Electron Micrograph Library

DNA & DNA-Protein Complexes

Institute for Molecular Virology. University of Wisconsin - Madison.

  • 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.
     
  • 0003a.jpg or 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
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  • 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 & Inman.

  • 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 replication.
    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 supercoiled. RepLam.jpg:,
    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. 0019a.jpg
    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.

  • Aberrant Replicative intermediates & repair of DNA lesions. reprep.htm

  • 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 ( 0029i.jpg).
    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
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  • 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 Inman: rbinman@wisc.edu

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    Original version: 3 May 1994.       Most recent update: 30 April 2005.