Binding of RecA to DNA occurs in three stages: first, the presynapsis, when RecA is polymerized on ssDNA forming a right-handed nucleoprotein filament; second, the synapsis, when the presy
Trang 1single-stranded DNA
Analysis by stepwise increase in ligand complexity
Irina P Bugreeva, Dmitry V Bugreev and Georgy A Nevinsky
Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Novosibirsk, Russia
Homologous recombination, required for the
mainten-ance of genetic diversity and DNA repair, is one of the
most important molecular genetic processes In
Escheri-chia coli, a pivotal role in homologous recombination is
played by RecA protein, which is responsible for search
for homologous DNA sequences and strand transfer
between them [1] RecA is an ATP-dependent
DNA-binding protein consisting of 352 amino acids
(37.8 kDa) [1] Binding of RecA to DNA occurs in three
stages: first, the presynapsis, when RecA is polymerized
on ssDNA forming a right-handed nucleoprotein
filament; second, the synapsis, when the presynaptic
complex binds dsDNA and actively searches for
homo-logy with the ssDNA; and third strand exchange, when
a new DNA duplex is formed and one of the strands formerly in dsDNA is released as ssDNA Thus, a RecA filament assembles on DNA at the first stage; this process is more efficient with ssDNA Binding of RecA
to ssDNA must be nonspecific, but the protein displays some preferences for binding poly(dT) and GT-rich sequences [2–5]
In the presence of ATP or its nonhydrolysable thio analog (ATPcS), RecA forms a right-handed filament
of 100 A˚ diameter and 95 A˚ pitch [6] The filament
is assembled cooperatively in the 5¢)3¢ direction (in respect to the ssDNA) [7] DNA in such complex is stretched by 50%, with the internucleotide distance increasing to 5.1 A˚ [8] If RecA binds to dsDNA, the
Keywords
RecA; DNA recognition mechanism
Correspondence
G A Nevinsky, Laboratory of repair
enzymes, Institute of Chemical Biology and
Fundamental Medicine, 8, Lavrentieva Ave.,
630090, Novosibirsk, Russia
Fax: +7 3832 333677
Tel: +7 3832 396226
E-mail: nevinsky@niboch.nsc.ru
(Received 8 January 2005, revised 24
February 2005, accepted 31 March 2005)
doi:10.1111/j.1742-4658.2005.04693.x
RecA protein plays a pivotal role in homologous recombination in Escheri-chia coli RecA polymerizes on single-stranded (ss) DNA forming a nucleo-protein filament Then double-stranded (ds) DNA is bound and searched for segments homologous to the ssDNA Finally, homologous strands are exchanged, a new DNA duplex is formed, and ssDNA is displaced We report a quantitative analysis of RecA interactions with ss d(pN)n of var-ious structures and lengths using these oligonucleotides as inhibitors of RecA filamentation on d(pT)20 DNA recognition appears to be mediated
by weak interactions between its structural elements and RecA monomers within a filament Orthophosphate and dNMP are minimal inhibitors of RecA filamentation (I50¼ 12–20 mm) An increase in homo-d(pN)2)40 length by one unit improves their affinity for RecA (f factor) approximately twofold through electrostatic contacts of RecA with internucleoside phos-phate DNA moieties (f 1.56) and specific interactions with T or C bases (f 1.32); interactions with adenine bases are negligible RecA affinity for d(pN)n containing normal or modified nucleobases depends on the nature
of the base, features of the DNA structure The affinity considerably increa-ses if exocyclic hydrogen bond acceptor moieties are present in the baincrea-ses We analyze possible reasons underlying RecA preferences for DNA sequence and length and propose a model for recognition of ssDNA by RecA
Abbreviations
EMSA, electrophoretic mobility shift assay; ODN, deoxyribooligonucleotide(s); SILC, stepwise increase in ligand complexity; ss-,
single-stranded; ds-, double-stranded.
Trang 2parameters of the resulting filament are the same as
for ssDNA, and the DNA duplex in the filament is
unwound as compared with B-DNA [9,10] In the
absence of ATP RecA forms a more compact inactive
filament of 64 A˚ pitch and 2.1 A˚ internucleotide
dis-tance [11]
After the filament is formed, the second DNA
bind-ing site of RecA can bind dsDNA In addition, ssDNA
can be bound there, even more efficiently than
dsDNA After strand exchange, the second RecA
DNA binding site binds the displaced strand following
new DNA duplex formation [12]
Binding of dsDNA to a RecA filament is followed
by search of homology between the appropriate strands
and then by strand exchange The mechanism of this
process is still unclear It was hypothesized that ssDNA
could invade through the minor or major groove of the
duplex and displace the respective strand [1]; if the
inva-sion occurs through the major groove, formation of a
peculiar DNA triplex (R-form DNA) was proposed
[13,14] An alternative mechanism (melting–annealing
model) for the homology search involves only formation
of canonical Watson–Crick pairs after melting of the
duplex and annealing of its appropriate strand to the
incoming strand [15] As DNA in the filament is
consid-erably stretched and unwound, the bases could be easily
extruded from the helix to be ‘examined’ for homology
with the incoming strand
Howard-Flanders proposed a triple helix as a
tran-sient, or even a stable, intermediate in the reaction [16]
However, all recent efforts have failed to detect such
a structure as a stable intermediate Instead, several
groups have described a stable synaptic complex
con-sisting of three strands and RecA, in which strand
exchange has already taken place [17,18] In this
com-plex, the incoming ssDNA is part of the new duplex and
the leaving strand has not yet been released Leaving
aside such an early triplex, one can jump forward and ask
what are the steps leading to such a poststrand exchange
intermediate? One can envision several slower
confor-mational changes, such as homology recognition via
base flipping (melting) and switching (annealing) [19]
DNA binding by RecA is thought to be mediated by
amino-acid residues from two protein loops, L1
(resi-dues 157–164) and L2 (resi(resi-dues 195–209) [1,20] Both
these regions are rather conserved among bacterial
RecA proteins but not between bacterial, archaean and
eukaryotic RecA homologs In addition, DNA could
interact with several RecA tyrosine residues (Tyr65,
Tyr103, Tyr264) [21–23], as well as with Lys183 [23,24],
Arg243 [22] and residues 233–242 [24] This list all
but exhausts the available information regarding
RecA–DNA interactions To our knowledge, there have
been no quantitative studies on general parameters of and individual contacts within the forming nucleo-protein filament
Our laboratory has designed a novel approach to analysis of protein⁄ nucleic acid interactions, based
on stepwise increase in ligand complexity (SILC approach) SILC produces quantitative estimates of the contributions of individual structural elements of DNA or RNA molecules into the affinity of enzymes
to such extended ligands [25–27] We have applied SILC to analyze DNA binding by a number of DNA polymerases [25–27], DNA repair enzymes [28– 31], EcoRI restriction endonuclease [32], HIV-1 integ-rase [33], and type I DNA topoisomeinteg-rases [34,35] In all these instances, virtually every nucleotide unit within the DNA binding cleft (10–20 base pairs cov-ered by the protein globule) interacts with the enzyme through weak additive electrostatic, hydro-phobic or van der Waals contacts to various struc-tural elements of the ligands, with electrostatic interactions of internucleoside phosphate moieties contributing most to the affinity (reviewed in [25– 27]) These nonspecific contacts provide high affinity (Kd¼ 10)5)10)8m) of all enzymes for specific and nonspecific DNA A transition from nonspecific to spe-cific DNA usually leads to formation of spespe-cific contacts and increase of the affinity by 1–2 orders of magnitude (up to Kd¼ 10)8)10)10m), while the reaction rate (kcat) is enhanced by 5–8 orders Thus, specificity of DNA-dependent enzymes is not of thermodynamic nature (the enzyme-substrate complex formation) but mostly originates from the following stage of enzyme-induced adjustment of DNA conformation and from chemical steps (kcat) of catalysis [25–27]
Quantitative studies concerning the efficiency of interactions between a RecA filament and DNA are
a prerequisite for understanding the nature of RecA filamentation; however, no such information is available
so far SILC is a very promising approach for obtaining the appropriate data Here we present a SILC analysis
of RecA interactions with ssODN of different structures and lengths and estimate the contribution of individual DNA elements in its affinity for a RecA filament
Results
Filamentation of RecA on ssDNA and its inhibition
In the presence of ATP or ATPcS RecA is polymer-ized on DNA forming a nucleoprotein filament We have studied the stability of a RecA filament formed with different individual 5¢-[32P]d(pN)n (n¼ 2–20) by
Trang 3electrophoretic mobility shift assay (EMSA) The
com-plexes between RecA and short individual d(pN)n
(n¼ 2–15) were easily disassembled, confirming the
lit-erature data on their low stability [36,37] Individual
5¢-[32P]d(pN)16)20 formed detectable complexes with
RecA under the condition used (data not shown), and
the best of them d(pT)20 was used for the rest of the
study At the RecA monomer: ODN ratio of 10 : 1,
almost all d(pT)20 was in the filament, in agreement
with the known RecA monomer interaction with three
nucleotide units of ssDNA [1]
As the interactions with short ODNs are of low
affin-ity, they are undetectable by EMSA and many other
widely used physicochemical techniques [27] However,
interactions of enzymes with low-affinity ligands can be
easily followed by observing inhibition of appropriate
enzymatic activity by these ligands (reviewed in [25–27])
In the case of short ODN interacting with RecA
mono-mers or forming short unstable filaments, the respective
ODN ligands should inhibit RecA filamentation on
d(pT)20 In addition, at high concentration short ODN
can compete with d(pT)20for the filament formed on this
substrate We have shown that the addition of any short
ODN causes a decrease in the amount of 5¢-[32P]d(pT)20
detectable in the RecA filament complex Concentration
dependencies of RecA-d(pT)20 complex formation on
the inhibitor concentration had regular hyperbolic
shapes (Fig 1), indicating that RecA filamentation on
d(pT)20 and its inhibition by short ODN, including
orthophosphate (I50¼ 0.5 m) and various dNMPs
(I50¼ 12–20 mm) as minimal ligands, obey formally
canonical steady-state equations of complex formation
The apparent values of I50(Fig 1) were used to
charac-terize the relative efficiency of RecA interactions with
various ODN; these data are summarized in Table 1
The Gibbs free energy characterizing enzyme-ligand
complex formation can be presented as a sum of DG
values for each individual contact:
DG0¼ DG0
1þ DG0
2þ ::: þ DG0
n with DG0i ¼ RT ln Kdi
ð1Þ where Kdi is the contribution of an individual contact
to the overall affinity [38] It follows from the
additi-vity of Gibbs free energies that the overall Kd(Kd¼ KI)
value characterizing complex formation is the product
of the Kdvalues for individual contacts:
DG0¼ RT ln Kd¼ RT ln½Kd1Kd2:::Kdn; and
Kd¼ Kd1Kd2:::Kdn
ð2Þ
To assess possible additivity of the interactions of
ODN with RecA filament, the data from Table 1 were
analyzed as logarithmic dependencies of I50for d(pN)n
(0£ n £ 20, n ¼ 0 corresponds to orthophosphate, Pi) Affinity of d(pN)n ligands to RecA increased mono-tonously in the d(pN)2–d(pN)20 interval, d(pT)n and d(pC)n producing nearly identical results (Fig 2) Dependencies of lgI50 on n were linear at 2£ n £ 20 (Fig 2), indicating that the affinity of RecA to each of the nucleotide units of d(pN)20is additive
Interestingly, experimentally estimated affinities of dNMP (I50¼ 12–20 mm) were somewhat higher than that for corresponding d(pN)2 (40–47 mm, Table 1) This phenomenon, also observed for some other enzymes, arises from greater conformational freedom
of individual dNMP (or short ODN) compared with the same ligands as elements of long DNA [25–27] Considerable stretching and unwinding of DNA in a RecA filament is associated with energetic costs required for sugar-phosphate backbone deformation and stacking disruption [6] Mononucleotides are not subject to such restrictions and thus can bind RecA more efficiently Extrapolation of the log dependencies for d(pT)n and d(pC)n to n¼ 1 (Fig 2) gives lower
A
B
Fig 1 Dependence of the relative level of inhibition of RecA filam-entation on [ 32 P]d(pT)20on the concentration of d(pT)10inhibitor (A) Reaction products separated by EMSA in polyacrylamide gel (B) Band intensities in (A) quantified by Cherenkov counting and plotted against inhibitor concentrations Lane 1, filamentation without the inhibitor; lane 10, reaction mixture without RecA; d(pT)10 inhibitor added at 0.05 m M (lane 2), 0.1 m M (lane 3), 0.2 m M (lane 4), 0.3 m M (lane 5), 0.5 m M (lane 6), 0.6 m M (lane 7), 0.8 m M (lane 8) and 1 m M (lane 9) The upward shift in free oligonucleotide position appears due to a time lag in loading different reaction mixtures onto a running gel.
Trang 4affinity values for RecA binding single d(pT) and
d(pC) units (I50¼ 63 mm) within longer d(pN)n
(Fig 2; Table 1) Thus, this value of I50¼ 63 mm is
a better parameter to characterize RecA affinity for
the higher-affinity nucleotide unit of d(pN)n in
com-parison with the remaining (n–1) nucleotide units
within d(pT)n or d(pC)n, which have lower affinity for
the filament (490 mm, see below)
I50 values are usually related to the KI values [38]
For example, in the case of competitive inhibition, they
are related through the equation I50¼ aKI (a¼
1 + [S]⁄ KS; KS is KMor Kd for substrate), where the
coefficient a depends on the affinity and concentration
of a substrate, d(pT)20in our case Therefore, the ratio
of KI values for two different inhibitors, KI(2)⁄ KI(1), is
equal to the ratio of apparent I50 values for these
inhibitors, I50(2)⁄ I50(1), and the ratio of these values
gives the Kd value characterizing a difference of the
enzyme contacts between the first and the second
inhibitors (Eqns 1 and 2) [38]
From the slope of the lgI50 vs n dependency
(Fig 2) the factor (f) reflecting an increase in
affin-ity of the enzyme for d(pN)n upon a one-unit
increase in the ligand length can be calculated as:
f¼ 10–[lgI 50 (n ¼ 20) ⁄ –lgI 50 (n ¼ 2)] ⁄ 18 (exact average values
of lgI50 were calculated using the log curves) From the slopes of the curves for d(pT)n and d(pC)n (Fig 2), the value f¼ 2.04 was calculated for the
f factor As 1⁄ f(n) ¼ I50(n)⁄ I50(n + 1)¼ KI(n)⁄
KI(n + 1)¼ Kd(n)⁄ Kd(n + 1), interaction of a RecA filament with any of the 19 units of d(pT)20 or d(pC)20 is characterized by Kd¼ KI¼ 1 ⁄ f ¼ 0.49 m Comparison of this value with I50 for free dNMP determined experimentally I50(experimental)¼ 12–20 mm, Table 1 or for a dNMP unit within d(pN)n by extra-polation to n¼ 1 I50(extrapolated)¼ 63 mm; Fig 2 shows that the affinity of RecA for one of the units
or d(pT)20 or d(pC)20 is 8–41-fold higher than for any of the remaining 19 units Extrapolation of the log dependencies for d(pT)n and d(pC)n to n¼ 0 gives I50(extrapolated)¼ 15 mm for a single internucleo-side phosphate group of d(pN)n, approximately 3.3-fold lower than the experimental I50¼ 0.5 m for free orthophosphate Overall, the affinity of a RecA filament for d(pT)n and d(pC)n at 2£ n £ 20 may
be described as I50[d(pN)n]¼ I50(d(pN)2)· (1 ⁄ f)n)2¼
Table 1 I50values for interactions of different ligands with the high-affinity DNA-binding center of E coli RecA filament.
Ligand (inhibition of d(pT)20) I50, M * –lgI50 Ligand (inhibition of d(pT)20) I50, M –lgI50
One internucleoside phosphate
within d(pC) n and d(pT) n **
within d(pA) n **
One (pT)-unit of d(pT)n** 6.3 · 10)2 1.20 One (pC)-unit of d(pC)n** 6.3 · 10)2 1.20
I50determined using d(pT)40
as substrate
*Error in I 50 values was 10–30%; means of 3–4 measurements are given; **The values of I 50 determined by extrapolation of lg-curves to
n ¼ 0 for Pi and n ¼ 1 for dNMPs (Fig 2); §d(pR), deoxyribosephosphate; ***R is a tetrahydrofuran analog of abasic deoxyribose.
Trang 5I50(d(pN)2)· (f)2–n, when at 1£ n £ 20 as
I50[d(pN)n]¼ I50(dNMP, extrapolated)· (1 ⁄ f)n)1,
where I50(dNMP, extrapolated)¼ 63 mm reflects
the contribution of the high-affinity nucleotide init
within longer d(pN)n, and f (2.04) describes an
increase in affinity due to a one-unit increase in
d(pN)n length
The logarithmic dependence for d(pA)n (Fig 2) can
be broken in two nearly linear segments with different
slopes at 2£ n £ 6–7 and 6–7 £ n £ 20 For the first
segment, f¼ 2.12 (Kd 0.47 m), and for the second,
f¼ 1.32 (Kd 0.76 m) Interestingly, the affinity of
RecA for d(pA)20 is 240-fold lower than for d(pT)20
(Table 1, Fig 2) This observation agrees well with
lower stability of a RecA⁄ d(pA)20 complex during
EMSA Extrapolation of the logarithmic dependency
for d(pA)n towards higher n suggests that only for
d(pA)40)45 the I50 value will be comparable with that
for d(pT)20; empirically, complexes of RecA with
d(pA)n are stable during electrophoresis from this
length onward (data not shown)
It can be clearly seen in Fig 2 that the nature of
protein–DNA interactions was nearly the same for
different d(pN)nat 1£ n £ 10 The next 10 DNA units were bound better in pyrimidine ODN A decrease in the interaction efficiency at n > 7–8 for d(pA)n could mean that the structure of DNA complex with the first three RecA monomers may be important for the assembly of the next monomers
The data shown in Fig 2 suggest that the further elongation of d(pT)n (n > 20) should also be accom-panied with a monotonous increase in their affinity
To investigate this possibility, we used a 5¢-[32P]d(pT)40
substrate and analyzed inhibition of RecA filamenta-tion by d(pT)20)40 (Table 1) The apparent I50 values for d(pT)20 determined with [32P]d(pT)20 and [32P]d(pT)40 as substrates were nearly the same (Table 1) Figure 2 (inset) shows that the lgI50 values for d(pT)20, d(pT)30, and d(pT)40apparently fall on a straight line This is consistent with an increase in RecA filament affinity with increasing ssDNA length The shallowing of the log dependence slope at n¼ 20–40 can be due to two reasons First, it cannot be excluded that correct determination of I50 values for d(pN)30)40 may be unreliable and the observed I50 val-ues are higher than real I50values On the other hand, the change in the slope of the log dependencies may reflect a decrease in the efficiency of RecA filament interaction with very long DNA due to ‘polymeric effect’ usually associated with increased mobility and flexibility of long polymeric structures with high con-formational freedom
Nature of RecA interactions with nucleic acids
It has been shown for many DNA-depending enzymes that strongest contacts they form with ssDNA are those with the internucleoside phosphate moieties; some enzymes also can interact with nucleobases [25– 27] Introduction of 5¢- or 3¢-terminal phosphate moiet-ies in ODN increased their affinity for RecA For instance, the apparent I50 value for d(Tp)7T (4.8· 10)3m) was about an order of magnitude higher than that for d(pT)8(5.0· 10)4m) and twofold higher than for d(Tp)8(2.5· 10)3m) Whereas the introduct-ion of a 3¢-phosphate moiety had an effect similar to that of the f factor nature (f ¼ 2.04) for pyrimidine ODN, the effect of a 5¢ phosphate was much more pronounced Although the negative charge at the ter-minal phosphates is one negative charge higher than at internucleotide phosphate moieties, this increase seems
to influence the filament affinity for the 5¢-terminal ODN phosphate to a larger extent than to the 3¢-ter-minal phosphate It is possible that the 5¢-ter3¢-ter-minal phosphate of ODN has more conformational freedom and can form additional contacts with the filament As
Fig 2 Affinity of RecA (logarithmic dependencies of apparent I50)
to homo-ODN of different lengths (n) determined using inhibition
of RecA filamentation on [32P]d(pT) 20 The I 50 values for different
d(pN)n (1 £ n £ 20) and oligonucleotides containing abasic units
or ethylated internucleoside phosphates are obtained using
[32P]d(pT) 20 and for d(pT) 20 )40 [32P]d(pT)40 (20 £ n £ 40, see the
inset): d(pT)n (open sircules; including the inset), d(pA)n (cross),
d(pC)n(triangles), Positions of –lgI50values for ethylated d[(pEt)T]10
and d[(pT) 2 (pR) 17 (pT)] (pR is a tetrahydrofuran analog of abasic
deoxyribose) are shown.
Trang 6the filament assembly on ssDNA occurs cooperatively
in the 5¢)3¢ direction [8], the increased affinity of RecA
to the 5¢-terminal phosphate of ODN may be
import-ant for better anchoring of ODN on the first RecA
monomer during the initiation of filamentation
Ethylation of internucleoside phosphate moieties
neutralizes their charges The affinity of a RecA
fila-ment for d(pT)10 (I50¼ 2.0 · 10)4m) was 25-fold
lower than for ethylated d[p(Et)T]10 (I50¼ 5.0 ·
10)3m) (Table 1), indicative of an important role of
negative charges of internucleoside phosphates for
RecA complexation with DNA
The affinity of RecA to d(pT)20(I50¼ 1.0 · 10)7m)
was 100-fold higher than to d[(pT)2(pR)17pT] (I50¼
9.5· 10)6m), a 20-mer lacking 17 out of 20
nucleo-bases (R is a tetrahydrofuran analog of abasic
deoxy-ribose) As was shown earlier [25–27], deoxyribose
moieties of DNA have little effect on its affinity for
proteins, while internucleoside phosphate groups make
the main contribution Taking this into account and
assuming that the lack of the bases did not influence
the filament interactions with the backbone, the
increase in affinity due to a single internucleoside
phosphate residue (electrostatic factor e) can be
esti-mated as e¼ (I50¼ 1.75 · 10)2m for d(pT)3)⁄ (I50¼
9.5· 10)6m for d[(pT)2(pR)17pT])1⁄ 17 ¼ 18421 ⁄ 17
¼ 1.56 (Kd¼ 0.64 m) As an increase in the affinity for
one (pT) unit (f¼ 2.04) is a product of its increase
due to an internucleoside phosphate group (factor e¼
1.56) and a T base (factor fT), fT can be calculated as
a ratio f⁄ e ¼ 1.31 Kd(T base)¼ 1 ⁄ 1.31 ¼ 0.76 (m)
The same value of fT can be calculated directly: fT¼
(I50¼ 9.5 · 10)6m for d[(pT)2(pR)17pT])⁄ (I50¼
1.0· 10)7m for d[(pT)20])1⁄ 17¼ 951⁄ 17¼ 1.31 The
affinity increases due to one C base (fC¼ 1.31) and
one T base (fT¼ 1.31) are the same Thus, RecA in
the filament forms weak additive contacts with each
internucleoside phosphate moiety and each base of
pyrimidine ODN, with the phosphates contribution
into the affinity being 1.2-fold more than that of
C or T bases
As the filament affinity for d(pA)20 (I50¼
2.4· 10)5m) was very similar to that for
d[(pT)2(pR)17pT] (I50¼ 9.5 · 10)6m) or for the
affin-ity calculated for a totally abasic oligomer d(pR)20(I50
2.1 · 10)5m), the filament probably interacts with
adenine bases in DNA very weakly if at all
RecA interactions with nucleobases
To evaluate the importance of exocyclic acceptor
moi-eties, we have compared the efficiency of RecA
filam-entation on d(pA)20 and d(pI)20, where in the latter,
the O6 acceptor moiety of hypoxanthine base substi-tutes for the exocyclic amino group of adenine The amount of d(pI)20incorporated in the filament was less than with d(pT)20 but d(pI)20formed a stronger com-plex with RecA than did d(pA)20(Fig 3)
RecA is a DNA-dependent ATPase, with the effi-ciency of ATP hydrolysis correlating with the stability and length of the RecA filament [36] Figure 4 shows that the extent of ATP hydrolysis correlates well with the efficiency of RecA filamentation on various d(pN)20, allowing us to use ATP hydrolysis to estimate the RecA filamentation efficiency and the stability of the resulting nucleoprotein filaments for a variety of DNA substrates
The highest values of ATP hydrolysis rate (expressed
as percentage of initial ATP) in the presence of differ-ent polynucleotides are summarized in Table 2 The results show that DNA substrates can be divided into three classes according to the efficiency of ATP hydro-lysis stimulation (Table 2) Although both guanine and hypoxanthine have an acceptor O6 and a donor NH1 moiety, poly(dG) was similar to poly(dA) in poor sti-mulation of ATP hydrolysis Deamination of poly(dG) and poly(dA) significantly increased the rate of ATP hydrolysis and the efficiency of filamentation Similar
A
B
Fig 3 Efficiency of RecA filamentation on 32 P-labeled d(pT)20, d(pA)20, and d(pI)20: electrophoretic mobility shift after 5 min of incubation (A) and time course of filamentation (B) d(pT) 20 (m), d(pI)20(d), d(pA)20(j).
Trang 7increase in ATP hydrolysis accompanied a switch from
poly(dAG) to a mixed deoxy(inosine⁄ xanthine)
poly-mer DNA containing both purines and pyrimidines
displayed wide variations in its interactions with
RecA For instance, poly(dAC) and poly(dTG) were
efficiently bound by RecA, poly(dAT) fell between
poly(dA) and poly(dT) ligands, and poly(dCG) promo-ted very little ATP hydrolysis The data in Table 2 indicate that purine poly(dN), even those containing exocyclic hydrogen bond acceptors, generally interacts with RecA and stimulates ATP hydrolysis less effi-ciently than pyrimidine polymers Perhaps the reason
is larger size of purine bases compared with pyrimi-dines, hindering binding of the former by RecA In addition, contacts formed by RecA could be important not only for the complex formation but also for con-formational changes in individual RecA monomers and their ATPase activity
Deamination of mixed polynucleotides with forma-tion of dI from dA, dX from dG, and dU from dC, caused an increase in the efficiency of interactions with RecA, especially for the poly(dCG)fi poly(dUX) transition Interestingly, RecA interaction with purine ligands was also improved by replacement of adenine exocyclic amino group with a halogen atom, also a hydrogen bond acceptor due to its lone electron pairs
Discussion
We have previously shown that the interaction of dif-ferent sequence-specific DNA enzymes (repair, topo-isomerization, restriction, integration enzymes) with each nucleotide unit of nonspecific ss- or ds-ODNs is usually a superposition of weak electrostatic and hydrophobic or van der Waals interactions with the individual structural elements [25–27] The interaction can be described by the power law:
Kd½dðpNÞn ¼ Kd½ðPiÞðeÞnðhCÞcðhTÞtðhGÞgðhAÞa;
where Kd[(Pi)] is the Kd for the minimal orthophos-phate ligand (or sometimes dNMPs), e is a factor reflecting an increase of affinity due to one internucleo-side phosphate group; hNare coefficients of increase in affinity due to hydrophobic and⁄ or van der Waals interactions of the enzyme with one of the bases: C, T,
G and A, the numbers of which in d(pN)nare equal to
c, t, g and a, respectively In addition, factor f reflect-ing increase in affinity due to one (pN)-unit is equal to (hN · e) When passing from one enzyme to another only the values of e (1.35–2.0) and hN(1.0–1.4) factors and Kd for orthophosphate (10-3-10-1m) or dNMP as minimal ligands are changed [25–27] As shown above,
a similar algorithm I50[d(pN)n]¼ I50(dNMP)· f1–ncan
be used for description of RecA filament interaction with ssODNs
Protein globules of various enzymes usually cover from 10 to 20 nucleotides of DNA and the affinity of the enzyme active center (or its specific site) for one
Fig 4 Time course of RecA filamentation on 32 P-labeled d(pT)20,
d(pC) 20 , and d(pA) 20 (A) and RecA-dependent [ 32 P]ATP[cP]
hydro-lysis stimulated by the same ODN (B) d(pT) 20 (m), d(pC) 20 (d),
d(pA)20(j).
Table 2 Highest levels of RecA-catalyzed ATP hydrolysis in the
presence of various poly(dN).
DNA
ATP
hydrolyzed (%) DNA
ATP hydrolyzed (%)
Trang 8nucleotide of d(pN)10)20 is usually significantly higher
(Kd¼ 10)3)10)1m) than for the remaining 9–19
nucleo-tides of DNA (Kd¼ 0.5–0.8 m) [25–27] RecA was no
exception, accepting free orthophosphate (I50¼ 0.5 m)
and various dNMPs (I50¼ 12–20 mm) as minimal
lig-ands (Table 1) These experimental I50 values for free
minimal ligands of RecA do not coincide with Kd
val-ues reflecting the affinity of a single internucleoside
phosphate (I50¼ 0.15–0.23 m) or a single d(pN) unit
(I50¼ 0.063–0.1 m) when they are structural elements
of longer d(pN)n (Table 1) Similarly to some other
enzymes [25–27], the latter I50 values were determined
by extrapolation of lg dependencies to n¼ 0 or n ¼ 1,
respectively (Fig 2; Table 1) Interestingly, the affinity
of a single internucleoside phosphate or a single d(pN)
unit of d(pN)n for RecA is comparable with the
affinity of these DNA structural elements in the case
of other enzymes [25–27]
Usually interactions of various enzymes with
mono-nucleotides of d(pC)n, d(pT)n, d(pG)n and d(pA)n are
additive and elongation of these d(pN)nby one
nucleo-tide unit results in an increase in the affinity by a factor
f of 1.4–2.0 [25–27] In principle, similar results were
observed for RecA in the case of all d(pN)n(see above)
The affinity of some enzymes for d(pN)n does not
always depend on the relative hydrophobicity of the
bases (f¼ 1) However, if the enzyme interacts with the
bases, the increase in affinity for such ODNs usually
follows the same order as the increase in the relative
hydrophobicity of the bases: C < T < G < A (hN¼
1.1–1.4) [25–27] The likely reason for this correlation
is the formation of very weak hydrophobic and⁄ or van
der Waals contacts of different efficiency and different
free energy gain upon transfer of these bases from
water to more hydrophobic DNA-binding sites of the
enzymes In a deviation from this empirical rule, RecA
binds more hydrophobic d(pA)20 approximately
240-fold less efficiently than d(pT)20and d(pC)20(Table 1)
A 25-fold decrease in the affinity of d[p(Et)T]10as
compared with d(pT)10(Table 1) has shown that
inter-nucleoside phosphate groups are important for RecA
filament interaction with ssDNA From the comparison
of I50 for d[(pT)2(pR)17pT] and d(pT)20 ( 100-fold)
the increase in affinity due to a single internucleoside
phosphate residue was estimated as the factor e¼ 1.56
The calculated I50 for totally abasic oligomer d(pR)20
( 2.1 · 10)5m) was found practically the same as I50
for d(pA)20 (2.4· 10)5m) (Fig 2, Table 1) This data
indicate that the filament probably does not or interact
very weakly with poly(dA) adenine bases and contacts
mostly with its phosphate groups The factor e (1.56) for
RecA is comparable with e factors for other enzymes:
uracil-DNA glycosylase (1.35), AP endonuclease (1.51),
DNA polymerases (1.52), Fpg (1.54), RNA helicase (1.61), topoisomerase I (1.67), EcoRI (2.0) and DNA ligase (2.14) [25–27] DG )0.4 kcalÆmol)1 corres-ponding to factor e¼ 1.56 is significantly lower than would be expected for strong electrostatic contacts (up
to)1.0 kcalÆmol)1), but comparable with the values for weak ion-dipole and dipole–dipole interactions [38] Thus, as in the case of the above-mentioned enzymes, the interaction of negatively charged internucleoside groups of ODNs with the RecA filament likely relies on dipolar electrostatic interactions rather than on electro-static interactions of immediately contacting groups From the ratio of factor f¼ 2.04 reflecting the increase in the affinity due to one (pN) unit of d(pT)n and d(pC)n and factor e¼ 1.56 showing the increase
in affinity due to a single internucleoside phosphate of these ODNs, the increases in affinity due to RecA interactions with a single T or C base were estimated
as the factors fT¼ fC¼ 1.31 Thus, RecA in the fila-ment forms weak additive contacts with each inter-nucleoside phosphate moiety and each base of pyrimidine ODN, with the phosphates contribution into the affin-ity being 1.2-fold more than that of C or T bases
As the relative affinity of RecA for d(pC)n, d(pT)n, and d(pA)ndoes not correlate with the relative hydro-phobicity of their bases and RecA does not interact with the bases of d(pA)n, it is reasonable to suggest that RecA could interact with C and T bases by form-ing specific bonds with appropriate amino acids rather than through nonspecific hydrophobic contacts Wittung et al reported that entalpy of Rec A bind-ing to ssDNA in the presence of [35S]ATP[cS] depends
on the base sequence with a clear preference to T than
to A and C bases [39] Similar results concerning higher affinity of RecA to poly(dT) than to poly(dA) and poly(dC) were demonstrated in the absence of cofactor [3] Thus, our data are in agreement with the preferential interaction of RecA with d(pT)n in com-parison with d(pA)n, but not with the data concerning d(pC)n However, data about interactions of RecA with poly(dC) reported in the literature are quite con-tradictory Amarahung et al observed that poly(dC) is
a very bad effector of ATPase activity of RecA [2] In contrast, McEttee and Weistock reported poly(dC) to
be the most efficient effector of the RecA ATPase activity [40] Binding of RecA to poly(dC) under a var-ity of conditions has been found to be worse than to other DNA sequences [40] Thus, the observed differ-ences for poly(dC) interaction with RecA cannot be easily explained
Unlike C and T bases, adenine possesses no exo-cyclic acceptors suitable for hydrogen bonding with RecA amino-acid residues Deamination of homo- and
Trang 9mixed polynucleotides with formation of dI from dA,
dX from dG or and dU from dC containing C¼ O
exocyclic hydrogen bond acceptors also promote
for-mation of more stable RecA⁄ ssDNA filament
com-plexes (Table 2) In addition, RecA had high affinity
to poly(dN) containing exocyclic acceptor halogen
atom instead amino group and to d(eA)n (eA, 1,N6
-ethenoadenine), in which a hydrogen bond donor
moi-ety at C6 is also replaced with an acceptor group (data
not shown) Thus, it can be suggested that the RecA
filament monomers possess in special positions of sites
for binding nucleobases hydrogen bond-donating
groups, which can form contacts with C¼ O exocyclic
acceptor groups at C6 of purines and C4 of
pyrimi-dines Figure 5 demonstrates schematically possible
hydrogen bonds of RecA with different DNA bases
Thus, NH2groups of G bases of ss poly(dG) can form
hydrogen bonds with an appropriate group in RecA
(for example, OH groups of Ser, Thr, Tyr, or acidic
amino acids) Oxygen atoms of G bases (Fig 5) can
interact, for example, with hydrogen atoms of
guanidi-nium groups of Arg residues (or NH2 groups of Lys
residues) Similar hydrogen bonds can be formed by C
and T bases, but there is no possibility for A bases to
form such bonds (Fig 5) which may be one reason for
the low affinity of RecA for d(A)20(Fig 2)
As mentioned above, specific interaction of RecA
with one C or T base leads to the increase in d(pN)n
affinity by a factor of 1.31 (DG ¼)0.16 kcalÆmol)1)
Interestingly, this DG value is significantly lower than
DG values (from )1 kcalÆmol)1 up to )6 kcalÆmol)1)
for strong hydrogen bonds which were observed
between enzymes and different small ligands [38]
However, a formation of very weak hydrogen bonds is
a common situation at recognition of lengthy DNA by
various enzymes [25–27] During formation of a
speci-fic complex of dsDNA with EcoRI, 12 specispeci-fic
hydro-gen bonds are formed, providing in total only about
two orders of affinity [32] This means that the energy
of every of these 12 bonds is rather low (DG
)0.23 kcalÆmol)1) and comparable with the energy
of weak additive nonspecific interactions (see above)
DG )0.28 kcalÆmol)1 is characterized each of five
pseudo-Watson–Crick hydrogen bonds formed by a
uracil residue with uracil DNA glycosylase [28]
Sim-ilar weak specific contacts with nucleotides of DNA
were observed for all other investigated sequence
speci-fic enzymes [25–35]
Altogether, the efficiency of RecA filament
inter-action with any individual nucleotide unit (I50¼ 0.5–
0.76 m) except one (I50 63–100 mm) is very low
Nevertheless, the additivity of RecA filament
inter-actions should provide extremely high affinity of the
filament to long ssDNA It is reasonable to suggest that the presence of exocyclic acceptor groups capable
of hydrogen bonding to the protein can be a critical factor accounting for the efficiency of ssDNA binding
by RecA Depending on the type of the nucleobase (purine or pyrimidine), the nature of RecA interaction with the bases and the conformation of RecA monomers may differ, which could play a key role in the search for homologous DNA One cannot exclude that interaction
of complex of RecA filament and ssDNA with dsDNA can lead to reorganization of firstly formed hydrogen bonds between protein and bases (Fig 5) and assist formation of new hydrogen bonds between C and G
or T and A bases of new DNA duplex
Experimental procedures
Materials
ATP, ATPcS, poly(N), and poly(dN) were purchased from
Fig 5 Proposed RecA amino-acid residue interactions with G, A, C and T bases of poly(dN) Impossibility of hydrogen bond formation
is marked (filled star).
Trang 10(2000 CiÆmmol)1), from Amersham Biosciences (Piscataway,
NJ, USA) Deaminated oligo- and polynucleotides were
synthesized as described in [41,42] To substitute amino
groups of different nucleobases in polynucleotides with
halogen atoms, the deamination reactions were performed
in the presence of 1 m of respective sodium halides
ODN were synthesized, purified and characterized as
described [43] All ODN were proven homogeneous by
ion-exchange and reverse-phase chromatography
Concentra-tions of the ODNs were determined from their absorption
at 260 nm using molar extinction coefficients calculated
according to [44] ODN were 5¢-labelled using bacteriophage
Electrophoreti-cally homogeneous E coli RecA protein was prepared as
described [45]
RecA filamentation
The reaction of RecA filamentation was carried out with
5¢-[32P]d(pT)20 or 5¢-[32P]d(pT)40 at 30C for 5 min The
[35S]ATP[cS], 0.1 lm 5¢-[32
filamenta-tion inhibitors were added in various concentrafilamenta-tions
d(pT)20)40 were obtained using 5¢-[32
a filamentation substrate The reactions were initiated by
P]d(pT)20,40
and one of the inhibitors Free 5¢-[32P]d(pT)20,40 was
electrophoresis in 10–20% nondenaturing polyacrylamide
gel [12] in TBE buffer The results were visualized by
auto-radiography, the bands were cut out from the gel and their
radioactivity determined by Cherenkov counting Affinity
values (inhibitor concentration producing a 50% decrease
in filamentation)
DNA-dependent ATPase activity of RecA
The efficiency of ATP hydrolysis by RecA in the presence
(pH 7.5) The standard reaction mixture (20 lL) included
1 mm DTT, 1 mm ATP, 4 lm RecA, and poly(dN) or
poly(N) in the concentration 0.1 mm nucleotides, or 70 lm
with-drawn and spotted on a TLC plate Vertical development
of the plate was performed in the ascending mode using 0.3
Cherenkov counting
Acknowledgements
The research was made possible in part by grants from the Program of Basic research of the Presidium of RAS ‘Presidium of the Russian Academy of Sciences (Molecular and Cell Biology Program 10.5)’, from the Russian Foundation for Basic Research, and from the Siberian Division of the Russian Academy of Sciences
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