5.6.2.1: DNA topoisomerase

display a low level of supercoil relaxation. Slow the rate of cleavage by a factor of 60 compared to the wild-type

R135A

form no detectable protein-DNA adduct during a 24 h incubation with the suicide substrate. The failure of this protein to relax supercoiled DNA is probably attributable to its inability to perform transesterification chemistry

R241A

Y294A

V617G

Camptotheca acuminata

B2DDA7

the mutant is resistant against topotecan

748878

D111N

2 entries

D113N

EcTOP, site-directed mutagenesis

D117N

site-directed mutagenesis, induction of mutant YpTOP1-D117N topoisomerase by arabinose is similar in the absence or presence of trimethoprim

727704

G116S

EcTOP 67 kDa N-terminal fragment, site-directed mutagenesis

G194A

40fold reduction in relaxation activity when compared to wild-type enzyme, DNA cleavage activity is 100fold lower than that of the wild-type enzyme

652567

G194R

no detectable relaxation activity and DNA cleavage

652567

H365A

whereas wild type enzyme shows pH-dependent cleavage of oligonucleotides, cleavage by the mutant enzyme is pH-independent. Cleaves DNA with a slower rate than the wild-type enzyme, but can cleave more substrate overall, mutant enzyme has a lower DNA binding affinity than the wild-type enzyme, mutant shows a shift in maximal relaxation to above pH 8.5, mutant enzyme is less able than the wild type enzyme to relax negatively supercoiled DNA

H365D |

mutant shows a shift in maximal relaxation to above pH 8.5, mutant enzyme is less able than the wild type enzyme to relax negatively supercoiled DNA

H365E |

mutant shows a shift in maximal relaxation to above pH 8.5, mutant enzyme is less able than the wild type enzyme to relax negatively supercoiled DNA

H365N

mutant shows a shift in maximal relaxation to above pH 8.5, mutant enzyme is less able than the wild type enzyme to relax negatively supercoiled DNA

H365Q

whereas wild type enzyme shows pH-dependent cleavage of oligonucleotides, cleavage by the mutant enzyme is pH-independent. Cleaves DNA with rates similar to the wild-type enzyme, mutant shows a shift in maximal relaxation to above pH 8.5, mutant enzyme is less able than the wild type enzyme to relax negatively supercoiled DNA

H365R |

mutant shows a shift in maximal relaxation to above pH 8.5, mutant enzyme is less able than the wild type enzyme to relax negatively supercoiled DNA

K13A

Investigation of the potential catalytic role of the serine and lysine residue conserved in the active site region of bacterial topoisomerase I Ser10 and Lys13 of Escherichia coli DNA topoisomerase I are mutated to alanines. 50-to 100fold reduction in relaxation activity, not affected in non-covalent DNA binding, mutations at Ser10 and Lys13 highly reduce the ability of topoisomerase I to support growth of the Escherichia coli cells, as expected from the loss of the relaxation activity. Ala13 is unable to make interactions with the acidic residues of the active site. Side chains of Ser10 and Lys13 are required for DNA cleavage activity, rates of cleavage are too low to be analyzed

K13R

Arg13 residue is a larger residue than lysine, and is unable to fit in the space assigned to Lys13. Reduced DNA binding affinity, possibly changing Ser10 to a threonine residue, and changing Lys13 to arginine allow retention of activity, relaxation activity assays show that introduction of a different side chain with similar functionality but different steric properties at these two positions result in even lower relaxation activitymutations at Ser10 and Lys13 highly reduce the ability of topoisomerase I to support growth of the Escherichia coli cells, as expected from the loss of the relaxation activity. Do not have severe changes in protein folding. Side chains of Ser10 and Lys13 are required for DNA cleavage activity, rates of cleavage are too low to be analyzed

M320R

P06612

site-directed mutagenesis

694442

R169

site-directed mutagenesis, the mutant shows reduced DNA cleavage and relaxation activity compared to the wild-type enzyme, the mutation to alanine changesd the selectivity of the enzyme for the base at the -4 position from a cytosine to an adenine

R173A

site-directed mutagenesis, the mutant displays similar sequence selectivity as the wild-type enzyme, but weaker cleavage and relaxation activity

R321F

site-directed mutagenesis, constants of oligonucleotide binding compared to the wild-type enzyme, the mutant shows reduced DNA cleavage and requirement for Mg2+

R321K

site-directed mutagenesis, constants of oligonucleotide binding compared to the wild-type enzyme, the mutant shows reduced DNA cleavage and requirement for Mg2+

R321L

site-directed mutagenesis, constants of oligonucleotide binding compared to the wild-type enzyme, the mutant shows reduced DNA cleavage and requirement for Mg2+

S10A

Investigation of the potential catalytic role of the serine and lysine residue conserved in the active site region of bacterial topoisomerase I Ser10 and Lys13 of Escherichia coli DNA topoisomerase I are mutated to alanines. 5-to 10fold reduction in relaxation activity, not affected in non-covalent DNA binding, mutations at Ser10 and Lys13 highly reduce the ability of topoisomerase I to support growth of the Escherichia coli cells, as expected from the loss of the relaxation activity. Ala10 is unable to form a hydrogen bond with phosphate 8. Side chains of Ser10 and Lys13 are required for DNA cleavage activity, yield of the cleaved complex observed is 30fold lower than the wild-type enzyme at each enzyme: substrate ratio even after 60 min of incubation

S10T

Reduced DNA binding affinity, possibly changing Ser10 to a threonine residue, and changing Lys13 to arginine allow retention of activity, relaxation activity assays show that introduction of a different side chain with similar functionality but different steric properties at these two positions result in even lower relaxation activitymutations at Ser10 and Lys13 highly reduce the ability of topoisomerase I to support growth of the Escherichia coli cells, as expected from the loss of the relaxation activity. Thr10 forms a hydrogen bond with phosphate 8. Do not have severe changes in protein folding. Side chains of Ser10 and Lys13 are required for DNA cleavage activity, rates of cleavage are too low to be analyzed

Y177A

site-directed mutagenesis, inactive mutant

Y177S

site-directed mutagenesis, inactive mutant

Y319F

site-directed mutagenesis, constants of oligonucleotide binding compared to the wild-type enzyme

D117N

Escherichia coli RFM475

site-directed mutagenesis, induction of mutant YpTOP1-D117N topoisomerase by arabinose is similar in the absence or presence of trimethoprim

A653P

known to be CPT resistant

682169

Cropo4

chimeric enzyme, human topo I having residues 631-717 replaced by residues 314-317 of Cre

690972

Cropo6

chimeric enzyme, human topo I having residues 631-717 replaced by residues 312-317 of Cre

690972

Cropo8

chimeric enzyme, human topo I having residues 631-717 replaced by residues 312-319 of Cre

690972

DELTA1-202

mutant enzyme is impaired in blunt end ligation but remains sensitive towards camptothecin in relaxation

662615

DELTA191-206

mutant is distinguished from wild-type enzyme by a decreased relaxation rate at distributive and an increased rate under processive conditions. Mutant is strongly impaired in blunt end ligation and insensitive towards camptothecin in relaxation

662615

E418K

the active site tyrosine. The Glu418Lys mutant is active and partially resistant to CPT in yeast cells expressed in yeast cells, display a camptothecin resistance that slowly decreases as a function of time

682169

E710G

site-directed mutagenesis, the mutation is not detectable in tissue samples from colorectal cancer patients

728293

F361S

camptothecin-resistant enzyme form

662623

F617N

site-directed mutagenesis

G365C

camptothecin-resistant Top1(G365C) mutant

682092

G622A

site-directed mutagenesis

G717D

the mutant enzyme is no more sensitive to camptothecin than the wild type enzyme

748187

hTopoI (deltaNL)

hTopoI deletion mutant. Considering the domain organization, hTopoI distinguishes itself from both Vaccinia virus TopoI and the tyrosine recombinases, by having rather extensive N-terminal and linker domains. Investigation of the resolution activity of hTopoI deleted in either one or both of these domains. Two hTopoI variants, hTopoI(deltaN) lacking aa 1-202, and hTopoI(deltaNL) lacking aa 1-202 and 660-688. Deleting the N-terminal and linker domains of hTopoI in order to mimic the domain organization of Vaccinia virus TopoI and the tyrosine recombinases inhibited resolution of the Holliday junction substrate. hTopoI(deltaNL) forms dimers

hTopoI(deltaN)

I623V

site-directed mutagenesis

K532A

K532R

cleavage and religation activity of the mutant enzyme are reduced, cleavage is reduced to a greater extent than religation

662238

K550Q

site-directed mutagenesis

K605Q

site-directed mutagenesis

K616M

site-directed mutagenesis

K616Q

site-directed mutagenesis

K616R

site-directed mutagenesis

K681A

site-directed mutagenesis, the mutation of Lys681, located on the tip of the linker domain, dramatically reduces the religation rate, the equilibrium of the mutant is shifted toward cleavage when compared to the wild-type enzyme. Mutation of residue 681 from lysine to alanine does not cause alterations in the linker domain secondary structure, and the main chain conformation of the mutated residue does not significantly change in relation with the neighbor residues, when compared to the wild-type protein

705975

L603V

site-directed mutagenesis

L617I

site-directed mutagenesis, the mutation is not detectable in tissue samples from colorectal cancer patients

728293

M621I

site-directed mutagenesis

M621S

site-directed mutagenesis

N618S

site-directed mutagenesis

N722S

camptothecin-resistant enzyme form, mutation leads to elimination of a water-mediated contact between the enzyme and topotecan

662623

Q613R

site-directed mutagenesis

R364K/G717R

the mutations lead to resistance towards inhibitors SN-38 and NSC 743400

748337

R621H

site-directed mutagenesis, the mutation is not detectable in tissue samples from colorectal cancer patients

728293

S10A/S112A/S394A

triple mutant 3A

693067

S10A/S21A/S112A/S394A

quadruple phosphomutant 4A

693067

S21A

site-directed mutagenesis

693067

T729A

mutations that confer camptothecin resistance, but do not interact directly with the drug or the DNA, oligonucleotide-directed mutagenesis

694447

T729E

camptothecin resistance, remarkable defect in DNA binding, oligonucleotide-directed mutagenesis

T729K

T729P

W203A/W205A/W206A

lacking processivity, exhibits resistance to camptothecin and is inactivated by urea. Indicates that the tryptophan anchor stabilizes the N-terminus of the functional domain and revents the loss of Top1 structure and function

W205G

Y337F

mutant enzyme is still able to bind single-stranded DNA but is unable to cleave the DNA substrate because of the absence of the active Tyr337

653375

Y552N

site-directed mutagenesis

Y612N

site-directed mutagenesis

Y723F

Y727F

catalytically inactive

748397

D325E

substantial level of resistance to camptothecin, 50% inhibition at 0.01 mM

660723

delta 175-262

N-terminal truncation performed on LdTop1S subunits

delta 180-262

N-terminal truncation performed on LdTop1S subunits

delta 182-262

N-terminal truncation performed on LdTop1S subunits

delta 200-262

N-terminal truncation performed on LdTop1S subunits

delta 210-262

N-terminal truncation performed on LdTop1S subunits

delta 39-635

in contrast to camptothecin, baicalein and luteolin fail to inhibit the religation step when the drugs are added to pre-formed enzyme substrate binary complex. This differential mechanism to induce the stabilization of cleavable complex with topoisomerase I and DNA by these selected flavones and camptothecin result in investigations of the effect of baicalein and luteolin on camptothecin-resistant mutant enzyme LdTOP1D39LS lacking 139 amino acids of the large subunit

682159

G185R

substantial level of resistance to camptothecin, 50% inhibition at 0.035 mM

660723

G185R/D325

parasites containing both mutations simultaneously show high level of resistance to camptothecin

660723

G185R/D325E

highly camptothecin-resistant with two substitutions in the core domain of the protein

678377

H453A

Very little relaxing activity detectable, activity of the H453A protein to be more than 100fold reduced compared with that of LdTOPIL-fus-S

679762

H453Q

Lower activity than LdTOPIL-fus-S, failing to completely relax the supercoiled DNA even after 40 min

679762

K352A

site-directed mutagenesis, the mutant shows highly reduced relaxation activity compared to wild-type enzyme, the cleavage religation equilibrium is shifted towards the cleavage

705597

K352R

site-directed mutagenesis, the mutant shows highly reduced relaxation activity compared to wild-type enzyme, the cleavage religation equilibrium is shifted towards the cleavage

705597

R314K

site-directed mutagenesis, the mutant shows onyl slightly affected relaxation activity and only a slightly slower rate of cleavage compared to wild-type enzyme

705597

Y327F

2 entries

D261A LdTOPIS

Site-directed mutagenesis studies identify Lys455 of LdTOPIL and Asp261 of LdTOPIS as two residues involved in subunit interaction. The reconstituted mutant enzymes LdTOPILK436A/S, LdTOPILN441A/S, L/LdTOPISK249A and L/LdTOPISN256A do not show any appreciable change in the relaxation pattern compared with the wild-type enzyme L/S. Reconstitution of L with LdTOPISD261A (SD261A) causes a 15fold decrease in the relaxation activity compared with L/S

GST-LdTOPIS delta 1-201

Generated N-and C-terminal-truncated deletion constructs of of L and S subunit. The heterodimerization between the two fragments is weak and therefore co-purified fragments show reduced DNA binding, cleavage and relaxation properties compared with the wild type enzyme

GST-LdTOPIS delta 1-210

constitute the minimal interacting fragment. Generated N-and C-terminal-truncated deletion constructs of of L and S subunit. The heterodimerization between the two fragments is weak and therefore co-purified fragments show reduced DNA binding, cleavage and relaxation properties compared with the wild type enzyme

GST-LdTOPIS delta 1-210/delta 255-262

GST-LdTOPIS delta 1-215

GST-LdTOPIS delta 1-80

GST-LdTOPIS delta 245-262

GST-LdTOPIS delta 255-262

K243A LdTOPIS

K436A LdTOPIL

K455A LdTOPIL

LdTOPIL delta 1-39

LdTOPIL delta 1-39/delta457-635

LdTOPIL delta 1-99

LdTOPIL delta 436-635

LdTOPIL delta 457-635

N256A LdTOPIS

N441A LdTOPIL

E215A

mutant activity with the mutation in the Topo-44 fragment (an N-terminal 44-kDa fragment of Topo-V) is active over a wider range of pH values, compared with wild-type enzyme. Mutant activity with the mutation in the Topo-44 fragment (an N-terminal 44-kDa fragment of Topo-V) is active over a wider range of pH values, compared with wild-type enzyme

E215H

mutant enzyme is active over a wider range of pH values, compared with wild-type enzyme, mutation in the Topo-44 fragment (an N-terminal 44-kDa fragment of Topo-V)

E215Q

mutant enzyme is active over a wider range of pH values, compared with wild-type enzyme, mutation in the Topo-44 fragment (an N-terminal 44-kDa fragment of Topo-V)

E215R

mutant enzyme is active over a wider range of pH values, compared with wild-type enzyme, mutation in the Topo-44 fragment (an N-terminal 44-kDa fragment of Topo-V)

H200A

mutant enzyme is slightly less active than wild type enzyme, mutation in the Topo-44 fragment (an N-terminal 44-kDa fragment of Topo-V)

H200E

mutant has low but detectable DNA relaxation activity at pH 5, mutation in the Topo-44 fragment (an N-terminal 44-kDa fragment of Topo-V)

H200N

mutant enzyme is slightly less active than wild type enzyme, mutation in the Topo-44 fragment (an N-terminal 44-kDa fragment of Topo-V)

H200Q

mutation in the Topo-44 fragment (an N-terminal 44-kDa fragment of Topo-V)

H200R

mutant enzyme shows relaxation activity over a pH range similar to the wild-type enzyme. In the Topo-78 backbone (an N-terminal 78-kDa fragment of Topo-V), qualitatively, H200R is as active as the wild type

K128R

mutant enzyme shows very minimal activity at pH 8

K218Q

the mutant is inactive in all of the pH conditions tested, mutation in the Topo-78 fragment (an N-terminal 78-kDa fragment of Topo-V)

R131A

mutant shows no detectable DNA relaxation activity, mutations in Topo-44 fragment (an N-terminal 44-kDa fragment of Topo-V) or in Topo-78 fragment (an N-terminal 78-kDa fragment of Topo-V)

R131K

conservative substitution that retains both the electrostatic and acid/base properties, except for the loss of the bidentate coordination possible through the guanidinium group of the arginine. Whereas the R131K mutant is purified successfully in the Topo-44 backbone, it results in degradation when expressed in the Topo-78 backbone

R144A

mutant enzyme shows no activity, mutation in Topo-78 fragment (an N-terminal 78-kDa fragment of Topo-V)

T226F

the mutation abolishes activity at all pH values in T44Y226F and shows only minimal activity at pH 8, mutation in the Topo-44 fragment (an N-terminal 44-kDa fragment of Topo-V)

Y226F

mutation abolishes DNA binding, mutation in the Topo-44 fragment (an N-terminal 44-kDa fragment of Topo-V)

D111A

A5U8X0

site-directed mutagenesis, mutation of the Mg2+-binding residue affects DNA relaxation activity, mutant D111A is dependent on Mg2+ for DNA cleavage and is compromised in religation

726811

D111N

site-directed mutagenesis, the mutation is lethal for the host

D113A

A5U8X0

site-directed mutagenesis

726811

E115A

A5U8X0

site-directed mutagenesis, mutation of the Mg2+-binding residue affects DNA relaxation activity, the mutant is compromised in religation

726811

G116S

site-directed mutagenesis, MtTOP1 TOPRIM motif mutation, the mutation inhibits DNA religation. The DNA cleavage activity of MtTOP1-G116 S is Mg2+-dependent

H139C

site-directed mutagenesis, the mutant can be labeled with fluorophores with no significant loss of relaxation activity, the mutant complements the temperature sensitive topA function of Escherichia coli strain AS17

K524C

L170C

T142C

Y174C

D111A

Mycobacterium tuberculosis H37Ra / ATCC 25177

site-directed mutagenesis, mutation of the Mg2+-binding residue affects DNA relaxation activity, mutant D111A is dependent on Mg2+ for DNA cleavage and is compromised in religation

D113A

Mycobacterium tuberculosis H37Ra / ATCC 25177

site-directed mutagenesis

E115A

Mycobacterium tuberculosis H37Ra / ATCC 25177

site-directed mutagenesis, mutation of the Mg2+-binding residue affects DNA relaxation activity, the mutant is compromised in religation

I420V

prepared by PCR-based mutagenesis

L530I

prepared by PCR-based mutagenesis

N421K

prepared by PCR-based mutagenesis

N722S

prepared by PCR-based mutagenesis

D710G

Ophiorrhiza pumila

the mutant is resistant against topotecan

748878

G721D

Single amino acid substitutions of yeast Gly721 alters Top1p sensitivity to camptothecin

G721E

Single amino acid substitutions of yeast Gly721 alters Top1p sensitivity to camptothecin

G721F

Single amino acid substitutions of yeast Gly721 alters Top1p sensitivity to camptothecin

G721L

Single amino acid substitutions of yeast Gly721 alters Top1p sensitivity to camptothecin

G721N

Single amino acid substitutions of yeast Gly721 alters Top1p sensitivity to camptothecin

G721Q

Single amino acid substitutions of yeast Gly721 alters Top1p sensitivity to camptothecin

G721V

Single amino acid substitutions of yeast Gly721 alters Top1p sensitivity to camptothecin

L720E

Single amino acid substitutions of Leu720 alters Top1p sensitivity to camptothecin

L720Q

Single amino acid substitutions of Leu720 alters Top1p sensitivity to camptothecin

N726D

2 entries

N726F

specific activity is about 5fold lower than that of wild-type enzyme, self-poisoning phenotype, mutation enhances the kinetics of DNA cleavage

N726H

self-poisoning phenotype, specific activity is about 5fold lower than that of wild-type enzyme, binding of DNA increases from 2.5fold at 50 mM KCl to 4fold at 100 mM KCl, 10fold increase in DNA cleavage activity

N726K

camptothecin resistance of cells expressing the mutant enzyme

N726Q

camptothecin resistance of cells expressing the mutant enzyme

N726S

mutant enzyme is resistant to camptothecin

651995

N726Y

mutation enhances the kinetics of DNA cleavage

T722A

self-poisoning phenotype, reduced rate of DNA religation relative to wild-type enzyme, specific activity of the mutant enzyme is slightly decreased, however protein binding of DNA and rates of enzyme-catalyzed

T722A/N726H

Thermotoga maritima DSM 3109

specific activity is about 5fold lower than that of wild-type enzyme, N726H mutation potentiates the cytotoxic activity of T722A, increase in DNA cleavage activity

Y727F

C559A/C561A

no detectable zinc is bound to the mutant enzyme and its fluorescence properties are modified, suggesting a change of conformation in the protein lacking zinc. GThe mutant enzyme exhibits an activity similar to that of the wild-type enzyme with the same optimal temperature for activity. Thermostability is not affected by the mutation. Decatenase activity of wild type and C559A/C561A mutant enzymes shows no significant difference. Cleavage positions obtained with the C559A/C561A mutant is similar to those observed with the wild type enzyme. Preference for cytosine at position -4 is retained, and cleavage intensities are comparable

748151

Y288F

Thermotoga maritima

P46799

totally inactive protein

748151

C559A/C561A

Y288F

Thermotoga maritima DSM 3109

totally inactive protein

D298A

relaxation rate is equivalent to that of the wild-type enzyme in absence and presence of Mg2+

E124A

relaxation rate is equivalent to that of the wild-type enzyme in absence and presence of Mg2+

E299A

relaxation rate is equivalent to that of the wild-type enzyme in absence and presence of Mg2+

F101A

mutant enzyme displayes an aberrant response to magnesium, rate of relaxation is lower by a factor of 4-5 in the presence of 5 mM magnesium than in its absence, the rate of relaxation in presence of NaCl plus magnesium is reduced by a factor of 10.20 compared to wild-type enzyme, affinity for 60-bp duplex is 8fold lower than that of wild-type enzyme

F297A

relaxation rate is equivalent to that of the wild-type enzyme in absence and presence of Mg2+

F88A

mutant enzyme displayes an aberrant response to magnesium, rate of relaxation is lower by a factor of 4-5 in the presence of 5 mM magnesium than in its absence, the rate of relaxation in presence of NaCl plus magnesium is reduced by a factor of 10-20 compared to wild-type enzyme, affinity for 60-bp duplex is 3fold lower than that of wild-type enzyme

K104A

relaxation rate is equivalent to that of the wild-type enzyme in absence and presence of Mg2+

K107A

relaxation rate is equivalent to that of the wild-type enzyme in absence and presence of Mg2+

K108A

relaxation rate is equivalent to that of the wild-type enzyme in absence and presence of Mg2+

K220A

supercoil relaxation by mutant enzyme in presence of NaCl and no divalent cation is slower by a factor of 5 than relaxation by wild-type enzyme, mutant enzyme is inhibited by including Mg2+ with NaCl, rate of relaxation in presence of NaCl plus Mg2+ is reduced by 20fold compared to wild-type enzyme

K220H

K220Q

K220R

supercoil relaxation by mutant enzyme in presence of NaCl and no divalent cation is slower by a factor of 2 than relaxation by wild-type enzyme, neither stimulated nor inhibited by Mg2+, rate of relaxation in presence of NaCl plus Mg2+ is reduced by 8fold compared to wild-type enzyme

N103A

relaxation rate is equivalent to that of the wild-type enzyme in absence and presence of Mg2+

N228A

relaxation of supercoiled DNA in presence of NaCl and no divalent cation is slower by a factor of 8 than relaxation by the wild-type enzyme

N228D

relaxation of supercoiled DNA in presence of NaCl and no divalent cation is slower by a factor of 40 than relaxation by the wild-type enzyme, mutant enzyme is inhibited by Mg2+, oligonucleotide cleavage is slower than that of wild-type enzyme by a factor of 200

N228Q

relaxation of supercoiled DNA in presence of NaCl and no divalent cation is slower by a factor of 5 than relaxation by the wild-type enzyme, not stimulated by including magnesium with NaCl

N228S

relaxation of supercoiled DNA in presence of NaCl and no divalent cation is slower by a factor of 2 than relaxation by the wild-type enzyme

N99A

relaxation rate is equivalent to that of the wild-type enzyme in absence and presence of Mg2+

Q116A

relaxation rate is equivalent to that of the wild-type enzyme in absence and presence of Mg2+

R90A

relaxation rate is equivalent to that of the wild-type enzyme in absence and presence of Mg2+

R97A

relaxation rate is equivalent to that of the wild-type enzyme in absence and presence of Mg2+

S204A

mutant protein relaxes supercoiled DNA slowly and is strongly inhibited in the presence of salt plus magnesium, the rate of single-turnover religation by covalently bound S204N is indistinguishable from wild-type within the limits of the assay

T296A

relaxation rate is equivalent to that of the wild-type enzyme in absence and presence of Mg2+

Y274F

vaccinia topoisomerase mutant, lacks active site tyrosine nucleophile but binds to DNA containing specific recognition sequence, like wild type vTopo

682013

A383P

mutant enzyme has wild-type levels of relaxation and cleavage activity

662426

C737Y

Q7CIL8

site-directed mutagenesis

D116N/W866L

Q7CIL8

site-directed mutagenesis

D117N

Q7CIL8

site-directed mutagenesis

D117N/C608G

Q7CIL8

site-directed mutagenesis

D117N/C668F

Q7CIL8

site-directed mutagenesis

D117N/C737Y

Q7CIL8

site-directed mutagenesis

D119N

Q7CIL8

site-directed mutagenesis

D80N/G94S/D117N

Q7CIL8

YpTOP39 has D80N, G94S, and D117N mutations

E121Q

Q7CIL8

site-directed mutagenesis

G116S

mutant enzyme shows more than 400fold loss of relaxation activity

G122S

M326D

substitution mutant

M326E

substitution mutant

M326K

substitution mutant

M326P

substitution mutant

M326R

overexpression of the M326R mutant YTOP resulted in 4 log loss of viability

M326S

substitution mutant

M326T

substitution mutant

M326V

mutant enzyme has wild-type levels of relaxation and cleavage activity

662426

R327F

site-directed mutagenesis, the mutation leads to the SOS response and has a strong cell killing effect when the mutant is overexpressed in Escherichia coli and induced with 0.2% arabinose, the mutant shows reduced DNA cleavage and requirement for Mg2+

R327I

R327L

R327S

R327W

T265M

site-directed mutagenesis, the mutant does not show a phenotype

V559I

site-directed mutagenesis, the mutant does not show a phenotype