Electron Micrograph Library
DNA & DNA-Protein Complexes
Institute for Molecular Virology. University of Wisconsin -
If there are any problems please contact Ross Inman: firstname.lastname@example.org
Copyright © 1994-2005: These
images can be freely used for educational or research purposes, but are
not in the public domain and are not to be used for any type of
0000a.jpg: RuvB protein bound to ds
linear DNA. Lisa Iype, Cox & Inman.
0001a.jpg and 0001b.jpg: RecA(K72R)-mediated
recombinational intermediates formed between M13mp8.52 ssC DNA and a
short dsL region from M13mp8.52 DNA containing an additional 52bp
medial heterologous insert. In this case the mutant RecA protein is
unable to bypass the medial heterology and the reaction stalls when
strand exchange reaches the heterology. However RecA(wt) protein is
able to bypass such heterology in a similar reaction ( 0001c.jpg ) to produce a completed product. An
exceptional RecA(K72R)-mediated intermediate is shown in 0001d.jpgwhere apparently the strand exchange
started from both ends of the linear substrate, but again the reaction
was stalled by the medial heterology. Method: cyto-C. Samples AMT
crosslinked. Shan,Cox & Inman. JBC 1996 271 5712-5724
0002a.jpg and 0002b.jpg: RecA-mediated
recombinational intermediates. The exchange has been blocked by 375bp
distal heterology. The resulting intermediates have completed strand
exchange up to the distal heterology, resulting in circles with a long
ssDNA tail and a short dsDNA tail (corresponding to the distal 375bp
heterology). When such a reaction is followed by the addition of RuvAB
proteins, the short dsDNA tail is apparently unwound to yield a single
stranded tail as shown in 0002c.jpg or 0002d.jpg. An exceptional type is shown in 0002e.jpg where an intermediate has been caught in
the act of reverse strand exchange. Method: cyto-C. Samples AMT
crosslinked. Lisa Iype, Inman & Cox JBC 1995 270 19473-19480.
0003b.jpg: RecA-mediated recombinational intermediate between a
gapped circle and ds linear DNA. Strand exchange has progressed very
close to the end of the ss region within the gapped circle. Reactions
can progress past the ss region into the duplex part of the gapped
circle resulting in a 4-strand exchange reaction and a Holliday
junction ( 0003c.jpg). Method: cyto-C. Samples
AMT crosslinked. Shan, Cox & Inman. JBC 1996 271 5712-5724
0005a.jpg: RecA-mediated recombinational
intermediate between a ss circle and two ds linear fragments, both
homologous with two different sections of the circle. A further
example is shown in 0005b.jpg. Method: cyto-C.
Wendy Bedale & Cox 1995
. 0006a.jpg:Single stranded DNA bound
with SSB. On the left side of this image is a dsL DNA molecule and
on the right are examples of the same DNA sequence but in a ssC form
after binding with SSB protein. It can be seen that the apparent DNA
length is much shorter and this is because SSB can loop ssDNA around
itself and therefore cause an apparent shortening of the DNA. 0006b.jpg shows similar SSB bound circles but now
the SSB protein has been gold-tagged using SSB antibody to which was
added protein A\gold. See Gold labeling protocol
for details of the gold labeling technique. Maria Schnos
0007a.jpg: RecA protein bound to form(X)
DNA. Maria Schnos.
0008a.jpg: RecA-mediated recombinational
product from a ss circle and a ds linear molecule containing 1037bp
medial heterology which was bypassed with the help of RuvAB proteins.
The heterology leads to a 1037b ssDNA loop within the product. Another
example, involving only 375bp of medial heterology is shown in 0008b.jpg. Method: cyto-C. Lisa Iype,
Elizabeth Wood, Inman & Cox JBC 269 1994 24967.
0009a.jpg: RecA-DNA filament + RecA
antibody + Gold(10nm)-protein A. Another example is shown in 0009c.jpg under higher gold labeling conditions. 0009d.jpg shows that when the same experiment is
performed in the absence of RecA antibody, no gold label is observed on
the RecA-DNA filament. Method: See Gold
labeling protocol. Maria Schnos.
0010a.jpg and 0010b.jpg:
Reovirus "spider". Reovirus was prepared under conditions of gentle
crosslinking, viral disruption and partial denaturation of the ds RNA.
It turns out that at least 7 of the 10 reovirus RNA segments have unique
ends attached to the residual reovirus proteins. A simpler example of
this technique, applied to phage P2, is shown in
0020a.jpg. Method: cyto-C. Maria Schnos, Nibert & Inman.
0011a.jpg: 10nm Gold particle conjugated
to Z- DNA antibody which in turn is bound to a 23bp d(G-C) Z-DNA
segment within plasmid DNA. The photo shows a plasmid with a single
gold particle, also two plasmids held together at the Z-DNA tracts with
gold and another plasmid with just the antibody bound. 0011b.jpg shows a further example, in this case
one plasmid has gold label and the other does not. When such molecules
are cut with restriction enzyme, it can be shown that the gold label is
situated at a unique position corresponding to the Z-DNA tract. Method:
cyto-C. John Jackson & Inman. Gene 1989 84 221-226.
0012a.jpg: RecF coated dsC DNA in
presence of gamma(S)-ATP; Just before spreading, extra DNA was
added. On the left and right are shown poorly and completely coated
plasmid circles. Method: Alcian. Brian Webb, Cox
0013b.jpg: RecR + dsL DNA + ATP. Very
little binding. RecR multimers can be seen in background and have
visible fine structure in some cases.
0014a.jpg: RecA + dsC DNA + gamma(S)-ATP.
Often, under these preparative conditions, the filaments appear to be
supercoiled ( 0014b.jpg).
0015a.jpg: Flock of Maxwell's demons
carrying out examination of dsL and ssC DNA. Method: cyto-C. Inman.
0016a.jpg: SSB coated ssC PhiX174 DNA.
Also shown is a dsL PhiX174 molecule. SSB causes a large apparent
decrease in the contour length of the DNA. Method: X-linked
and cyto-C. Maria Schnos.
Bacteriophage lambda DNA. This genome contains over 48.5 Kbp
DNA. The molecule has been spread under partial denaturing conditions
in order to see the unwound A+T rich regions which are located on the
right half of this genome. 0025a.jpg: Method:
Cyto-C at high pH. Maria Schnos & Inman.
0017a.jpg: Bacteriophage lambda DNA
replicative intermediate isolated from infected E. coli. Mode of
replication is bidirectional and the ss connections arising from
synthesis on the lagging strand templates are situated in a trans
position across the daughter strands. The replicative intermediate has
been partially denatured to allow a determination of the origin of
In the following example bacteriophage lambda DNA replicative
intermediates were crosslinked with psoralen + UV to prevent complete
separation of strands after denaturation of the DNA. Negatively
supercoiled DNA is much more resistant to denaturation then relaxed
DNA. As can be seen in this example, after denaturation, the daughter
strands are highly denatured (but held together by the crosslinking)
whereas the remaining parental DNA is essentially native. This shows
therefore that such replicative intermediates have two domains, the
daughter DNA is relaxed but the parental section is negatively
Maria Schnos & Inman. (J.Mol.Biol. 1987. 193, 377). Initiation of
bacteriophage Lambda replication requires that the initiation protein O
binds to the ori region of DNA and that the origin region be negatively
supercoiled, The plasmid pOri2 contains two lambda origin regions and
when O protein is reacted with this plasmid, both ori regions are
brought together by O protein. 0018a.jpg:
Similar experiments to the above, using real Lambda replicative
intermediates, give similar but more complicated results. O protein
brings both daughter origins together. In the following molecule,
bidirectional replication has just begun and the origins\O protein
position can be seen just a little below the mid point of the daughter
segments. The growing point at the top has begun to branch migrate
(perhaps as a result of the isolation process). SSB protein was added
prior to O protein to avoid complications to do with O protein also
binding to ssDNA segments within intermediates.
Aberrant Replicative intermediates & repair of DNA
If SSB is omitted then in some cases, both origins and both growing
points are all bound together at a single position by O protein.
The next image shows another lambda replicative intermediate reacted
with protein O, in which replication has proceeded much further around
the DNA circle. Again both daughter origin regions are held together by
O protein. 0019b.jpg
Maria Schnos & Inman.
0020a.jpg and 0020b.jpg:DNA
released from Bacteriophage P2. DNA can be released from
bacteriophage P2 by gently cross-linked and spreading under conditions
that tend to disrupt phage heads but leave phage tails intact. Under
these conditions it is possible to demonstrate close proximity of a DNA
end with the proximal end of the tail. The DNA end involved can be
determined from the partial denaturation pattern and is found to always
be the left end of the P2 genome. This DNA end is presumably already
part way into the tail tube and the implication can be made that this
end is the first to enter the bacteria upon infection. Much more
complicated examples of this technique, using reovirus (which contains
10 internal dsRNA segments), are shown in 0010a.jpg
and 0010b.jpg. Method: cyto-C. Dhruba
Chattoraj & Inman.
Recombinant DNA molecules resulting from reaction with the Avian
Myeloblastosis virus integrase (IN). In
0026a.jpg: are shown examples of the initial unreacted substrate DNA
and an end-to-end dimeric recombinant (A) and a Y-type recombinant
intermediate, the top right arm corresponds to a monomeric unit while
the other arms together arise from another monomer (B). This sample was
partially denatured before spreading to produce denatured sites which
were an aid in identification of the recombinational intermediate
components. (C) Y-type recombinant; an extreme example of that shown in
(B) where the recombinant junction is very close to a molecular end thus
yielding a very short third arm. The above examples show components of
the reaction mixture after removal of IN protein. In 0026b.jpg is shown intermediates with protein
still intact. Supercoiled plasmid was incubated with IN protein, then
crosslinked with HCHO and linearized with ScaI before spreading. (A)
Example of an interaction within a single DNA molecule leading to a
loop. (B) Interaction between two DNA molecules mediated by the IN
complex. Duane Grandgenett, Inman,Vora & Fitzgerald. J Virology 67,
2628-2636 (1993) Method: cytoC
RecO and RecR proteins prevent RecA filament disassembly from
the 5' end of ss linear DNA. When RecA is added to circular ssDNA
followed by incubation in the presence of SSB and an ATP regeneration
system, the circles are completely coated with RecA and only a minor
number have filament imperfections in which short regions can be seen
to not contain bound RecA. Examples of both the major and minor types
are shown in the following micrograph ( 0027a.jpg).
In contrast, when linear ssDNA is treated in the same way, RecA again
coats the DNA but almost always the coat does not extend completely to
one of the ends. These single stranded ends tend to get longer when the
reaction is incubated for extended times and as a result the regions
coated with RecA decrease in length. This disassembly occurs at 5' DNA
ends whereas RecA assembly proceeds 5' to 3' with respect to the DNA. ( 0027b.jpg or 0027c.jpg).
The single stranded ends can be shown to contain SSB by reacting SSB
antibody with the complex followed by protein A-gold (
0027d.jpg or 0027e.jpg). See Gold protocol for details of gold labeling. If
linear filaments, containing the SSB-coated ss ends, are now
additionally incubated with RecO-RecR proteins, the DNA is found to be
completely bound with protein and the SSB-coated single stranded DNA
ends are no longer observed ( 0027f.jpg). Qun
Shan, Julie Bork, Inman and Cox. J. Mol. Biol. 1997 265 519-540.
RecA-mediated 4-strand exchange that exhibits an interesting DNA
unwinding activity. 0028.htm.
RecF protein bound to DNA prevents the propagation of a RecA
filament. When RecA protein is added to a gapped DNA circle in the
presence of SSB and a regeneration system, the RecA initiates
cooperative binding within the single stranded gap, a RecA filament
then propagates 5' to 3' across the ss region and rapidly extends into
the dsDNA within the circle. The final result is a completely
filamented circle ( 0029a.jpg).
If RecF protein is added to gapped circular DNA it binds non
cooperatively and within any one sample there can be a wide variety of
such complexes; some circles possess just a few randomly bound proteins
while others have many. Furthermore as the ratio of RecF/DNA is
increased, more binding can be observed and eventually this leads to
circles that are essentially fully bound by protein; yielding an
amorphous type of filament that lacks the striations that are so
obvious in RecA filaments. The increased binding of protein is shown in
the following series ( 0029b.jpg and 0029c.jpg and 0029d.jpg.
When gapped circles are prebound with RecF and RecR and then reacted
with RecA, full RecA filamentation is prevented. The effect requires
RecF and is enhanced if RecR is also present. As the ratio of
RecFR/DNA is increased, the attenuation of RecA filamentation is
increased and eventually no RecA is bound, as shown in the following
micrographs ( 0029e.jpg and
0029f.jpg and 0029g.jpg). Gold antibody
labeling shows that the regions not filamented by RecA contain both
RecF ( 0029h.jpg) and RecR (
These results, along with other data, support the idea that randomly
bound RecFR prevents passage of the leading edge of a growing RecA
filament; perhaps by directly blocking its propagation. Evidence
suggests that this blocking effect is stronger on ds than ss DNA. This
property of RecFR complexes may play an important role in the
prevention of RecA filamentation of normal ds DNA that is in the
vicinity of a site requiring recombinational repair of a ss region
containing a lesion. Such a property would have the effect of
conserving RecA molecules by preventing their tendency to filament
adjacent regions of a chromosome that are not in need of repair. Brian L
Webb, Cox and Inman. Cell 1997 91 347-356.
Interaction between Integration Host Factor (IHF) and the DNA
Replication Origin of Plasmid R6K
Binding of IHF to a fragment of double stranded DNA containing the
gamma replication origin produces a very tight fold in the DNA as shown
in the following micrographs. The origin region is situated almost
midway along these DNA fragments. The diagram shows the DNA path within
the protein complex. IHF-DNAs.jpg
Marcin Filutowicz and Ross B. Inman. J. Biological Chemistry (1991)
266 24077- 24083
The bacteria Deinococcus radiodurans, besides belonging to a
group of very ancient bacteria, displays a remarkable resistance to a
variety of damaging agents. In particular it is known to easily survive
1,700,000 rads of ionization radiation !
In bacteria, RecA type proteins are often implicated in repair of DNA
lesions and it is of interest to study this type of protein in
Deinococcus. The images below simply show that the Deinococcus RecA
behaves very similarly to RecA isolated from E. coli. rdreca1.gif shows a reaction between RecA,SSB
and linear dsDNA from PhiX174. rdreca2.gif
shows a reaction between RecA,SSB and circular ssDNA from PhiX174. In
the second image, at low contrast, can be seen a few condensed circular
ssDNA molecules reacted with SSB rather than with RecA. J. Kim et al. J.
Bact. 2002, Vol 184 p1649-1660.
If there are any problems contact Ross
Electron Micrograph Library
Original version: 3 May 1994. Most recent update: 30 April 2005.