sequence are numbered.
B, formation of RecA protein complexes with oligonucleotides
containing a chi-site for chemical modification experiments. After the
formation of presynaptic complexes of RecA protein on oligonucleotides
containing a chi-site two short unlabeled oligonucleotides
complementary to the chi-site and control sequences were added. The
resulting complexes were subjected to treatment with DMS.
View larger version (12K):
[in a new window]
Fig. 2.
DMS modification of oligonucleotides
containing a chi-site in complexes with RecA reveals phasing of RecA
monomers. Dependence of the modification pattern on the position
of the chi-site. Scans 1-6 present the results of DMS modification of
RecA protein complexes with oligonucleotides of series I. The chi-site
sequence regions are underlined, guanines in the control
sequence are numbered (see also Fig. 1).
Chi-site-containing oligonucleotides used for formation of complexes
with RecA protein
in phase location (3 nucleotides apart from each other), the G-1 and
G-2 residues are always characterized by similar reactivity changes. On
the other hand, the reactivity of G-4, in accordance with its out
of phase location relative to the G-3, G-2, and G-1 residues does
not correlate with the reactivity of any of these 3 residues, but also
exhibits a periodic variation depending on the position of the chi-site
(compare scans 1 and 4).
View larger version (20K):
[in a new window]
Fig. 3.
Phasing of RecA monomers in both directions
from chi. Panels A and B present patterns of
modification of RecA protein complexes with oligonucleotides of series
II and III, respectively. The chi-site sequence regions are
underlined, guanines in the control sequences are
numbered.
View larger version (18K):
[in a new window]
Fig. 4.
A phosphorylated 5'-end influences the
pattern of modification. Dependence of the modification patterns
on oligonucleotide length. Oligonucleotides of different lengths were
used for complex formation (series III).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (22K):
[in a new window]
Fig. 5.
Possible phases of the positioning of RecA
monomers on DNA. Each RecA monomer interacts with 3 nucleotides of
the DNA. Individual nucleotide binding sites in RecA monomers are
designated by arrows of different length to emphasize their
different abilities to increase the reactivity to DMS. Nucleotides
interacting with the site allowing strongest modification are presented
in black. Panels A, B, C, and
D present different possible modes or phases of RecA
monomers on DNA. Depending on the phase, different modification
patterns (presented in the lower part of the
panels) are generated.
Each Nucleotide Binding Site within a RecA Monomer Is Distinct and Distinguishable-- The exact structure of the DNA binding domain of RecA protein remains to be established. Unfortunately, there are no data that could resolve the different sites on RecA that interact with each of the 3 individual nucleotides bound to each monomer. As is evident from the variation of G-base reactivity toward DMS at the different sites in the RecA monomer, our data presented here and obtained previously (5) show that individual nucleotides bind to distinct and distinguishable sites in each RecA monomer. In addition, that DNA sequences can influence the phasing of RecA implies the non-equivalence of the nucleotide binding sites and some base preference of the binding at each of the three sites.
We do not suppose that the phasing of RecA is mediated exclusively by the chi-site sequence. Because the nucleotide binding centers of a RecA monomer differ in their affinity for different bases, thermodynamically favorable modes of RecA monomer arrangements ("phasing") should exist for RecA complexes on a wide range of non-uniform DNA sequences. Obviously, this phasing is more facile on sequences that exhibit a modulation of base sequence with a period of 3 nucleotides (for example, in regions of DNA with a strong codon bias). In preliminary experiments we have confirmed that a number of randomly chosen sequences can influence the phasing of RecA monomers to varying degrees (not shown). A detailed investigation of this question requires further study and would be more informative with a DNA modification agent that is not base-specific.
In a bacterial cell RecA acts as a component of a rather complex protein machine (for review, see Ref. 11) and other components of the recombination/repair system may also influence the phasing of RecA.
Organization of the Presynaptic Nucleoprotein Filament and Possible Biological Role of RecA Phasing-- The physical non-equivalence of individual nucleotide binding sites in a RecA monomer raises the issue of the functional organization of recombinational protein-DNA complexes. The main function of RecA protein is to promote the recognition of homology between similar but not identical DNA molecules. Nucleotide bases bound at the 3 distinct sites of the RecA monomer may tolerate non-homology in a partner DNA quite differently, either before or during strand exchange. Such a triplet organization bears a striking resemblance to the structure of the protein coding regions in DNA, where the informational value of a nucleotide depends on its position inside a codon and, consequently, also exhibits a periodic variation with a period of 3 nucleotides. Additional studies are needed to answer the question of whether this is just a coincidence or a feature of functional importance in genetic processes promoted by RecA.
We suggest a relationship between the phasing of RecA monomers with some functionally important features in DNA. For example, there are several facts about chi and the distribution (location) of G and T bases in coding regions of DNA that allow us to speculate about a mechanism by which the RecA monomers on ss-DNA can be phased with the protein coding frames of this DNA. Chi is not only a recombinational hotspot but is also one of the most overrepresented sequences in E. coli (12) with an extremely strong tendency to be phased relative to the protein coding frame (13). The 3 nucleotide periodicity in the arrangement of G and T bases is a property of the codon bias of E. coli DNA (13, 14) in general and particularly in the vicinity of chi (15). The propensity of DNA sequences to influence the positioning of RecA demonstrated here together with the observations that both chi and the GT-islands of preferred pairing (6) are in phase with open-reading frames allows us to suggest that in presynaptic complexes RecA monomers may be phased with respect to the protein coding frame.
RecA phasing with respect to open-reading frames and the ability of RecA to significantly decrease the fidelity of heteroduplex formation (16, 17) compared with that of heteroduplex formation in the absence of protein might have an important role in permitting recombination between homologous but not identical protein encoding DNAs with different codon usage. If, for example, the phasing allows this "anti-proofreading" decrease in fidelity to be mostly at the third position in a codon, the wobble position could be more easily assimilated in genetic crosses. Such a decrease in fidelity in the third position of a codon could maximize the probability of exchanges between functionally active genes with different DNA sequences but encoding identical protein sequences as takes place, for example, in the process of horizontal gene transfer.
This may not be the only possible role of the phasing of RecA monomers.
Because of the plurality and complexity of the processes RecA protein
participates in it is difficult to foresee all possible consequence of
this phenomenon. Nevertheless, we suggest that the DNA
sequence-dependent positioning of RecA monomers may be an
important structural and functional feature of recombinational protein-DNA cofilaments.
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ACKNOWLEDGEMENTS |
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We thank Drs. Vlad Malkov, Oleg Voloshin, and Igor Panyutin for useful discussions and Drs. Peggy Hsieh, Howard Nash, and Susan Gottesman for critically reading the manuscript. We are also grateful to George Poy for oligonucleotide synthesis and Linda Robinson for technical help.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by Grants 01-04-49690-a from the Russian Foundation for Basic Research and INTAS-96-1290 from INTAS.
To whom correspondence should be addressed. Fax: 301-496-9878;
E-mail: camerini@ncisun1.ncifcrf.gov.
Published, JBC Papers in Press, November 7, 2001, DOI 10.1074/jbc.M108871200
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ABBREVIATIONS |
|---|
The abbreviations used are:
ss, single-stranded;
DMS, dimethyl sulfate;
ATPS, adenosine
5'-O-(3-thiotriphosphate);
ds, double-stranded.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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