Role of Specific Amino Acid Residues in T4 Endonuclease V That Alter Nontarget
Simon G. Nyaga, M. L. Dodson, and R. Stephen Lloyd*
Sealy Center for Molecular Science and The Department of Human Biological Chemistry and Genetics,
The UniVersity of Texas Medical Branch, GalVeston, Texas 77555-1071
ReceiVed August 30, 1996; ReVised Manuscript ReceiVed January 22, 1997X
ABSTRACT: Endonuclease V is a pyrimidine dimer-specific DNA glycosylase-apurinic (AP)1lyase which,
in ViVo or at low salt concentrations in Vitro, binds nontarget DNA through electrostatic interactions and
remains associated with that DNA until all dimers have been recognized and incised. On the basis of the
analyses of previous mutants that effect this processive nicking activity, and the recently published cocrystal
structure of a catalytically deficient endonuclease V with pyrimidine dimer-containing DNA [Vassylyev,
D. G., et al. (1995) Cell 83, 773-782], four site-directed mutations were created, the mutant enzymes
expressed in repair-deficient Escherichia coli, and the enzymes purified to homogeneity. Steady-state
kinetic analyses revealed that one of the mutants, Q15R, maintained an efficiency (kcat/Km) near that of
the wild-type enzyme, while R117N and K86N had a 5-10-fold reduction in efficiency and K121N was
reduced almost 100-fold. In addition, K121N and K86N exhibited a 3-5-fold increase in Km, respectively.
All the mutants experienced mild to severe reduction in catalytic activity (kcat), with K121N being the
most severely affected (35-fold reduction). Two of the mutants, K86N and K121N, showed dramatic
effects in their ability to scan nontarget DNA and processively incise at pyrimidine dimers in UV-irradiated
DNA. These enzymes (K86N and K121N) appeared to utilize a distributive, three-dimensional search
mechanism even at low salt concentrations. Q15R and R117N displayed somewhat diminished processive
nicking activities relative to that of the wild-type enzyme. These results, combined with previous analyses
of other mutant enzymes and the cocrystal structure, provide a detailed architecture of endonuclease
V-nontarget DNA interactions.
The exposure of DNA to ultraviolet light (UV) of short
wavelength (295 nm or less) causes the formation of several
biologically important photoproducts, the most prevalent of
these lesions being cyclobutane pyrimidine dimers and the
6-4 dipyrimidine adducts (Taylor, 1994). Both prokaryotic
and eukaryotic cells have mechanisms either for the removal
of these lesions (nucleotide excision repair or enzyme-
catalyzed photoreversal) or for damage avoidance (recom-
bination). In a limited number of organisms, a base excision
repair pathway also exists for initiating the removal of these
lesions [reviewed by Lloyd and Linn (1993), Lloyd (1993),
and Lloyd and Van Houten (1995)]. One of these base
excision repair enzymes is T4 endonuclease V. It has a
narrow substrate specificity for cis-syn cyclobutane pyri-
midine dimers, although recent studies have shown that it
can incise trans-syn dimers and Fapy A adducts at a rate
approximately 1% that of the cis-syn dimer (Smith &
Taylor, 1993; Dizdaroglu et al., 1996). Biochemical and
genetic investigations have identified the active site of
endonuclease V as the R-amino group of the N-terminal
threonine residue (Schrock & Lloyd, 1991, 1993; Dodson
et al., 1993). Nucleophilic attack results in the cleavage of
the glycosyl bond of the 5′-pyrimidine of the dimer, resulting
in an imino intermediate. The enzyme can either dissociate,
leading to an abasic site, or proceed by a ?-elimination
reaction to result in an R,?-unsaturated aldehyde (Dodson
et al., 1993, 1994).
However, prior to catalysis, it is essential for endonuclease
V to locate the specific damaged site within large amounts
of undamaged DNA, referred to in this paper as nontarget
DNA. This problem of site-specific target location is
common to many DNA interactive proteins involved in
numerous cellular processes such as gene expression, genome
replication, RNA transcription, restriction/modification, and
DNA repair [reviewed in von Hippel and Berg (1989) and
Lloyd and Van Houten (1995)]. In Vitro, this protein-
nontarget DNA interaction has been shown to be dominated
by electrostatic interactions and to be sensitive to the
monovalent salt concentration of the solution, such that below
40 mM the target search is processive, while at higher salt
concentrations the search becomes three dimensional or
distributive. The biological importance of this nontarget
DNA search mechanism has been demonstrated in numerous
studies in which reductions in nontarget DNA binding result
in decreased abilities of these mutants to enhance UV
survival in repair-deficient Escherichia coli (E. coli) (Dowd
& Lloyd, 1989a,b, 1990; Nickell et al., 1991, 1992; Augus-
tine et al., 1991). Insights into what residues are important
in this protein-DNA interaction have come from both
mutagenesis of selected basic amino acids and the solving
of the crystal structure of endonuclease V (Morikawa et al.,
1992). The structure which was resolved at 1.6 Å revealed
the existence of a number of positively charged amino acid
†This work was supported by U.S. Public Health Service Grants
ES04091 and ES06676 and American Cancer Society Grant FRA 381.
* To whom correspondence should be addressed. Telephone: 409-
772-2179. FAX: 409-772-1790. E-mail: firstname.lastname@example.org.
XAbstract published in AdVance ACS Abstracts, March 15, 1997.
1Abbreviations: AP, apurinic/apyrimidinic; UV, ultraviolet; EDTA,
ethylenediaminetetraacetic acid; FPLC, fast protein liquid chromatog-
raphy; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel
Biochemistry 1997, 36, 4080-4088
S0006-2960(96)02218-0 CCC: $14.00© 1997 American Chemical Society
residues that lie on the internal curvature of the comma-
shaped enzyme molecule.These residues also form a
groove-like depression which can accommodate one strand
of B-form DNA. Recently, a cocrystal structure has been
solved using a catalytically incompetent form of the enzyme,
complexed with dimer-containing DNA (Vassylyev et al.,
1995). Table 1 summarizes the published data on mutants
that displayed altered nontarget DNA binding.
analyses of these data, coupled with a closer examination of
the crystal structures, revealed that additional amino acid
residues may be involved in nontarget DNA binding. Thus,
we hypothesized that the creation of specific mutant enzymes
might yield additional information on the architecture of
interaction between endonuclease V and undamaged DNA.
In addition to amino acid residues that may be important
for nontarget DNA binding, it should be noted that numerous
basic and aromatic amino acids have also been implicated
in dimer-specific binding. These include an extensive study
by Doi et al. (1992), who demonstrated a critical binding
role for R3, R22, R26, R117, and K121, and from our
laboratory in which a key role in dimer-specific binding was
suggested for W128, Y129, and K130 (Recinos & Lloyd,
1988; Stump & Lloyd, 1988; Lloyd & Augustine, 1989).
However, the focus of this current study was to concentrate
our efforts on residues which might differentially affect
nontarget DNA from target DNA binding.
MATERIALS AND METHODS
Materials. Phenyl-Sepharose CL-4B, heparin Sepharose
and Mono S matrices, and column chromatographic supplies
were purchased from Pharmacia. Prestained protein molec-
ular weight markers were purchased from BRL. Duplex
cis-syn thymine dimer containing 12-mer and 49-mer
oligonucleotides containing a site-specific cis-syn thymine
dimer and its complementary oligonucleotide were generous
gifts from Drs. Colin Smith and John-Stephen Taylor
(Washington University, St. Louis, MO). Kodak X-ray films
(X-Omat) were purchased from Amersham. [γ-32P]ATP
(6000 Ci/mmol) and [R-32P]ATP (6000 Ci/mmol) were
purchased from DuPont-NEN. Sequenase and Sequenase
version 2.0 DNA sequencing kits were purchased from
United States Biochemical Corp. EcoRI and ClaI restriction
enzymes, T4 polynucleotide kinase, and T4 DNA ligase were
purchased from New England Biolabs, Beverly, MA. pBR322
and the mutagenic oligonucleotides (23-mers) were prepared
and synthesized, respectively, in the NIEHS Center Molec-
ular Biology Core at UTMB. Oligonucleotide sizing markers
(8-32 bases) were purchased from Pharmacia. Bio-Gel P-10
(90-180 µm) matrix was purchased from Bio-Rad.
Oligonucleotide Site-Directed Mutagenesis of the denV
Gene. The E. coli strains, phage, and plasmids used in this
study are described in Table 2.
The structural gene, denV, encoding endonuclease V and
the transcription terminator sequences were previously
constructed behind the λOLPRpromoter in M13mp18 (Re-
cinos & Lloyd, 1986; Recinos et al., 1986). Single-stranded
M13 DNA was isolated from E. coli strain UT481 (Zoller
& Smith, 1983) that had been previously infected with M13
mp18 OLPR denV. Mutagenic oligonucleotides were de-
signed from the published denV sequence (Radany et al.,
1984; Valerie et al., 1984) to alter independently the codons
for amino acids at positions Q15(CAA) to R15(CGT), K121-
(AAA) to N121(AAC), K86(AAG) to N86(AAC), and R117-
(CGT) to N117(AAC). The full sequences of the four 23-
mer oligonucleotides, designed from the denV sequence, with
the altered codons being underlined, were as shown: (1)
Q15R, 5′ C CAT TAA GTG ACG GTC AGC CAA T 3′;
(2) K121N, 5′ G TTG TGC AAT GTT TTC ATC TAA A
3′; (3) R117N, 5′ T TTC ATC TAA GTT AGC TTG TGA
T 3′; (4) K86N, 5′ C TGT AGT ATC GTT GAT ATT AAA
Table 1: Summary of Endonuclease V Mutants Previously Shown
To Display Altered Nontarget DNA Binding
mutant functional effect
diminished nontarget DNA binding
AP-specific nicking and low levels of dimer-specific nicking;
no enhancement in cellular survival of repair-deficient E. coli
diminished nontarget DNA binding
no enhancement in cellular survival of repair-deficient E. coli
diminished nontarget DNA binding
substantial level of in Vitro dimer and AP-specific nicking;
unable to complement repair-deficient E. coli
reduced nontarget DNA binding
unable to complement repair-deficient E. coli
increased electrostatic affinity for nontarget DNA
wild-type levels of apurinic DNA-specific nicking activities
enhanced nontarget DNA binding activity
reduced nontarget DNA binding
enhanced nontarget DNA binding but with diminished
Table 2: E. coli, Phage, and Plasmid Constructs Used in This Study
strain, plasmid, or phage genotype or phenotypesource
UT481Met thy ∆(Lac-pro) hsdRBamHI hsdM+supD Tn10/F′
traD36 proAB lac Iqz∆ M15
uVrA6 recA13 arg+thr-1 leu-6 thi-1 supE44 lacY1galK2 ara-14
xyl-5 mtl-1 proA2 his-4 argE3 str-31 tsx-33 sup-37
F-∆(merC-mrr) leu supE44 ara14 galK2 lacY1 proA2 rpsL20
(Strr) xyl-5 mtl-1 recA13
C. Lark, University of Utah
A. Ganesan, Stanford University
T. Wood, University of Texas
Amprλ OLPRλ 45galK+
Amprλ OLPRendonuclease V+λ 45galK+
Amprλ OLPR λ 45galK+endonuclease V (Arg-15)
Amprλ OLPR λ 45galK+endonuclease V (Asn-121)
Amprλ OLPR λ 45galK+endonuclease V (Asn-86)
Amprλ OLPR λ 45galK+endonuclease V (Asn-117)
Endonuclease V Mutants Defective in Binding Nontarget DNA
Biochemistry, Vol. 36, No. 14, 1997 4081
The oligonucleotides were synthesized and purified as
described (Lloyd et al., 1986). The purified oligonucleotides
were annealed as primers to the M13mp18 OLPR denV
single-stranded circular DNA template and extended using
Sequenase 2.0 (Zoller & Smith, 1983).
subsequently sealed by T4 DNA ligase. Following the
primer extension reaction, the resultant relaxed covalently
closed circular DNAs were used to transform E. coli UT481.
Plaques containing M13mp18 OLPR-denV with the desired
mutations were selected by differential hybridization utilizing
32P-end-labeled mutagenic oligonucleotides as probes (Ben-
ton & Davis, 1977; Recinos & Lloyd, 1986). Phage from
the initial positive plaques were selected and purified.
Single-stranded DNAs were prepared from second round
screening of positive plaques, and the mutations were
confirmed by DNA sequence analysis (Sanger et al., 1977).
Double-stranded replicative form (RF) M13mp18 DNAs
containing the desired mutations were isolated (Zoller &
Smith, 1983) and the mutant denV genes liberated by a ClaI
restriction digestion. These fragments were subcloned into
the E. coli expression vector pGX2608 (Recinos & Lloyd,
1986) and transformed into E. coli HB101. The colonies
were selected by ampicillin resistance and differential
hybridization using a denV gene-specific probe. Plasmid
DNAs were prepared from the positive colonies, and the
correct orientation of the denV fragments was confirmed by
diagnostic restriction digestions with EcoRI and ClaI (Re-
cinos & Lloyd, 1986). Plasmids containing the wild-type
and the mutant denV genes in the correct orientation were
transformed into the recombination and excision repair-
deficient E. coli AB2480 (uVrA-, recA-) and used for
establishing cultures (inoculation) for protein purification.
Intracellular Accumulation of the Mutant Endonuclease
V Proteins. E. coli AB2480 strains containing the plasmids
encoding the wild-type and mutated denV genes were grown
to stationary phase at 30 °C in Luria-Bertani (LB) medium
supplemented with 100 µg/mL ampicillin (Recinos & Lloyd,
1986). The cells were harvested, resuspended at a constant
optical density, and analyzed for the accumulation of
immunoreactive endonuclease V by SDS-PAGE and sub-
sequent Western blot analyses (Laemmli, 1970; Towbin et
al., 1979; Burnette, 1981; Higgins & Lloyd, 1987).
Mutant Protein Purification. The wild-type and mutant
proteins were expressed in E. coli AB2480 utilizing the
λOLPRhybrid promoter within the pGX2608. Cultures of
each mutant (3 L) were grown at 30 °C for 16 h in LB broth
media supplemented with 100 µg/mL ampicillin.
proximately 3 × 1012cells were pelleted by centrifugation
at 7000 rpm for 10 min at 4 °C and resuspended in 240 mL
of 20 mM Tris-HCl (pH 7.5), 10 mM EDTA, 200 mM
potassium chloride, and 10% (v/v) ethylene glycol (buffer
A). The resuspended cells were then lysed by sonication
(Branson Sonifier 450, Danbury, CT), and the cellular debris
was removed by centrifugation in a Sorvall GSA rotor
(10 000 rpm for 20 min at 4 °C). Complete cell lysis was
confirmed by examination of samples before and after
sonication using a light microscope (Zeiss, Thornwood, NY).
The supernatant was passed through a 500 mL single-
stranded DNA-agarose column (Schaller et al., 1972) that
was equilibrated with buffer A. After the column was
extensively washed, the bound proteins were eluted with a
continuous salt gradient starting with buffer A to buffer A
supplemented with 1.5 M potassium chloride (buffer B). This
The nick was
was followed by a 500 mL wash with buffer B. The fractions
were monitored for the presence of mutant endonuclease V
by Coomassie brilliant blue staining, silver staining, and
Western blot analyses. The fractions containing the highest
concentrations of mutant endonuclease V proteins were
pooled. Saturated ammonium sulfate was added to a final
concentration of 700 mM.
The samples were then applied to a 20 mL phenyl-
Sepharose column which had been equilibrated with excess
25 mM sodium phosphate (pH 6.8), 1.0 mM EDTA, 10%
(v/v) ethylene glycol, and 700 mM ammonium sulfate (buffer
C). The bound proteins were washed with approximately
150 mL of buffer C. A continuous salt gradient starting with
buffer C to buffer C without ammonium sulfate (buffer D)
was used to elute the bound proteins. The flow through and
sample fractions were monitored as described above. Wild-
type and all mutant proteins eluted in the flow through.
These fractions were dialyzed against three buffer changes
at 4 °C with buffer consisting of 25 mM sodium phosphate
(pH 6.8), 1 mM EDTA (pH 8.0), and 10% (v/v) ethylene
glycol (buffer E). These samples were applied to a 50 mL
Mono S column (FPLC) that had been previously equili-
brated with buffer E. After the column was washed with
buffer E, a continuous salt gradient starting with buffer E to
buffer E supplemented with 1.0 M potassium chloride (buffer
F) was run, and 2.0 mL fractions were collected. The flow
through and fractions were assayed for nicking activity using
a duplex 5′-end-labeled 49-mer oligonucleotide containing
a cis-syn pyrimidine dimer. The purity of the fractions was
determined by silver staining of SDS-polyacrylamide gels.
The fractions containing the highest concentration of
mutant endonuclease V were pooled and dialyzed as previ-
ously described. The proteins were applied to a 20 mL
heparin-Sepharose column which had previously been
equilibrated with buffer E. The column was washed with
buffer E, and the bound proteins eluted by a continuous salt
gradient starting with buffer E to buffer E supplemented with
1.5 M sodium chloride (buffer G). The column was washed
with buffer G. Fractions (5-8 mL) were collected and
assayed for activity and purity as described earlier. One of
the mutants, K86N, was further purified using a Bio-Gel size
exclusion matrix P-10 gel (Bio-Rad). The mutant enzyme
was extensively dialyzed as described earlier and passed
through a P-10 gel column (5 cm × 1 cm) which had
previously been equilibrated with buffer E. Fractions (2 mL)
were collected and assayed for purity and activity by silver
staining of SDS-polyacrylamide gels and pyrimidine dimer
nicking of a duplex 5′-end-labeled 49-mer oligonucleotide,
Pyrimidine Dimer Nicking ActiVities of the Mutants on
49-mer. Pyrimidine dimer-specific nicking activities of the
enzymes were assayed using a duplex 49-mer oligonucleotide
containing a cis-syn cyclobutane pyrimidine dimer. The
49-mer oligonucleotide (single stranded) had the sequence
AATCATGGTCATATGGTCATAGCT 3′ in which the two
boldfaced T’s (TT) represent the site of the cis-syn
cyclobutane pyrimidine dimer. Approximately 0.3 ng of the
49-mer oligonucleotide was labeled on the 5′ end, using
[γ-32P]ATP, and annealed to an excess of the complementary
oligonucleotide. The oligonucleotide was incubated at 37
°C with increasing concentrations of the mutant and the wild-
type enzymes in high salt buffer [25 mM sodium phosphate
4082 Biochemistry, Vol. 36, No. 14, 1997
Nyaga et al.
(pH 6.8), 1 mM EDTA, 100 mM NaCl, and 100 µg/mL
BSA]. The reactions were stopped with the addition of
oligonucleotide loading buffer [95% (v/v) formamide, 20 mM
EDTA, 0.02% (w/v) bromphenol blue, and 0.02% (w/v)
xylene cyanol]. The products were separated on a 15%
polyacrylamide gel by electrophoresis for 4 h at 800 V. The
amounts of the nicked products were determined by Phos-
phorImager analyses (Molecular Dynamics, Sunnyvale, CA).
Steady-State Kinetics of Pyrimidine Dimer DNA Glyco-
sylase for Wild-Type and Mutant Endonuclease V. A 12-
mer oligonucleotide (substrate) with the sequence 5′ GCAC-
GAATTAAG 3′ (bases in boldface represent the position
of the thymine dimer) which was annealed to its complement
was used for these analyses. The oligonucleotide was labeled
on the 5′ end with [γ-32P]ATP using T4 polynucleotide
kinase. The short length of these oligonucleotides and the
presence of the cyclobutane pyrimidine dimer mandated
experimentation to determine the maximum temperature at
which the DNA was maintained as a duplex molecule.
The temperature which did not allow denaturation of the
12-mer oligonucleotide was determined as follows: 4 nM
(22.4 µL) 5′-end-labeled duplex 12-mer was preincubated
for a minimum of 30 min at various temperatures (10, 15,
20, and 25 °C) in buffer containing 25 mM sodium phosphate
(pH 6.8), 1 mM EDTA, 100 mM NaCl, 100 µg/mL BSA,
and 5% (v/v) glycerol. Wild-type endonuclease V (20 pM)
was added to a total reaction volume of 280 µL and
incubation allowed to continue at the respective temperatures.
Aliquots (40 µL), were taken at various time points, 0 (before
addition of the enzyme), 3, 6, 9, 12, and 15 min. The
aliquots were added to tubes containing 0.4 µL of 10% (w/
v) SDS and 1 µL of 1 M piperidine. The tubes were placed
in a dry ice-ethanol bath and then heated to 90 °C for 30
min. Piperidine treatment ensured that all the AP sites were
converted to single-strand breaks.
separated by electrophoresis through polyacrylamide gels
containing 8 M urea and the products quantitated by
PhosphorImager analyses. The initial rates were determined
graphically from the slopes of the kinetic data. The natural
logarithms of the initial rates for the different temperatures
plotted against the reciprocal of the absolute temperature (K)
yielded the Arrhenius plot (see Figure 2).
Various concentrations of the 12-mer oligonucleotide
ranging from 1 to 50 nM were incubated at 15 °C separately
with limiting concentrations of wild-type or mutant endo-
nuclease V (see Table 3) in the presence of 5% (v/v) glycerol
in a total reaction volume of 280 µL. Preincubation was
done at 15 °C for a minimum of 30 min in the absence of
the enzymes. The reactions were carried out in buffer
containing 25 mM sodium phosphate (pH 6.8), 1 mM EDTA,
100 mM NaCl, and 100 µg/mL BSA. Aliquots (40 µL) were
taken at different time points, 0, 3, 6, 9, 12, and 15 min.
The reactions were terminated by the addition of 0.4 µL of
10% (w/v) SDS and 1 µL of 1 M piperidine. The tubes
were placed in a dry ice-ethanol bath and then heated to
90 °C for 30 min. The samples were spun, and an equal
volume of oligonucleotide loading buffer was added. The
products were separated through a 15% polyacrylamide gel
containing 8 M urea. Electrophoresis was carried out at
constant voltage (800 V) for 3-4 h. The wet gels were
exposed and analyzed by PhosphorImager. Vmaxand Kmfor
the wild-type and the mutant enzymes were obtained from
Lineweaver-Burk plots. Previously, we have shown that
The products were
this preparation of wild-type endonuclease V contains 50%
active molecules based on sodium borohydride trapping
experiments (McCullough et al., 1996). All mutants were
assumed to be 100% active, and thus the kinetic parameters
reported represent minimal values.
Pyrimidine Dimer-Specific Plasmid Nicking Assay. Form
I pBR322 DNA, 120 µL (500 µg/mL), in buffer containing
25 mM sodium phosphate (pH 6.8) and 1 mM EDTA was
diluted to 100 µg/mL in either low salt buffer [25 mM
sodium phosphate (pH 6.8), 1 mM EDTA, 100 µg/mL BSA]
or high salt buffer [25 mM sodium phosphate (pH 6.8), 1
mM EDTA, 100 mM NaCl, and 100 µg/mL BSA] to a total
volume of 600 µL. The DNA was irradiated by 254 nm
short-wave UV light at 100 µW/cm2so as to generate
approximately 25 dimers per plasmid DNA (Gruskin &
Lloyd, 1986). Dilutions of wild-type or mutant endonuclease
V (see Figure 3 legend) were added to 3.5 µg of UV-
irradiated or unirradiated pBR322 to a total reaction volume
of 70 µL so as to react 0.5 µg of DNA per time point.
Incubation was carried out at 37 °C, and 10 µL aliquots were
taken at 0, 1, 2.5, 5, 15, and 30 min. The reactions were
stopped by the addition of an equal volume of electrophoresis
loading buffer [50 mM Tris-HCl (pH 8.0), 40% (w/v)
sucrose, 20 mM EDTA, 2% (w/v) SDS, and 0.02% (w/v)
bromphenol blue].The various forms of DNA were
separated by electrophoresis through 1% agarose gels. The
gels were stained with ethidium bromide and the band images
captured and analyzed by the VISAGE Bioimage electro-
phoresis gel analysis system (Bio Image, Ann Arbor, MI).
Correction for the decreased intercalation of ethidium
bromide in form I DNA was accomplished by multiplying
the integrated optical density (IOD) values for form I DNA
by a factor of 1.42 (Lloyd et al., 1978). In control experi-
ments in which irradiated plasmid DNAs were incubated with
wild-type or mutant enzymes, less than 2% conversion of
form I to form II DNA was observed with no conversion to
form III DNA. The small amount of nicking is likely to
arise due to apurinic sites in the form I plasmid preparation.
Electrostatic Potentials for Wild-Type and Mutant Forms
of T4 Endonuclease V. Mutant enzyme structures were
modeled on the wild-type enzyme using the Look program
(Molecular Applications Group). Electrostatic potentials at
the molecular surface were calculated and displayed with
GRASP (Nicholls et al., 1991).
RESULTS AND DISCUSSION
Intracellular Accumulation and Purification of the Wild-
Type and Mutant Endonuclease V Proteins. In order to
ensure that the expression of mutant proteins did not affect
the intracellular accumulation, the steady-state intracellular
levels of the wild-type enzyme and each mutant enzyme were
compared using SDS-polyacrylamide gel electrophoresis
and Western blot analyses of crude cell lysates (Figure 1A).
K86N (lane 2), R117N (lane 3), and K121N (lane 4)
accumulated to levels comparable to wild type (lane 6), while
Q15R (lane 5) accumulated poorly (lane 6). Lane 1 contains
the negative control (pGX2608-denV-), and therefore, no
endonuclease V was present. Other transformations of the
Q15R mutant into E. coli AB2480 (uVrA-recA-) resulted
in relatively higher accumulation (data not shown).
The wild-type and the mutant enzymes were purified by
five chromatographic steps: single-stranded DNA-agarose,
phenyl-Sepharose, Mono S (FPLC), heparin-Sepharose and
Endonuclease V Mutants Defective in Binding Nontarget DNA
Biochemistry, Vol. 36, No. 14, 1997 4083
Bio-Gel P-10. All the proteins were purified to apparent
homogeneity as revealed by silver staining of SDS-
polyacrylamide gel (Figure 1B). The proteins were deter-
mined to be catalytically active by the ability to nick a duplex
49-mer oligonucleotide containing a cis-syn cyclobutane
pyrimidine dimer (data not shown).
concentrations were determined to be as follows: wild type,
40 nmol/mL; Q15R, 20 pmol/mL; R117N, 650 pmol/mL;
K86N, 20 pmol/mL; and K121N, 93 pmol/mL.
Steady-State Kinetics of Pyrimidine Dimer DNA Glyco-
sylase for Wild-Type and Mutant Endonuclease V. In order
to determine the basic steady-state kinetic parameters for all
of the purified enzymes, we chose to use a short, 12-mer
oligonucleotide that contained a single site-specific cyclobu-
tane pyrimidine dimer. This oligonucleotide was constructed
by J. S. Taylor at Washington University and was designed
on the basis of the footprint of endonuclease V on a 49-mer
substrate that also contained a site-specific dimer (Latham
et al., 1995). Those experiments revealed an asymmetric
positioning of the dimer within the footprint. Due to both
the short length of the substrate and the presence of the dimer
lesion, we first needed to establish the appropriate conditions
under which the 12-mer remained duplex.
The final protein
Reaction mixtures (without enzyme) were preincubated
for at least 30 min at 10, 15, 20, and 25 °C to ensure that
temperature equilibrium had been established. Endonuclease
V (wild type) was added, and aliquots were taken at various
times and treated with piperidine to convert all glycosylase
and glycosylase-AP lyase nicked products to a uniquely
sized DNA fragment. Previous reports had indicated that
the glycosylase reaction was favored at low temperatures
over the combined glycosylase/AP lyase reaction (Seawell
et al., 1980). The initial rates of incision were plotted as a
function of temperature (K) (Figure 2). The 10, 15, and 20
°C reactions produced a linear Arrhenius plot, while the 25
°C rate deviated significantly. These data were interpreted
to mean that the duplex DNA began melting between 20
and 25 °C. Therefore, we chose to conduct all steady-state
experiments at 15 °C.
Table 3 summarizes the kinetic data for wild-type and
mutant endonuclease V. Q15R and R117N exhibited near
wild-type Kmvalues whereas K121N and K86N showed a
4-8-fold increase in Kmvalues respectively relative to the
wild type. All the mutants had decreased (2-35-fold)
catalytic activities (kcat) with K121N showing a considerable
decrease (35-fold) relative to that of the wild type. All the
mutants showed decreases (2-84-fold) in efficiencies (kcat/
Km) with the most severely affected being K86N and K121N
(9- and 84-fold, respectively).
importance of K86 and K121 in both substrate binding and
The cocrystal structure of T4 endonuclease V and its
substrate (Vassylyev et al., 1995) revealed that the side chains
of R3, H16, R117, and K121 are involved in polar interac-
tions with the two phosphates of the pyrimidine dimer and
also that K86 is involved in substrate recognition. This is
These data indicate the
FIGURE 1: (A) Western blot analyses of endonuclease V proteins.
Total cellular proteins were accumulated within the stationary phase
E. coli AB2480 (uVrA-recA-) grown at 30 °C. Samples (50 µL)
of crude lysates (1.0 mg/mL) in 10 mM Tris-HCl (pH 8.0) and 1
mM EDTA were boiled in an SDS-containing buffer for 5 min
before being loaded and separated by SDS-PAGE. The lane
assignments are as follows: (1) pGX2608-denV-, (2) K86N, (3)
R117N, (4) K121N, (5) Q15R, (6) wild type, and (7) prestained
molecular mass markers. The arrow shows the positions of wild-
type or mutant forms of endonuclease V. (B) Silver staining of
wild-type and mutant endonuclease V. Purified wild-type and
mutant endonuclease V (20-50 µL) was mixed with an equal
volume of SDS loading buffer. The samples were boiled for 5-10
min and then separated by SDS-PAGE. The proteins were stained
by silver salts. Lanes: (1) WT endonuclease V, (2) K86N, (3)
R117N, (4) K121N, (5) Q15R, and (6) molecular mass markers.
The arrow shows the positions of wild-type or mutant forms of
FIGURE 2: Arrhenius plot for wild-type endonuclease V. A duplex
5′-end-labeled 12-mer oligonucleotide (4 nM) was preincubated at
various temperatures (10, 15, 20, and 25 °C) prior to the addition
of wild-type endonuclease V (20 pM). Aliquots were taken at 0
(before the addition of enzyme), 3, 6, 9, 12, and 15 min and
analyzed by SDS-PAGE containing 8 M urea. The Arrhenius plot
was generated from a plot of natural logarithms of the initial rates
versus the reciprocal of the absolute temperature (K).
Table 3: Steady-State Kinetics of Pyrimidine Dimer DNA
Glycosylase for Wild-Type and Mutant Endonuclease V
2.26 × 106
1.42 × 106
5.8 × 105
2.64 × 105
2.69 × 104
4084 Biochemistry, Vol. 36, No. 14, 1997
Nyaga et al.
consistent with decreased substrate binding of K86N and
K121N, as well as the decreased catalytic activities of
R117N, K86N, and K121N observed in this study. Previous
studies by Doi et al. (1992) of R117Q and K121Q demon-
strated decreased binding affinities while only K121Q
showed an increased kcat. The modest discrepancies observed
between kcatvalues for K121Q (Doi et al., 1992) and K121N
(this study) may be due to a number of factors including the
lengths or conformation of the side chains of glutamine
versus that of asparagine. The longer side chain of glutamine
compared to that of asparagine may provide the right distance
for the interaction with the phosphates of the pyrimidine
dimer. The differences that are reported in kcatand Kmvalues
for wild-type endonuclease V (6-fold and 10-fold, respec-
tively) may stem from differences in the experimental
conditions by the two laboratories. In our study, a 12-mer
oligonucleotide containing an asymmetrically positioned
dimer was used, as opposed to a 14-mer oligonucleotide with
a centrally placed dimer, and our reactions were carried out
at 15 °C as opposed to 37 °C. This latter point may be
especially relevant since the Arrhenius plot (Figure 2)
indicated the possibility of denaturation of the 12-mer at
temperatures above 20 °C.
Analyses for ProcessiVe Nicking ActiVity. Although the
mutant enzymes showed decreased catalytic activities, it was
still possible to determine whether these mutations had
significantly affected nontarget DNA binding. In order to
accomplish this, the processive nicking activity of endonu-
clease V was monitored using UV-irradiated supercoiled
(form I) plasmid DNA containing multiple pyrimidine dimers
per plasmid (Lloyd et al., 1980). For in Vitro analyses, low
salt conditions are essential to manifest the full processivity
of the wild-type enzyme, and these same conditions were
used to assay the relative processivity of the altered proteins.
Experimentally, when supercoiled (form I) plasmid DNA is
incised, form II (nicked circular) DNA is produced, and form
III (linear) DNA is generated when two nicks occur in close
proximity on complementary strands.
formation of form III DNA that is diagnostic of the
processive nicking activity such that form III DNAs ac-
cumulate linearly throughout a kinetic reaction and that form
I DNA is still present as form III accumulates (Lloyd et al.,
1980; Gruskin & Lloyd, 1986). In contrast, in analyses of
reactions in which a distributive search mechanism is
operational, there is a long delay in the appearance of form
III DNA molecules because breaks are being put into the
plasmid population on a random basis. Thus, all form I DNA
disappears prior to the accumulation of significant amounts
of form III DNA. The data in Figure 3 show the kinetic
analyses performed at low salt conditions (closed symbols)
It is the rate of
FIGURE 3: Kinetic analysis of pyrimidine dimer-specific nicking of UV-irradiated pBR322 DNA at low and high salt concentrations.
pBR322 DNA containing multiple dimers per plasmid was incubated at 37 °C under low (40 mM) or high (140 mM) salt concentrations
with the wild-type or mutant enzymes. Aliquots were taken at various time points (0, 1, 2.5, 5, 15, and 30 min) and analyzed for the
presence of different topological DNA forms. The amounts of enzymes used were as follows: WT, 1.0 ng (low salt) and 2.5 ng (high salt);
Q15R, 1.5 ng (low salt) and 1.2 ng (high salt); R117N, 12 ng (low salt) and 1.2 ng (high salt); K86N, 0.7 ng (low salt) and 1.3 ng (high
salt); K121N, 30 ng (low salt) and 60 ng (high salt). The panels are as follows: (A) wild type, (B) Q15R, (C) R117N, (D) K86N, and (E)
K121N. The symbols used are as follows: form I (closed circle, low salt; open circle, high salt); form III (closed triangle, low salt; open
square, high salt).
Endonuclease V Mutants Defective in Binding Nontarget DNA
Biochemistry, Vol. 36, No. 14, 1997 4085
for the wild-type enzyme (Figure 3A) and the four mutant
enzymes: Q15R (Figure 3B), R117N (Figure 3C), K86N
(Figure 3D), and K121N (Figure 3E).
The wild-type enzyme (Figure 3A) shows the characteristic
loss in form I DNA with concomitant increases in forms II
(not shown) and III. It is this linear increase in form III
DNA while there is still a high percentage of form I DNA
that is the hallmark of processivity (Lloyd et al., 1980;
Gruskin & Lloyd, 1986). The data for two of the mutants,
R117N and Q15R, revealed characteristics of both processive
and distributive mechanisms: evidence for processivity in
that there was a significant accumulation in the percentage
of form III DNA while there was still high levels of form I
DNA and evidence for a distributive character in that there
was a significant time lag in the appearance of the form III
DNAs. Together, these data suggest that the R117N and
Q15R display a limited processive nicking activity, such that
dissociation occurs prior to incision of all dimers within the
plasmid. These data are qualitatively similar to the incision
activity of the UvrABC complex on UV-irradiated plasmid
DNA, in which it was estimated that UvrABC initiates the
repair over 2-3 kb segments of DNA prior to dissociation
(Gruskin & Lloyd, 1988a,b).
In contrast to the two mutants described above, both the
K86N and the K121N showed severe reductions in proces-
sive nicking activities, in that no form III DNAs accumulated
throughout the extent of the kinetic analyses. The 30 min
time point (Figure 3E) shows complete conversion of form
I to form II DNA (not shown), and yet no form III DNA
was present. In additional experiments, greater concentra-
tions of these enzymes were used, and form III DNA only
appeared long after the complete disappearance of form I
DNAs (data not shown). Thus, we can conclude that these
two mutations (K86N and K121N) have severely affected
the nontarget DNA binding activity in each mutant.
To further demonstrate that each of these mutants has
decreased affinity for nontarget DNA, similar reactions were
carried out, except under high salt conditions (open symbols)
(Figure 3). The wild-type enzyme (Figure 3A) clearly shows
a distributive reaction mode in that no form III DNA
accumulates until after all form I DNA has been incised.
This suggests that multiple single-strand breaks were ran-
domly introduced into the population without any significant
percentage being in close proximity on complementary
strands. All four of the mutants displayed complete loss of
form I DNA with no increase in form III DNA, thus
providing strong evidence that they also use a distributive
search mechanism at high salt concentrations.
In order to assess the magnitude of the effect that changes
in the salt concentration have on the processive nicking
activity, a rate comparison was made for the rate of loss of
form I DNA (-ln mass fraction of form I) at the two different
salt concentrations. Data were normalized relative to the
incision activity per nanogram of input enzyme (Figure 4
FIGURE 4: Comparative analyses of kinetic rates of incision at low and high salt concentrations. The negative natural logarithms of the
mass fraction form I DNA remaining at each time point for data in Figure 3 were determined and normalized relative to the amounts of
each enzyme used in each reaction. The closed circles show the dimer nicking rates obtained in low salt, while open circles show the results
of dimer nicking rates in high salt conditions. Panels: (A) wild type, (B) Q15R, (C) R117N, (D) K86N, and (E) K121N.
4086 Biochemistry, Vol. 36, No. 14, 1997
Nyaga et al.
and Table 4). For the wild type, a comparison of rates at
the high salt versus low salt reaction conditions revealed a
dramatic difference in the apparent rate of incision. How-
ever, this apparent rate difference is merely a manifestation
of the wild-type enzyme’s processivity, such that many
breaks are put in one plasmid prior to dissociation. This
interpretation is also consistent with significant accumulation
of form III DNA while form I DNA was still present.
Qualitatively similar data were generated for mutants Q15R
and R117N in which a comparison of the -ln form I DNA
is made at the high and low salt concentrations (Panels B
and C of Figure 4, respectively). In contrast, the K86N
(Figure 4D) mutant had identical slopes at both salt
concentrations, indicating a distributive search mechanism
at either salt concentration. Furthermore, the slope of the
K121N was greater at low salt than at high salt (Figure 4E).
These data suggest that the loss of charge at K121 severely
restricts access or correct orientation of the enzyme with
respect to the DNA, such that at high salt concentrations,
few productive enzyme-DNA encounters occur. Thus, the
rate at which single pyrimidine dimers can be located is in
fact enhanced under low salt conditions. Calculations of the
relative ratios of the slopes are given in Table 4. It is clear
that the processivity of R117N and Q15R is close to that of
the wild type while that of the K86N and K121N is very
Analyses of Electrostatic Potentials. In order to further
understand the molecular interaction between the residues
of endonuclease V and nontarget DNA, electrostatic poten-
tials along the surfaces of wild-type and the mutant forms
of endonuclease V were calculated and displayed using
Graphical Representation and Analysis of Surface Properties
(GRASP, Columbia University) as shown in Figure 5. In
this display, the blue color represents positive charges while
red represents negative charges. The R117N mutant (Figure
5C) appears to have retained wild-type electrostatic potentials
(Figure 5A). The position of R117 in the cocrystal structure
reveals that it is approximately in the middle of R-helix 3 as
it connects the acidic face with the basic DNA interactive
face (Vassylyev et al., 1995). Side views of the cocrystal
structure reveal that it is not directly on the concave surface
of the DNA interacting face, although phosphate contacts
can be made through water. It is this relative position which
may minimize its interaction with nontarget DNA.
addition, Q15R appears to have an increase in positive
potential in the active site region (Figure 5B) as had been
predicted. This may explain the wild-type activities observed
in kcatand Km(Table 3) and nontarget DNA binding (Figures
3A-C and 4A-C) for both Q15R and R117N.
K121N (Figure 5E) and K86N (Figure 5D) appear to have
experienced a reduction in the positive potential as shown
by the distribution and the intensity of the blue color relative
to that of the wild type. The distribution and intensity of
the positive potential around the active site (E23 shown in
red at the center of each panel in Figure 5) in the K121N
mutant seem to be the most adversely affected of all the four
mutants relative to the wild type. Like R117, K121 is also
contained in R-helix 3 but is part of the overall basic face
of endonuclease V that contacts DNA. In contrast to R117,
its (K121) relative position on the positively charged face
of endonuclease V readily explains the reduction in nontarget
Table 4: Comparative Rates of Pyrimidine Dimer-Specific Nicking
rates of incision
enzymes high salt low salt
ratio of incision rates,
high salt/low salt
to wild type (%)
FIGURE 5: Electrostatic potentials for wild-type and mutant forms
of T4 endonuclease V. Electrostatic potentials along the surfaces
of both the wild-type and mutant forms of T4 endonuclease V were
calculated and displayed using the GRASP program. Panels: (A)
wild type, (B) Q15R, (C) R117N, (D) K86N, and (E) K121N. The
color codes are blue for positive and red for negative potentials.
The active site (E23) is shown as a red structure in the center of
Endonuclease V Mutants Defective in Binding Nontarget DNA
Biochemistry, Vol. 36, No. 14, 1997 4087
DNA binding when its site was mutated. The K86N mutant Download full-text
(Figure 5D) clearly lacks a significant portion of its positive
charges at the bottom left corner (position of K86). These
observations may explain the reductions in kcat (Table 3),
increases in Km (Table 3), and significant reductions in
nontarget DNA binding (Figures 3D,E and 4D,E) for both
K86N and K121N relative to that of the wild type (Table 3,
Figures 3A and 4A). Our results for K86N are significantly
different than those reported by Doi et al. (1992) in which
K86 was replaced with Q86; this mutant exhibited wild-type
pyrimidine dimer glycosylase activity. At the present time
we do not understand the significant differences between
these two mutants since both glutamine and asparagine are
neutral amino acids. Additionally, inspection of the elec-
trostatic potential reveals that the nature of the active site
(E23) was not changed to any appreciable degree as shown
in red at the center of each panel. This is an indication that
the observed activity difference (kcat, Km, and nontarget DNA
binding) between the wild type and the mutants is not likely
to be due to a perturbation of the structure of the active site
but rather due to the change of the nontarget DNA binding
All these data, taken together, suggest that critical contact
points exist between the enzyme and DNA at positions K86
and K121. Previous site-directed mutagenesis and X-ray
diffraction studies have shown the critical nature of the
residues at R3, R22, and R26 in nontarget DNA binding
(Dowd & Lloyd, 1989a,b; Morikawa et al., 1992). Col-
lectively, these data reveal that the interaction of endonu-
clease V with nontarget DNA is a path along the outside of
the enzyme, including at least these five basic residues (R3,
R22, R26, K86, and K121). These data also suggest that,
for nontarget DNA interactions, there are no significant
contacts made by R117 and Q15. Our previous studies also
showed that another residue, K33, on the basic face of the
enzyme did not make nontarget DNA contacts (Dowd &
Lloyd, 1990). The steady-state kinetic results are consistent
with those obtained from UV-irradiated plasmid DNAs in
which K86 and K121 were residues shown to be critical in
nontarget DNA binding. Thus, these studies have enabled
us to have a clear understanding of the molecular interactions
between endonuclease V and nontarget DNA.
We thank the NIEHS Center Molecular Biology Core for
synthesis of the oligonucleotides. We also extend our sincere
thanks to Ms. Judy Daniels for her excellent assistance in
the preparation of the manuscript.
Augustine, M. L., Hamilton, R. W., Dodson, M. L., & Lloyd, R.
S. (1991) Biochemistry 30, 8052-8059.
Benton, W. D., & Davis, R. W. (1977) Science 196, 180-182.
Burnette, W. N. (1981) Anal. Biochem. 112, 195-203.
Dizdaroglu, M., Zastawny, T. H., Carmical, J. R., & Lloyd, R. S.
(1996) Mutat. Res. 362, 1-8.
Dodson, M. L., Schrock, R. D., & Lloyd, R. S. (1993) Biochemistry
Dodson, M. L., Michaels, M. L., & Lloyd, R. S. (1994) J. Biol.
Chem. 269, 32709-32712.
Doi, T., Recktenwald, A., Karaki, Y., Kikuchi, M., Morikawa, K.,
Ikehara, M., Inaoka, T., Hori, N., & Ohtsuka, E. (1992) Proc.
Natl. Acad. Sci. U.S.A. 89, 9420-9424.
Dowd, D. R., & Lloyd, R. S. (1989a) J. Mol. Biol. 208, 701-707.
Dowd, D. R., & Lloyd, R. S. (1989b) Biochemistry 28, 8699-
Dowd, D. R., & Lloyd, R. S. (1990) J. Biol. Chem. 265, 3424-
Gruskin, E. A., & Lloyd, R. S. (1986) J. Biol. Chem. 261, 9607-
Gruskin, E. A., & Lloyd, R. S. (1988a) J. Biol. Chem. 263, 12728-
Gruskin, E. A., & Lloyd, R. S. (1988b) J. Biol. Chem. 263, 12738-
Higgins, K. M., & Lloyd, R. S. (1987) Mutat. Res. 183, 117-121.
Laemmli, U. K. (1970) Nature 227, 680-685.
Latham, K. A., Taylor, J. S., & Lloyd, R. S. (1995) J. Biol. Chem.
Lloyd, R. S. (1993) in Nucleases, pp 445-454, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY.
Lloyd, R. S., & Augustine, M. L. (1989) Proteins: Struct., Funct.,
Genet. 6, 128-138.
Lloyd, R. S., & Linn, S. (1993) in Nucleases, pp 263-316, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Lloyd, R. S., & Van Houten, B. (1995) in DNA Repair Mecha-
nisms: Impact on Human Diseases and Cancer (Voss, J. M.
H., Ed.) pp 25-66, Landes Co., Austin, TX.
Lloyd, R. S., Haidle, C. W., & Hewitt, R. R. (1978) Cancer Res.
Lloyd, R. S., Hanawalt, P. C., & Dodson, M. L. (1980) Nucleic
Acids Res. 8, 5113-5127.
Lloyd, R. S., Recinos, A., III, & Wright, S. T. (1986) Biotechniques
McCullough, A. K., Scharer, O., Verdine, G. L., & Lloyd, R. S.
(1996) J. Biol. Chem. 271, 32147-32152.
Morikawa, K., Matsumoto, O., Tsujimoto, M., Katayanagi, J.,
Ariyoshi, M., Doi, T., Ikehara, M., Inaoka, T., & Ohtsuka, E.
(1992) Science 256, 523-526.
Nicholls, A., Sharp, K. A., & Honig, B. (1991) Proteins 11, 281-
Nickell, C., Anderson, W. F., & Lloyd, R. S. (1991) J. Biol. Chem.
Nickell, C., Prince, M. A., & Lloyd, R. S. (1992) Biochemistry 31,
Radany, E. H., Naumovaki, L., Love, J. D., Gutekunst, K. A., Hall,
D. H., & Friedberg, E. C. (1984) J. Virol. 52, 846-856.
Recinos, A., III, & Lloyd, R. S. (1986) Biochem. Biophys. Res.
Commun. 138, 945-952.
Recinos, A., & Lloyd, R. S. (1988) Biochemistry 27, 1832-1838.
Sanger, F., Mikley, S., & Coulson, A. R. (1977) Proc. Natl. Acad.
Sci. U.S.A. 74, 5463-5467.
Schaller, H. C., Nu ¨sslein, F. J., Bonhoeffer, C. K., & Nietzschmann,
I. (1972) Eur. J. Biochem. 26, 474-481.
Schrock, R. D., III, & Lloyd, R. S. (1991) J. Biol. Chem. 266,
Schrock, R. D., III, & Lloyd, R. S. (1993) J. Biol. Chem. 268, 880-
Seawell, P. C., Smith, C. A., & Ganesan, A. K. (1980) J. Virol.
Smith, C. A., & Taylor, J.-S. (1993) J. Biol. Chem. 268, 11143-
Stump, D. G., & Lloyd, R. S. (1988) Biochemistry 27, 1839-1843.
Taylor, J.-S. (1994) J. Am. Chem. Soc. 27, 76-82.
Towbin, H., Staehelin, T., & Gordon, J. (1979) Proc. Natl. Acad.
Sci. U.S.A. 76, 4350-4354.
Valerie, K., Henderson, E. E., & deRiel, J. K. (1984) Nucleic Acids
Res. 12, 8085-8096.
Vassylyev, D. G., Kashiwagi, T., Mikani, Y., Ariyoshi, M., Iwai,
S., Ohtsuka, E., & Morikawa, K. (1995) Cell 83, 773-782.
von Hippel, P. H., & Berg, O. G. (1989) J. Biol. Chem. 264, 675-
Zoller, M. J., & Smith, M. (1983) Methods Enzymol. 100, 468-500.
4088 Biochemistry, Vol. 36, No. 14, 1997
Nyaga et al.