Vol.
174,
No.
January
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
31, 1991
Pages
660-666
HMG 14 AND PROTAMINE ENHANCE LIGATION OF LINEAR DNA TO FORM LINEAR MULTIMERS: PHOSPHORYLATION OF HMG 14 AT SER 20 SPECIFICALLY INHIBITS INTERMOLECULAR DNA LIGATION Lowell
G. Shellin, Buffalo
Received
November
5,
Nancy W. Futile
VA Medical
Center
and Stephen and SUNY
W. Spaulding
at Buffalo,
NY
1990
HMG 14 and protamine can be used to enhance intermolecular ligation of low concentrations of linear DNA. Adding HMG 14 (50 moles per mole DNA) caused 50% of blunt-ended DNA to form predominantly dimers, and all cohesive-ended DNA to form multimers (>6-mer) in response to T4 ligase. Protamine was maximally effective at 40:1, producing mostly dimers and trimers. Adding higher concentrations of HMG 14 did not affect the ligation pattern of cohesive-ended DNA, while higher concentrations of protamine inhibited the formation of multimers. Phosphorylation of HMG 14 at Ser 20 by Ca”-phospholipid dependent protein kinase abolished the ability of HMG 14 to stimulate intermolecular ligation, but did not substantially interfere with intramolecular ligation, or the binding of HMG 14 to linear or circular DNA as assessed by gel mobility. Thus Ser 20, which is located in the amino terminal DNA-binding domain of HMG 14, appears to modulate DNA-DNA interactions. 0 1991 AcademicPress, Inc.
The high mobility HMG
14 & 17 bind electrostatically
their amino terminal DNA
group protcins
14 & 17 prcfcrcntially to the phosphate
regions (2-6).
Their
(4,7), as might have been expected
proteins
(5).
denaturation
(4,6,7).
also bind to nuclear
proteins.
the acidic residues
the DNA
useful in performing
enhance
duplex, protecting
regions.
are also thought
blunt-end
ligation
ligations
binding
at Ser 6 and at 24 DNA
(4).
HMG
to be electrostatic
14 & 17 (8,9), via
We now present evidence that indicate
of linear DNA
the DNA binding without
DNA
substantially
in dilute solution.
domain, afrecting
inhibits
HMG
which could be Phosphorylating
the ability of HMG
14 to
its ability to bind to DNA.
METHODS 1. Prenaration of Ser 20 Phosnho-HMG 14 Ca++-phospholipid dependent protein kinase (C-kinasc) has previously been shown to specifically phosphorylate HMG 14 on Ser 20 (10). Pure calf thymus HMG 14 (200 ug) was 0006-291X/91
$1.50 660
of
it from thermal
the ability of T4 ligase to form linear multimcrs,
14 at Ser 20, a site within intermolecular
does not result in the unwinding
that phosphorylation
These interactions
(1).
of DNA via basic residues in
14 to naked DNA and to nuclcosomal
in their C-terminal
14 (and protamine)
promote
to DNA
Our recent studies indicate of HMG
backbone
if they had been acting as single-strand
In fact, they actually stabilize
reduces the binding
HMG
binding
associate with active chromatin
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
incubated with rat liver C-kinase type 2, 30 uM ATP, 25 mM Tris (pH 7.4) 6.25mM Mg acetate, 0.3 mM CaCl,, 0.25 mM EGTA, phosphatidyl scrine (30udml) and diacylglycerol (1.25ug/ml), final volume 2.4 ml for SO minutes at 3OC, incorporating slightly more than 1 mole/mole HMG 14 (9-11). The reaction mixture was then rcchromatographed on Mono-S to remove the kinase and unreactcd ATP (12). Native HMG 14 elutes at 0.42 M NaCl,(4) whereas after phosphorylation with C-kinasc. its clution position was shifted to 0.38 M NaCl. On 15% polyacrylamide acid-urea gels, the Scr 20 phospho-HMG 14 ran slower than native HMG 14 and no contaminating native HMG 14 was detected upon silver staining (4.12). The peak in OD,,, at 0.38 M coincided with a sharp peak in radioactivity incorporated from gamma [32P]-ATP, indicating that the shift in position was due to phosphorylation by the Ckinase (12). We also phosphorylated HMG 14 at Ser 6 and Ser 6.24 with CAMP-dependent V8 protcase peptides of these different forms of A-kinase as previously described (12). phosphorylated HMG 14 were separated on a 15% polyacrylamide/lS% glycerol gel (13). Autoradiography of the gel showed specific labeling on separate fragments for each form, as previously reported using tryptic pcptide fragments (10). The amounts of HMG 14 in the native and phosphorylated preparations were determined by o-phthalaldehyde fluorescence (12). 2. Retardation of the Electronhoretic Mobilitv of Double-stranded DNA bv HMG 14 The details of the DNA gel-shirt assay have been described (4). Briefly, mixtures of different forms of pBR322 DNA were preincubated with various concentrations of native HMG 14 or Ser 20 phospho-HMG 14 Car 15 min. at 37C in lx TBE (89 mM Tris HCl, 89 mM Boric Acid, 2mM EDTA) (see figure legends for details). The mobility each of the DNA.5 (linear, nicked circular, or negatively supercoilcd) observed in the presence of a given concentration of HMG 14 was compared on 1% agarose gels to the mobility of the same form of DNA measured when no HMG 14 was present. The ratio of the mobilities with and without HMG 14 was then subtracted from one lo indicate the pcrccnt retardation. 3. Ligation of Linear DNA with Blunt or with Cohcsivc Ends pBR322 DNA was linearized with Pvu II to produce blunt-ended DNA, or with Hind III to produce four-base 5’ protruding (cohesive) ends, and purified by phenol extraction and ethanol precipitation. This DNA was used at a concentration loo low to permit intermolecular ligation by itself (6 x 10e9 molts/L (14)). After prcincubation with various concentrations of either native HMG 14. Ser 20 phospho-HMG 14, Ser 6 phospho-HMG 14, Ser 6,24 diphospho-HMG 14 or salmon sperm protamine (Sigma)(in 50 mM Tris HCI. pH 7.8, 10 mM MgCl,, 20mM DTT, 1 mM ATP, 50ug/ml BSA) at 24C for 15 min. 3 units T4 ligase were added and the incubation continued, generally for 1 hr at 24C as rccommendcd by NEN-Biolabs for their T4 DNA ligase. Ligation was cstimatcd from plots of the disappearance of the linear monomeric DNA and the appearance of DNA multimcrs as a function of the molar concentration of the protein being studied. The amount of DNA in the various bands was assessed by scanning photographic negatives with a Beckman DUgB spectrophotomcter. RESULTS 1. Effect of HMG Increasing blunt-ended formed
the concentration DNA
linear
production
14 on the Tntermolccular
(Fig. 1A).
multimers
related
While
to hcptamcrs) occurred
the formation show that HMG
14 promoted
DNA
with Blunt and Cohesive ends the intermolecular
HMG
at molar ratios around
of multimcrs
ligation
14 per mole DNA,
from Pvu II-linearized
pBR322 1OO:l.
of the DNA by HMG
(see below). 661
of
T4 ligase
DNA
Maximal
Ratios greater
larger than dimers (not shown).
14 effectively saturates linear pBR322
this result suggests that the inhibition
to some type of saturation
case with cohesive-ended
of DNA
In the presence of 50 molts
multimcrs
than 4OO:l actually inhibited shift assays (see 3, below) ratio of 4OO:l.
of native HMG
(dimcrs
of blunt-ended
Ligation
DNA
observed at ratios >400:1
Gel at a may be
14, this does not appear to be the
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
123456789
Fieure
1. EFFECT OF NATIVE AND SER 20-PHOSPHORYLATED HMG 14 IN PROMOTING THE LIGATION OF LINEAR PBR322 DNA A. Blunt-ended DNA: HMG 14 or Ser 20-phosphorylated HMG 14 were incubated with Pvu II-cut pBR322 DNA in a final volume of 50~1 containing lx T4 ligation buffer for 15 at 24C. Three units of T4 ligase were then added and the mixture was incubated for 1 hr at 24C. Samples (IS@) were subjected to electrophoresis in 0.8% GTG agarose (Nusieve) gels containing lx TBE buffer for 19 h at 20C (65 V). Gels were stained with lpg/mI ethidium bromode and photgraphed with Polaroid 665 positive/negative film. Lanes 1: lambda phage DNA cut with Hind III as DNA size marker; lanes 2-9: Pvu II-cut pBR322 DNA linear DNA; lanes 3-9: treated with T4 ligase (3 units/reaction); lanes 4-6: ligated in the presence of native HMG 14 at molar ratios of 5&l, 100~1 and 200~1; lanes 6-9: ligated in the presence of Ser 20 phospho-HMG 14 at molar ratios of 155:1, 31O:l and 62O:l. ” L” = linear pBR322 DNA B. Cohesive-ended DNA: HMG 14 or Ser 20-phosphorylated HMG 14 were incubated with Hind III-cut pBR322 DNA as described above. Lane l-8: Hind III pBR322 DNA (cohesive-ends); lanes 2-8: with T4 ligase; lanes 3-5: HMG 14 at molar ratios of 50:1, 1OO:l and 2OO:l; lanes 6-8: Ser 20-phospho-HMG 14 at molar ratios of 155:1, 310~1 and 62O:l. “N” = nicked circular DNA. Bands running faster than N are covalently closed circular DNA with successively greater linking numbers (20).
Self-annealing DNA
of ds-DNA
with cohesive ends occurs even at low DNA
is longer than about 225 bp, assuming its flexibility
is not reduced
ionic strength
buffers, etc, and assuming that there are no constraints
Adding
14 promoted
HMG
whereas 1B).
it inhibited
intermolecular
intramolecular
ligation
formation
of linear pBR322
of covalently
At molar ratios as low as 451, the CCC DNA
molecular
ligation
of Hind III-linearized
multimers
(predominantly
hexamers)
detected
after short incubations
production
of the multimers.
high concentrations
of HMG
were produced.
(
174,
No.
January
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
31, 1991
Pages
660-666
HMG 14 AND PROTAMINE ENHANCE LIGATION OF LINEAR DNA TO FORM LINEAR MULTIMERS: PHOSPHORYLATION OF HMG 14 AT SER 20 SPECIFICALLY INHIBITS INTERMOLECULAR DNA LIGATION Lowell
G. Shellin, Buffalo
Received
November
5,
Nancy W. Futile
VA Medical
Center
and Stephen and SUNY
W. Spaulding
at Buffalo,
NY
1990
HMG 14 and protamine can be used to enhance intermolecular ligation of low concentrations of linear DNA. Adding HMG 14 (50 moles per mole DNA) caused 50% of blunt-ended DNA to form predominantly dimers, and all cohesive-ended DNA to form multimers (>6-mer) in response to T4 ligase. Protamine was maximally effective at 40:1, producing mostly dimers and trimers. Adding higher concentrations of HMG 14 did not affect the ligation pattern of cohesive-ended DNA, while higher concentrations of protamine inhibited the formation of multimers. Phosphorylation of HMG 14 at Ser 20 by Ca”-phospholipid dependent protein kinase abolished the ability of HMG 14 to stimulate intermolecular ligation, but did not substantially interfere with intramolecular ligation, or the binding of HMG 14 to linear or circular DNA as assessed by gel mobility. Thus Ser 20, which is located in the amino terminal DNA-binding domain of HMG 14, appears to modulate DNA-DNA interactions. 0 1991 AcademicPress, Inc.
The high mobility HMG
14 & 17 bind electrostatically
their amino terminal DNA
group protcins
14 & 17 prcfcrcntially to the phosphate
regions (2-6).
Their
(4,7), as might have been expected
proteins
(5).
denaturation
(4,6,7).
also bind to nuclear
proteins.
the acidic residues
the DNA
useful in performing
enhance
duplex, protecting
regions.
are also thought
blunt-end
ligation
ligations
binding
at Ser 6 and at 24 DNA
(4).
HMG
to be electrostatic
14 & 17 (8,9), via
We now present evidence that indicate
of linear DNA
the DNA binding without
DNA
substantially
in dilute solution.
domain, afrecting
inhibits
HMG
which could be Phosphorylating
the ability of HMG
14 to
its ability to bind to DNA.
METHODS 1. Prenaration of Ser 20 Phosnho-HMG 14 Ca++-phospholipid dependent protein kinase (C-kinasc) has previously been shown to specifically phosphorylate HMG 14 on Ser 20 (10). Pure calf thymus HMG 14 (200 ug) was 0006-291X/91
$1.50 660
of
it from thermal
the ability of T4 ligase to form linear multimcrs,
14 at Ser 20, a site within intermolecular
does not result in the unwinding
that phosphorylation
These interactions
(1).
of DNA via basic residues in
14 to naked DNA and to nuclcosomal
in their C-terminal
14 (and protamine)
promote
to DNA
Our recent studies indicate of HMG
backbone
if they had been acting as single-strand
In fact, they actually stabilize
reduces the binding
HMG
binding
associate with active chromatin
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
incubated with rat liver C-kinase type 2, 30 uM ATP, 25 mM Tris (pH 7.4) 6.25mM Mg acetate, 0.3 mM CaCl,, 0.25 mM EGTA, phosphatidyl scrine (30udml) and diacylglycerol (1.25ug/ml), final volume 2.4 ml for SO minutes at 3OC, incorporating slightly more than 1 mole/mole HMG 14 (9-11). The reaction mixture was then rcchromatographed on Mono-S to remove the kinase and unreactcd ATP (12). Native HMG 14 elutes at 0.42 M NaCl,(4) whereas after phosphorylation with C-kinasc. its clution position was shifted to 0.38 M NaCl. On 15% polyacrylamide acid-urea gels, the Scr 20 phospho-HMG 14 ran slower than native HMG 14 and no contaminating native HMG 14 was detected upon silver staining (4.12). The peak in OD,,, at 0.38 M coincided with a sharp peak in radioactivity incorporated from gamma [32P]-ATP, indicating that the shift in position was due to phosphorylation by the Ckinase (12). We also phosphorylated HMG 14 at Ser 6 and Ser 6.24 with CAMP-dependent V8 protcase peptides of these different forms of A-kinase as previously described (12). phosphorylated HMG 14 were separated on a 15% polyacrylamide/lS% glycerol gel (13). Autoradiography of the gel showed specific labeling on separate fragments for each form, as previously reported using tryptic pcptide fragments (10). The amounts of HMG 14 in the native and phosphorylated preparations were determined by o-phthalaldehyde fluorescence (12). 2. Retardation of the Electronhoretic Mobilitv of Double-stranded DNA bv HMG 14 The details of the DNA gel-shirt assay have been described (4). Briefly, mixtures of different forms of pBR322 DNA were preincubated with various concentrations of native HMG 14 or Ser 20 phospho-HMG 14 Car 15 min. at 37C in lx TBE (89 mM Tris HCl, 89 mM Boric Acid, 2mM EDTA) (see figure legends for details). The mobility each of the DNA.5 (linear, nicked circular, or negatively supercoilcd) observed in the presence of a given concentration of HMG 14 was compared on 1% agarose gels to the mobility of the same form of DNA measured when no HMG 14 was present. The ratio of the mobilities with and without HMG 14 was then subtracted from one lo indicate the pcrccnt retardation. 3. Ligation of Linear DNA with Blunt or with Cohcsivc Ends pBR322 DNA was linearized with Pvu II to produce blunt-ended DNA, or with Hind III to produce four-base 5’ protruding (cohesive) ends, and purified by phenol extraction and ethanol precipitation. This DNA was used at a concentration loo low to permit intermolecular ligation by itself (6 x 10e9 molts/L (14)). After prcincubation with various concentrations of either native HMG 14. Ser 20 phospho-HMG 14, Ser 6 phospho-HMG 14, Ser 6,24 diphospho-HMG 14 or salmon sperm protamine (Sigma)(in 50 mM Tris HCI. pH 7.8, 10 mM MgCl,, 20mM DTT, 1 mM ATP, 50ug/ml BSA) at 24C for 15 min. 3 units T4 ligase were added and the incubation continued, generally for 1 hr at 24C as rccommendcd by NEN-Biolabs for their T4 DNA ligase. Ligation was cstimatcd from plots of the disappearance of the linear monomeric DNA and the appearance of DNA multimcrs as a function of the molar concentration of the protein being studied. The amount of DNA in the various bands was assessed by scanning photographic negatives with a Beckman DUgB spectrophotomcter. RESULTS 1. Effect of HMG Increasing blunt-ended formed
the concentration DNA
linear
production
14 on the Tntermolccular
(Fig. 1A).
multimers
related
While
to hcptamcrs) occurred
the formation show that HMG
14 promoted
DNA
with Blunt and Cohesive ends the intermolecular
HMG
at molar ratios around
of multimcrs
ligation
14 per mole DNA,
from Pvu II-linearized
pBR322 1OO:l.
of the DNA by HMG
(see below). 661
of
T4 ligase
DNA
Maximal
Ratios greater
larger than dimers (not shown).
14 effectively saturates linear pBR322
this result suggests that the inhibition
to some type of saturation
case with cohesive-ended
of DNA
In the presence of 50 molts
multimcrs
than 4OO:l actually inhibited shift assays (see 3, below) ratio of 4OO:l.
of native HMG
(dimcrs
of blunt-ended
Ligation
DNA
observed at ratios >400:1
Gel at a may be
14, this does not appear to be the
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
123456789
Fieure
1. EFFECT OF NATIVE AND SER 20-PHOSPHORYLATED HMG 14 IN PROMOTING THE LIGATION OF LINEAR PBR322 DNA A. Blunt-ended DNA: HMG 14 or Ser 20-phosphorylated HMG 14 were incubated with Pvu II-cut pBR322 DNA in a final volume of 50~1 containing lx T4 ligation buffer for 15 at 24C. Three units of T4 ligase were then added and the mixture was incubated for 1 hr at 24C. Samples (IS@) were subjected to electrophoresis in 0.8% GTG agarose (Nusieve) gels containing lx TBE buffer for 19 h at 20C (65 V). Gels were stained with lpg/mI ethidium bromode and photgraphed with Polaroid 665 positive/negative film. Lanes 1: lambda phage DNA cut with Hind III as DNA size marker; lanes 2-9: Pvu II-cut pBR322 DNA linear DNA; lanes 3-9: treated with T4 ligase (3 units/reaction); lanes 4-6: ligated in the presence of native HMG 14 at molar ratios of 5&l, 100~1 and 200~1; lanes 6-9: ligated in the presence of Ser 20 phospho-HMG 14 at molar ratios of 155:1, 31O:l and 62O:l. ” L” = linear pBR322 DNA B. Cohesive-ended DNA: HMG 14 or Ser 20-phosphorylated HMG 14 were incubated with Hind III-cut pBR322 DNA as described above. Lane l-8: Hind III pBR322 DNA (cohesive-ends); lanes 2-8: with T4 ligase; lanes 3-5: HMG 14 at molar ratios of 50:1, 1OO:l and 2OO:l; lanes 6-8: Ser 20-phospho-HMG 14 at molar ratios of 155:1, 310~1 and 62O:l. “N” = nicked circular DNA. Bands running faster than N are covalently closed circular DNA with successively greater linking numbers (20).
Self-annealing DNA
of ds-DNA
with cohesive ends occurs even at low DNA
is longer than about 225 bp, assuming its flexibility
is not reduced
ionic strength
buffers, etc, and assuming that there are no constraints
Adding
14 promoted
HMG
whereas 1B).
it inhibited
intermolecular
intramolecular
ligation
formation
of linear pBR322
of covalently
At molar ratios as low as 451, the CCC DNA
molecular
ligation
of Hind III-linearized
multimers
(predominantly
hexamers)
detected
after short incubations
production
of the multimers.
high concentrations
of HMG
were produced.
(
