binds to several regions in cos DNA (10, 11) and whose properties resemble those of ... Analysis of the protein sequences of the two IHF subunits has revealed.
Volume 17 Number 5 1989
Nucleic Acids Research
Integration host factor (IHF) stimulates binding of the gpNul subunit of X terminase to cos DNA
Gayle Shinder and Marvin Gold
Department of Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada Received October 21, 1988; Revised and Accepted January 31, 1989
ABSTRACT The X terminase enzyme binds to the cohesive end sites (cos) of multimeric replicating X DNA and introduces staggered nicks to regenerate the 12 bp single-stranded cohesive ends of the mature phage genome. In vitro this endonucleolytic cleavage requires spermidine, magnesium ions, ATP and a host factor. One of the E. coli proteins which can fulfill this latter requirement is Integration Host Factor (IHF). IHF and the gpNul subunit of terminase can bind simultaneously to their own specific binding sites at cos. DNase I footprinting experiments suggest that IHF may promote gpNul binding. Although no specific gpNul binding to the left side of cos can be detected, this DNA segment does play a specific role since a cos fragment that does not include the left side or whose left side is replaced by non-cos sequences, is unable to bind gpNul unless either spermidine or IHF is present. Binding studies on the right side of cos using individual or combinations of gpNul binding sites I, II and III indicate that binding at sites I and II is not optimal unless site III is present. INTRODUCTION The bacteriophage X terminase enzyme plays a key role in the maturation and packaging of the viral DNA. Terminase is composed of the products of the two X genes Nul and A (1), and is responsible for binding and cutting immature DNA at the cohesive end site (cos) (2, 3, 4). The binding and cutting regions have been termed cos B and cos N respectively (3, 4). Experiments with hybrids of X and phage 21 have shown that the Nul subunit of terminase interacts with cos DNA (5). Moreover, DNase I footprinting experiments have revealed three gpNul binding sites to the right of the annealed cohesive ends (6; Figure 1). A CG to TA transition at base pair +154 (-1 and +1 are the 6th and 7th base pairs respectively of the lambda nucleotide sequence (7)) within site I results in the defective growth of phage carrying this mutation (termed cos 154) in IHF-deficient E. coli (8) as well as the inability of gpNul to fully protect this site from DNase I cleavage (6).
© I RL Press
2005
Nucleic Acids Research vitro, cos-cleavage requires spermidine, magnesium ions, and ATP as well as an E. coli protein (9). Two proteins were found that could each fulfill this host factor requirement; Terminase Host Factor (THF) and Terminase Host Factor, which can be Integration Host Factor (IHF) (10). purified from IHF deficient hosts, is a 22K heat stable, basic protein that binds to several regions in cos DNA (10, 11) and whose properties resemble those of the prokaryotic type II DNA-binding proteins or "histone-like" proteins such as HU (12, 13). IHF is a 20K protein which is composed of two subunits encoded by the genes himA and hip (himD) and is required for the site-specific recombination event in lysogenic integration and excision (14). Analysis of the protein sequences of the two IHF subunits has revealed similarities to the type II DNA-binding proteins (15). In addition to its role in X integration and excision, IHF also regulates translation initiation, and transcription of cII in vitro (16, 17, 18), as well as in vitro transcription from PE and PCM of bacteriophage Mu (19). More recently it has been shown to be required for the recombination event that controls phase variation in E. coli (20), and for activating phage fl DNA replication (21). The various roles for IHF in E. coli have been reviewed in some detail (13, 22). IHF has been purified (23, 24) and has been shown to recognize and bind to a specific DNA sequence, i.e. (T/C)N(T/C)AANNNNTTGAT(A/T) (25, 26). The cos region of X contains six sequences (Figure 1) that closely resemble this consensus and these each have different affinities for IHF (8, 27). DNase I protection experiments have shown that IHF strongly protects I1, but only weakly protects I2 at higher IHF concentrations. At IO', I3, and I4, there was weak protection of some base pairs at high IHF concentrations, while at IO there was no detectable protection (27). In the present study we show that IHF probably fulfills its host factor role by promoting the binding of gpNul to some of the latter's three specific sites on the right side of cos. If the left side of cos is removed and/or replaced by other DNA, then IHF or spermidine becomes absolutely essential for We have also made a preliminary investigation into the gpNul binding. question of whether all three sites on the right side of cos can bind gpNul independently or if at least two sites, and therefore the possibility of cooperative interactions, are essential. In
MATERIALS AND METHODS a) Proteins And Nucleic Acids The purification and properties
2006
of gpNul are described elsewhere (28).
Nucleic Acids Research 13
14
11
12 A
H
'Ac
10'
10
*.
Hp
Hp
Hc B
1 61 231 4-23-
Figure 1:
The cos region of bacteriophage lambda. The large arrow the annealed cohesive ends; I, II and III are the three gpNul binding sites; IO, IO', Il, I2, I3, and 14 are the sequences resembling the IHF consensus (25); solid line, X DNA, stippled bar, pUC9 DNA; H, HindIII; Hc, HincII; Hp, HpaII; A, AvaII; B, BamHI. The numbers represent the size of each restriction fragment in base pairs. represents
pWP14
Hd
Hd
H
Hp
Hp
H
D
1H/D
B
+E/Hp +
H
FILL IN
Hp 3' RECESSED ENDS
B D 11BE
pUCO B/Hc
9C HC
pUC9 E/Hc
pGS87 \ /
Hc/H
p/Hc
Hd
Figure 2: Construction of plasmids pGS4, pGS6 and pGS87. Details of the plasmid constructions have been discussed in Materials and Methods. A, AvaII; Hd, HindIII; B, BamHI; Hc, HincII; D, DraI, Hp, HpaII; H, HaeIII. I, II and Arrows above these sites denote the III are the same as in Figure 1. direction of the putative recognition sequence.
2007
Nucleic Acids Research The construction of plasmids pWP14 and pWP32 (cos 154 mutant) has been described (6) and a partial restriction map of the insert is shown in Figure 1. To end-label the fragments for binding studies, pWP14 or pWP32 DNA was
cleaved with BamHI, extracted once with phenol, and then precipitated with ethanol. The 3'-end was labeled using DNA polymerase I (Klenow fragment) from Bethesda Research. Laboratories according to Maniatis et al. (29). After labeling, the DNA was extracted once with phenol, precipitated with ethanol and then cleaved with HindIII. The 423 bp fragment was isolated from a 6% polyacrylamide gel by electroelution. The DNA was precipitated with ethanol and resuspended in water. The 161 bp BamHI/HpaII fragment was isolated by cleaving the 423 bp fragment with HpaII and isolating the 161 bp fragment from an 8% polyacrylamide gel. The 231 bp BamHI/AvaII fragment was isolated by cleaving the 423 bp fragment with AvaII and electroeluting the fragment from an 8% polyacrylamide gel. Plasmid pGS87 was constructed as shown in Figure 2. Plasmid pWP14 was cleaved with EcoRI and HindIII at two restriction sites within the pUC9 polylinker region (30). The cos fragment was isolated and then cleaved with !paII. The 3'-recessed ends of the HpaII sites were filled in using dGTP and dCTP and DNA polymerase I (Klenow fragment) according to Maniatis et al. (29). The 171 bp EcoRI/HpaII fragment was isolated and cloned into EcoRI/HincII cleaved pUC9 DNA to generate plasmid pGS87. Plasmid pGS4 (Figure 2) was constructed by cleaving the BamHI/HindIII cos fragment from pWP14 (6) with HaeIII and cloning the 56 bp BamHI/HaeIII fragment containing gpNul binding site I into BamHI/HincII cleaved pUC9 DNA (30). Plasmid pGS6 (Figure 2) was constructed by cleaving the BamHI/HindIII cos fragment from pWP14 with DraI and HaeIII. The 55 bp DraI/HaeIII fragment containing gpNul binding site II was cloned into the HincII site of pUC9. The fragments from pGS4 and pGS6 were end-labeled as described above. b) DNase I Footprinting Footprinting was carried out essentially as described by Galas and Schmitz (31). The 40 4l reaction mixture consisted of 50 mM KC1, 50 mM Tris-HCl pH7.5, 5% glycerol, 1 mM 0-mercaptoethanol, 10 to 50 fmoles of end-labeled DNA, and where indicated 6 mM spermidine, 2 mM Mg 2+, and 1.5 mM ATP. The amount of gpNul added varied between 3 and 16 pg depending on the preparation used; however, the same preparation was used in any comparative experiments described below. Between 0.03 and 1.5 jg of IHF in a volume of 1 4l was used per reaction (IHF was a gift from Dr. H. Nash of the NIH) and was stored and
2008
Nucleic Acids Research
A
C
B
1 2
1
2
3
4
1
2
3
4
5
6
7
- i_lii7
1:1 ,'T!*
.*.*** '
A. DNase I footprint of gpNul on the BamHI/HindIII cos fragment. Figure 3: Lane 1: 0 Ag gpNul; Lane 2: 16 pg gpNul. B. DNase I footprint of gpNul on the BamHI/HpaII cos fragment in the absenee and presence of spermidine, magnesium ions, and ATP. This DNA fragment is lacking the entire left side of cos and the first 36 bp on the right side. Lanes 1 and 2: the absence of spermidine, magnesium ions, and ATP; Lanes 3 and 4: the presence of these components. Lanes 1 and 3: 0 pg gpNul; Lanes 2 and 4: 16 ig gpNul. I, II, and III are the same as in Figure 1. C. DNase I footprint of gpNul on the BamHI/HpaII cos fragment - studies on the individual effects of spermidine, magnesium ions, and ATP. Lane 1: 0 jg gpNul; Lanes 2-7: 16 pg gpNul; Lanes 1 and 2: spermidine, magnesium ions, and ATP; Lane 3: spermidine; Lane 4: magnesium ions; Lane 5: ATP; Lane 6: magnesium ions, and ATP; Lane 7: no spermidine, magnesium ions, or ATP. I, II, and III are the same as in Figure 1.
diluted in buffer containing 0.5M KCl. After 20 minutes at room temperature, heparin was added to a final concentration of 0.1 pg/ml and the reaction continued for 5 minutes at room temperature. DNase I was added to a final concentration of between 3.9 and 11.6 pg/ml (a 1 mg/ml stock solution of DNase I from Cooper Biomedical was diluted in a solution containing 0.56 M MgCl2, 2 mM CaCl2, 10% glycerol, 50 mM Tris-HCl, pH 7.5, 0.1 mg/ml bovine serum albumin, before use) and the contents of the tube were gently mixed at room temperature for 30 seconds. The reaction was stopped by the addition of 8 pl
2009
Nucleic Acids Research of a solution of 3 M ammonium acetate, 0.25 M EDTA, 0.15 mg/ml sonicated calf thymus DNA (Sigma) and was immediately extracted once with phenol. The DNA was precipitated with ethanol and resuspended in a solution of 80% formamide, 2% xylene cyanol, 50 mM TBE pH 8.3 (50 mM Tris, 50 mM borate, 1 mM EDTA). The reactions were electrophoresed on a denaturing 5% polyacrylamide gel beside the purine and pyrimidine sequencing reactions (32). The gel was dried and the DNA was visualized by autoradiography. RESULTS a) The Left Side Of Cos Is Important For gpNul Binding; The Effect Of
Spermidine. The gpNul subunit of bacteriophage X terminase binds to three sites on the right (+) side of cos (6; Figure 3A). Since DNase I footprinting (31) failed to show evidence of gpNul binding to the left (-) side, we studied binding to a truncated cos fragment lacking sequences to the left of binding site III, in order to ascertain if the sequence to the right of and including site III was The cos DNA previously used was a 423 bp for binding. sufficient BamHI/HindIII fragment derived from plasmid pWP14 (6; Figure 3A), and end-labeled at the BamHI site. The truncated fragment containing the three gpNul binding sites was isolated by cleaving the end-labeled BamHI/HindIII fragment with HpaII (Figure 1) generating an end-labeled 161 bp fragment. This HpaII site is located immediately 5' from gpNul binding site III. Figure 3B shows the results of a DNase I footprint experiment using this truncated Sites I, II and III were not protected from DNase I cleavage cos fragment. (compare lanes 1 and 2). Since it had previously been shown that in vitro cos-cleavage requires at least spermidine, magnesium ions (Mg 2+), and ATP (2), it seemed reasonable to add these components to the footprint reaction to see if they would stimulate binding. Binding in the presence of these components resulted in complete protection of sites I, II and III (Figure 3B, compare In addition, protection was observed at two small regions lanes 3 and 4). between sites I and II. These regions were also protected when DNase I footprinting experiments were performed on the 423 bp cos fragment (6; Figure 3A). It is clear from the above experiment that in the absence of sequences to the left of site III, gpNul will not protect sites I, II and III from DNase I cleavage unless spermidine, Mg2+, and ATP are added to the reaction. Each component was subsequently tested individually in the footprint reaction in order to determine which one(s) was responsible for stimulating gpNul binding
2010
A 1
12
.
i_ ws:_w B12
Nucleic Acids Research
3 4
_ _
XXF''SF ......
_
-
w|
.
is w
.$*.F
N-
1
=X
:
_ _
_
.
_
F
_ _
^
..
1-M
111
Z _
US::e::a
*S]
.,
"
:
lW
_ _ *}5BSS
w.A.
A&
Ai
..
..-
\sf
**
4*:
3n11lt
aiF
s e i.
^ s
.wt w
l
_.
£,,. _|S,-
,l
s
w .X A. DNase I footprint of gpNul on the EcoRI/PvuII fragment of Figure 4: pGS87 in the absence and presence of spermidine, magnesium ions, and ATP. This fragment has 199 bp of pUC9 sequences substituted for cos sequences to the left of gpNul binding site III. Lanes 1 and 2: absence of spermidine, magnesium ions, and ATP; Lanes 3 and 4: presence of the above components; Lanes 1 and 3: 0 ig gpNul; Lanes 2 and 4: 16 ug gpNul. B. DNase I footprint of gpNul on the BamHI/AvaII cos fragment. This fragment is composed of the entire right side of cos but only 34 bp on the left side. Lanes 1 and 2: presence of spermidine, magnesium ions, and ATP; Lanes 3 and 4: absence of the above components. Lanes 1 and 3: 0 jg gpNul; Lanes 2 and 4: 16 jg gpNul.
(Figure 3C). If only spermidine was included (Lane 3), the pattern of protection was indistinguishable from that obtained when all three components were added (Lane 2). If, however, only Mg2+ was added (Lane 4), protection was complete at sites II and III but only partial at site I. This experiment has been repeated several times and in some cases, there was no protection at site I. There was no protection at any of the sites in the presence of only ATP (Lane 5); however, if Mg 2+, and ATP were both present (Lane 6), the pattern of protection resembled that obtained in the presence of Mg2+ alone (Lane 4). These experiments indicate that spermidine is the component whose presence best stimulates gpNul binding to a cos fragment lacking sequences to the left of site III. If the same concentration of putrescine was substituted for spermidine, the three sites remained unprotected (data not shown).
2011
Nucleic Acids Research Although Mg2+ and ATP were not essential for binding, all subsequent experiments that were performed in the presence of spermidine also contained Mg and ATP. The above experiments, in addition to showing a requirement for spermidine/Mg2+ under certain conditions, also demonstrate the importance of sequences to the left of site III for gpNul binding. To answer the question of whether cos sequences were uniquely necessary, or if any DNA sequence to left of the site III could result in the regeneration of the spermidine/Mg -independent footprint, DNase I footprinting experiments were performed on a cos containing fragment from plasmid pGS87 (Figure 2). This plasmid was cleaved with EcoRI, 3'-end- labeled, and then cleaved with PvuII. The 370 bp EcoRI/PvuII fragment thus generated consisted of 199 bp of pUC9 DNA to the left of gpNul binding site III. DNase I footprinting experiments in the absence (Figure 4A, Lanes 1 and 2) and presence (Lanes 3 and 4) of spermidine/Mg2+ again showed the requirement (compare Lanes 2 and 4). It appears, therefore, that cos X sequences are essential for the generation of the spermidine/Mg 2+-independent footprint. Since the 161 bp cos fragment (Figure 1) lacked 36 bp on the right of the annealed cohesive ends in addition to the entire left side, it was unclear whether it was the left side alone that was necessary for spermidine/Mg 2+independent binding or just an intact right side. To answer this question, the 423 bp end-labeled BamHI/HindII fragment from pWP14 was cleaved with AvaII (Figure 1) to generate a 231 bp fragment containing the entire right side of cos but only 34 bp on the left side. Nuclease protection studies on this fragment again showed the requirement (Figure 4B, compare Lanes 2 and 4). It is therefore apparent that in the absence of spermidine/Mg2+ the left side of cos is important for binding of gpNul to sites on the right side. We had previously shown that gpNul protection of the three sites on the bottom strand of cos was not as clearly defined as on the top strand (6). The role of spermidine/Mg2+ presented above led us to repeat the DNase I protection studies on the 423 bp fragment (6) but this time in the presence of the polyamine. While there was no difference in protection pattern for the top strand from that obtained previously in the absence of spermidine/Mg2+, the bottom strand now showed a more clearly defined footprint (data not
shown). 2+ b) Integration Host Factor Can Effectively Substitute For Spermidine/Mg=DNA
2012
Spermidine is a polyamine which is effective in altering the structure of in solution (33, 34). E. coli's Integration Host Factor (IHF) which can
;Aew8i
-_F - . ,
Nucleic Acids Research
A
B12
4
3
____ _
DF
s
z
_
.u;Iu3
Ii
..I
_
>_
_
_
..
.
....
..
::: lr
....
::: grar
:':':
-
3=.
|
Gllm
'.B
_
::-:
w.}
3L ** |. . _ _ . DW,B F r
1w 1
_
,[. _
_
u
,
_ 111
X*** .
.*
XP
*:
.*
.w
N
' *< x a _ t s
I
Abi
*
Ix
.f..
A."p...j:Am.'
..
se
..
w
I
A. The Effect of IHF on the gpNul DNase I Protection Pattern of Figure 5: Lanes 1 and 2: presence of spermidine, magnesium the BamHI/HpaII fragment. ions, and ATP; Lanes 3, 4 and 5: absence of the above three components. Lane 1: no protein; Lane 2: 16 pg gpNul; Lane 3: 16 pg gpNul; Lane 4: 16 pg gpNul and 1.5 jg IHF; Lane 5: 1.5 pg IHF. > = enhanced cleavage at C63. I, II and III are the same as in Figure 1. IHF, IHF binding site. B. Effect of IHF on the binding of gpNul to the BamHI/HpaII fragment of cos 154 mutant DNA. Lane 1: no protein; Lane 2: 16 pg gpNul; Lane 3: 16 pg gpNul and 1.5 pg IHF; Lane 4: 1.5 pg IHF. > = enhanced cleavage at C63; o. enhanced cleavage at C14 . IIm and IIIm are the gpNul binding sites analogous to II and III in te wild-type. 4--> is the region analogous to I in the wild-type. IHF is the same as in A.
bind to sequences within cos (27) has been shown to bend DNA when it binds to lambda att P DNA (35), as well as to sites in IS1 and pBR322 DNA (36). We reasoned that if IHF can bend DNA when it binds, it might be able to replace Nuclease protection studies on the 161 bp BamHI/HpaII fragment spermidine. from pWP14 were performed with gpNul both in the presence (Figure 5A, Lane 2)
2013
Nucleic Acids Research and absence (Lane 3) of spermidine, Mg , and ATP. In addition, IHF was included in a reaction that was lacking all three of the above components In this latter reaction, the footprint was observed at all three (Lane 4). gpNul binding sites. In addition, a fourth region of protection (denoted IHF) was observed which corresponded to the region protected when IHF was the only This region includes the sequence that closely protein present (Lane 5). resembles the IHF consensus and is in fact an IHF-binding site (27). This experiment was repeated several times and in all cases the results were similar except that the degree of protection at site I varied from being In addition, similar results were obtained with the partial to full. BamHI/AvaII fragment containing only 34 bp on the left side of cos, as well as It is with the EcoRI/PvuII fragment from plasmid pGS87 (not shown). interesting to note that C63 (>) which is protected from DNase I cleavage by gpNul binding is not protected in the presence of IHF (Lane 4). Spermidine has no effect on gpNul binding in the presence of IHF (data not shown). These results demonstrate that IHF can substitute for spermidine in the stimulation of binding of gpNul to a cos fragment lacking X sequences to the left of the annealed cohesive ends. c) IHF Does Not Stimulate gpNul Binding To Site I Of The Cosl54 Mutant DNA At protein concentration ) within site II was almost completely protected at levels of 16 pg (Lane 2) and 24 pg (Lane 3) of gpNul, and only partial protection was observed at site I (the intensities of the bands decreased only about two to three fold as determined by a densitometric scan). It therefore appears as if site III is critical for complete protection at sites I and II. Figure 7B on the other hand, shows that at levels of 16 pg (Lane 2) and 24 pg (Lane 3) of gpNul, both sites II and III of the AvaII/HaeIII fragment of pWP14 (5' end-labeled at the AvaII site) were almost fully protected from DNase I cleavage. This latter result is in agreement with observations on the cosl54 mutant. The above experiments suggest that cooperativity does play a role in the binding of gpNul. However, since the absence of site III resulted in little protection of sites I and II while the absence of site I had no effect on protection at sites II and III, it is clear that there are differences in the interdependence of the three sites. Site III appears to be very important for
2016
Nucleic Acids Research
A
B3 ___-
A
-
M
* .....
f
iVl
I
:.Il
'ir..
W
4 lUl
xe
s
Figure 7: DNase I Protection by gpNul on DNA fragments containing two binding sites. A. BamHI/DraI fragment from pWP14. Lane 1: 0 jg gpNul; Lane 2: 16pg gpNul; Lane 3: 24 pg gpNul; > = protected base. B. AvaII/HaeIII fragment from pWP14. Lane 1: 0 pg gpNul; Lane 2: 16 pg gpNul; Lane 3: 24 pg gpNul.
binding at the other two sites while by itself it can bind gpNul but not very well. DISCUSSION We have shown that sequences on the left side of cos X influence the conditions necessary for the binding of gpNul to its three binding sites on the right side. If the left side sequences are not present, binding does not occur unless the polyamine spermidine or Mg2+ are added (Figures 3 and 4). Spermidine was the most effective (Figure 3C). An efficient substitute for spermidine is the E. coli protein, Integration Host Factor (IHF) (Figure 5A). IHF also appears to be able to stimulate binding of low levels of gpNul to
2017
Nucleic Acids Research cos; spermidine and/or Mg2 cannot promote the binding at sub-optimal gpNul The presence of IHF does not, however, promote binding of concentrations. gpNul at any level to the mutant site I in the cos 154 mutant DNA (Figure 5B). DNase I footprinting experiments on individual paired sequences suggest that there is cooperativity among the three gpNul binding sites (Figures 6 and 7). a) The Role Of The Left Side Of Cos, Spermidine, And IHF In gpNul Binding. The absence of the left side of cos results in the binding of gpNul Spermidine, a polyamine, has the becoming spermidine or IHF-dependent. ability to compact DNA (34), while DNA bending has been observed when the histone-like protein IHF binds to DNA (34, 36). Recently, IHF has been shown to induce a bend at its binding site (37; Figure 1). These findings suggest that both spermidine and IHF alter the DNA structure in some way so as to facilitate gpNul binding. Furthermore, it has been shown that binding of IHF to cos DNA results in enhanced DNase I cleavage of specific bonds (27; this work), an observation consistent with the idea of an altered DNA structure. In addition, binding of both IHF and gpNul to cos results in a region (base 63) that shows greater sensitivity to DNase I than when gpNul binds alone. This further suggests that IHF is responsible for altering the DNA structure. DNA bending is an important phenomenon in many high precision DNA transactions, and the stable protein-DNA structures that are formed have been called specialized nucleoprotein structures or 'snups' (38). These structures have been found at the origin of replication of bacteriophage X DNA (39), at the attP site of X DNA (40), and are involved in the transposition of bacteriophage Mu DNA in vitro (41). The transposition 'snup' structure or transpososome requires the 'histone-like' protein HU; however the latter's role has not yet been defined. Although X integration and excision require IHF, the attP 'snup' structure or 'intasome' can be formed without it. It is possible, however, that the structure may be more precise in the presence of IHF (40). The role of IHF in gpNul binding and ultimately cos cleavage may therefore be to promote the formation and/or stabilization of a specialized nucleoprotein structure. gpNul binds tightly to three sites on the right side of cos (as seen by DNase I protection) and weakly to the left side. Although the latter has not been observed in nuclease protection experiments, band retardation experiments with fragments containing only sequences on the left side reveal not a distinct complex, but a diffuse band indicative of This has also been protein-DNA interactions (unpublished observations). observed with some DNA fragments lacking cos sequences. gpNul that is weakly bound to the left side could interact with gpNul molecules bound on the right
2018
Nucleic Acids Research side to even further stabilize binding to the three binding sites. In the absence of certain sequences in the left side of cos, binding to the right side is not stabilized and therefore no footprint is observed unless components such as spermidine, Mg 2+, or IHF which can alter the DNA structure are added to promote gpNul interactions between the three binding sites and therefore strengthen the binding. Recently (42) spermidine has been shown to stabilize binding of E. coli DNA gyrase to the par locus of plasmid pSC101. It is noteworthy that although histone-like ribosomal DNA-binding proteins also stimulated binding, IHF was ineffective as was HU protein, suggesting that the two agents act in different manners. Also, the gyrase binding was carried out in 10 mM Mg , a concentration apparently too low to affect the DNA structure. This is in marked contrast to our results where 2 mM Mg2+ is almost as effective as spermidine or IHF alone. It is known that cationic metals do promote sequence-directed DNA bending (43) but according to these authors, Mg2+ is not very effective at the low concentrations used in this work. b) IHF Is Unable To Restore Full Binding Of gpNul To Site I Of The Cosl54 Mutant DNA The cos154 mutant which has a CG to TA transition at the tenth position of the 16 bp recognition sequence of site I is unable to grow in IHF deficient hosts (8) whereas wild-type X can grow, although with a reduced burst size, in the absence of IHF (44). In addition, the mutant DNA cannot be cleaved efficiently in vitro if Terminase Host Factor (THF) is substituted for IHF (Parris and Gold, unpublished observation). Nuclease protection experiments on this mutant DNA have shown that gpNul is unable to fully protect site I from DNase I cleavage whereas protection at sites II and III is unaffected (6). Since IHF appeared to be a requirement for growth and for DNA cleavage of this mutant, we hypothesized that IHF should stimulate gpNul binding to the mutant site I. This, however, was not the case (Figure 5B), and we must therefore conclude that the promotion of cosl54 DNA cleavage and packaging by IHF is via some mechanism other than stimulation of gpNul binding. If protein-protein interactions are essential for the stabilization of binding and ultimately for cos cleavage and packaging, the absence of gpNul from site I would reduce these critical interactions. IHF would counteract this effect by bending the DNA and interacting with gpNul at sites II and III. Other host factors may be unable to interact with gpNul as favourably as IHF does. It would be informative to make the analogous mutation at sites II and III to see if gpNul binding would likewise be affected, and if so, if IHF would restore
2019
Nucleic Acids Research It has not been possible in these experiments to decisively binding. determine whether THF can also stimulate binding of gpNul to cos. As shown previously (11), THF even at low concentrations effectively protects a very large region of DNA to the right of cos from nuclease digestion; this protection has precluded making meaningful observations on the effects on gpNul binding. c) Cooperative Binding Of gpNul To Its Three Sites On The Right Side Of Cos The data shown in Figures 6 and 7 indicate that gpNul binding sites II and III are interdependent and that optimal binding at site I is dependent on binding at site III. The observation that DNase I protection at sites II and III was independent of site I was first indicated in binding studies with the coslS4 mutant DNA (8), where full binding was still observed at sites II and III even though binding at site I was greatly reduced (6). Early studies on the terminase binding site, localized cos B to the region between +50 and +120 bp (3, 4) a region consisting only of gpNul binding sites II and III. These studies showed that when added in excess, DNA containing this region was able to bind and titrate terminase so that in vitro cos-cleavage of another cos fragment was inhibited. Thus, our results which show nuclease protection at sites II and III in the absence of site I, are in agreement with these early binding studies. In addition, Miwa and Matsubara (4) identified a 15 bp inverted repeat part of which corresponds to our putative gpNul recognition sequence (6) within sites II and III. Elimination of either of these sequences abolished binding (3, 4). The results presented in this paper which show that there is only partial or no protection in the absence of gpNul binding sites II or III, are also in agreement with these earlier findings. d) Conclusions There are many parallels between in vitro X site-specific recombination at IHF is believed to play a role in the attP and in vitro cos-cleavage. formation of higher order 'snup' structures at attP. Based on the many similarities between the two systems, we propose that IHF is playing a similar Electron microscopy shows bending of role in the cos cleavage reaction. cos-containing DNA in the presence of IHF (37). Terminase alone does not bend the DNA, nor does it alter the structure of IHF-bent cos DNA. Our results indicate that complex protein-protein and protein-DNA interactions are occurring when gpNul and IHF bind to cos X DNA. It is now important to look at the role of the A subunit of terminase to see if it plays a part in these This can be most effectively accomplished by using complex interactions.
2020
Nucleic Acids Research purified terminase holoenzyme. By determining how these proteins and the DNA interact, we hope to gain a better understanding of the DNA cleavage event which is central to X DNA packaging. ACKNOWLEDGMENTS This work was supported by a grant from the Medical Research Council of We appreciate the efforts of Dr. P. Sadowski in critically reading Canada. the manuscript and of Wendy Parris for assistance in preparing the gpNul. REFERENCES 1. Sumner-Smith, M., Becker, A. and Gold, M. (1981) Virology, 111, 642-646. 2. Becker, A. and Gold, M. (1978) Proc. Natl. Acad. Sci. USA, 75, 4199-4203. 3. Feiss, M., Widner, W., Miller, G., Johnson, G., and Christiansen, S. (1983) Gene 24, 207-218. 4. Miwa, T. and Matsubara, K. (1983) Gene, 24, 199-206. 5. Frackman, S., Siegele, D.A. and Feiss, M. (1985) J. Mol. Biol. 183,
6. 7. 8. 9. 10. 11. 12.
13. 14.
15. 16.
17. 18.
19. 20. 21.
225-238. Shinder, G. and Gold, M. (1988) J. Virol. 62, 387-392. Sanger, F., Coulson, A.R., Hong, G.F., Hill, D.F. and Petersen, G.B. (1982) J. Mol. Biol. 162, 729-773. Bear, S.E., Court, D.L. and Friedman, D.I. (1984) J. Virol. 52, 966-972. Gold, M. and Becker, A. (1983) J. Biol. Chem. 258, 14619-14625. Gold, M. and Parris, W. (1986) Nucl. Acids Res. 14, 9797-9809. Shinder, G., Parris, W. and Gold, M. (1988) Nucl. Acids Res. 16, 2765-2785. Rouviere-Yaniv, J. and Gros, F. (1975) Proc. Natl. Acad. Sci. USA, 72, 3428-3432. Drlica, K. and Rouviere-Yaniv, J. (1987) Microbiol. Rev. 51, 301-319. Weisberg, R. and Landy, A. (1983) in Lambda II, Hendrix, R.W., Roberts, J.W., Stahl, F.W. and Weisberg, R.A., Eds., pp 211-250, Cold Spring Harbor, New York. Flamm, E.L., and Weisberg, R.A. (1985) J. Mol. Biol. 183, 117-128. Hoyt, M.A., Knight, D.M., Das, A., Miller, H.I., and Echols, H. (1982) Cell, 31, 565-573. Mahajna, J. , Oppenheim, A.B., Rattray, A. and Gottesman, M. (1986) J. Bact. 165, 167-174. Peacock, S., Weissbach, H. and Nash, H.A. (1984) Proc. Natl. Acad. Sci. USA, 81, 6009-6013. Krause, H.M. and Higgins, N.P. (1986) J. Biol. Chem. 261, 3744-3752. Eisenstein, B.I., Sweet, D.S., Vaughn, V. and Friedman, D.I. (1987) Proc. Natl. Acad. Sci. USA, 84, 6506-6510. Greenstein,D., Zinder, N.D. and Horiuchi, K. (1988) Proc. Natl. Acad. Sci. USA, 85, 6262-6266.
22. Friedman, D.I. (1988) Cell, 55, 545-554.
23. Nash, H.A. and Robertson, C.A. (1981) J. Biol. Chem. 256, 9246-9253. 24. Nash, H.A., Robertson, C.A., Flamm, E., Weisberg, R.A. and Miller, H.I. (1987) J. Bact. 169, 4124-4127. 25. Craig, N.L. and Nash, H.A. (1984) Cell, 39, 707-716. 26. Gardner J.F. and Nash, H.A. (1986) J. Mol. Biol. 191, 181-189. 27. Xin, W. and Feiss, M. (1988) Nucl. Acids Res. 16, 2015-2030. 28. Parris, W., Davidson, A., Keeler, Jr., C.L. and Gold, M. (1988) J. Biol. Chem. 263, 8413-8419. 29. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning, 2021
Nucleic Acids Research
30. 31. 32. 33. 34. 35. 36. 37.
A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Vieira, J. and Messing, J. (1982) Gene 19, 259-268. Galas, D.J. and Schmitz, A. (1978) Nucl. Acids Res. 5, 3157-3170. Maxam, A.M. and Gilbert, W. (1980). Methods Enzymol. 65, 499-559. Krasnow, M.A. and Cozzarelli, N.R. (1982) J. Biol. Chem. 257, 2687-2693. Baeza, I., Gariglio, P., Rangel, L.M. Chavez, P., Cervantes, L., Arguello, C., Wong, C. and Montanez, C. (1987) Biochemistry 26, 6387-6392. Robertson, C.A. and Nash, H. (1988) J. Biol. Chem. 263, 3554-3557. Prentki, P., Chandler, M. and Galas, D.J. (1987) EMBO J. 6, 2479-2487. Kosturko, L.D., Daub, E. and Murialdo, H. (1989) Nucl. Acids Res. (in
press) 38. Echols, H. (1986) Science, 233, 1050-1056. 39. Dodson, M., Roberts, J., McMacken, R. and Echols, H. (1985) Proc. Natl. Acad. Sci. USA, 82, 4678-4682. 40. Better, M., Lu, C., Williams, R.C. and Echols, H. (1982) Proc. Natl. Acad. Sci. USA, 79, 5837-5841. 41. Surette, M.G., Buch, S.J. and Chaconas, G. (1987) Cell, 49, 253-262. 42. Wahle, E. and Kornberg, A. (1988) EMBO J. 7, 1889-1895. 43. Laundon, C.H. and Griffith, J.D. (1987) Biochemistry 26, 3759-3762. 44. Miller, H.I. and Friedman, D.I. (1977) in DNA Insertion Elements, Plasmids and Episomes, Bukhari, A.I., Shapiro, J.A. and Adhya, S.L., Eds., pp 349-356, Cold Spring Harbor, New York.
2022
