Page 23 - ebook

The mechanism of gap creation by a multifunctional
nuclease during base excision repair

Jungmin Yoo and Gwangrog Lee
School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, 61005, Republic of Korea

ABSTRACT
During base excision repair, a transient single-stranded DNA (ssDNA) gap is produced at the apurinic/apyrimidinic (AP) site. Exonuclease III, capable of performing both AP endonuclease and
exonuclease activity, are responsible for gap creation in bacteria. We used single-molecule fluorescence resonance energy transfer to examine the mechanism of gap creation. We found an AP
site anchor-based mechanism by which the intrinsically distributive enzyme binds strongly to the AP site and becomes a processive enzyme, rapidly creating a gap and an associated transient
ssDNA loop. The gap size is determined by the rigidity of the ssDNA loop and the duplex stability of the DNA and is limited to a few nucleotides to maintain genomic stability. When the 3′ end is
released from the AP endonuclease, polymerase I quickly initiates DNA synthesis and fills the gap. Our work provides previously unidentified insights into how a signal of DNA damage changes
the enzymatic functions.
INTRODUCTION
Base damage, the most common DNA lesion, is caused by alkylation, oxidation, deamination, and depurination/depyrimidination 1 . A series of enzymes
recognize and process various kinds of DNA base damage in sequence in a process called base excision repair (BER). In BER, all damaged bases
are cleaved and converted into apurinic/apyrimidinic (AP) sites by damage-specific glycosylases. Upon the removal of the damaged bases by
glycosylases, exonuclease III (ExoIII) precisely cut the phosphodiester backbone of DNA (Fig. 1). ExoIII in Escherichia coli and apurinic/apyrimidinic
endonuclease I and II (APE1 and APE2) in humans are ExoIII family members and the main AP endonucleases responsible for processing over 80%
of base damage 2, 3 . ExoIII is a multifunctional enzyme that functions as both an AP endonuclease at AP sites and an exonuclease in the absence of AP Fig. 1. Structure of ExoIII. Modeled DNA-enzyme complex
sites 4, 5 . It remains unknown how both enzymatic activities are coordinated during gap creation and how the gap size is limited to a few nucleotides for reconstituted in silico from ExoIII (PDB entry: 1AKO) and DNA
(PDB entry: 1DE8), showing the active site (green), the binding
optimal repair. We addressed how the dual function of ExoIII is regulated on different substrates and in different buffer conditions. of the protruding α helix into the DNA major groove (blue), and
AP site–specific binding (red).
METHODS Fig. 2. smFRET experimental scheme
before and after degradation by ExoIII and
an ssDNA mimicking the degradation
product (left). ExoIII converts the dsDNA
To monitor the activity of ExoIII in real time, we adopted single molecule fluorescence resonance between the donor (green) and the
energy transfer (smFRET) 6 . The donor (Cy3) and acceptor (Cy5) dyes were covalently acceptor (red) to ssDNA, resulting in an
efficiency.
FRET
connected to the 5’ non-hydrolyzed strand of a double-stranded (ds)DNA (left in Fig. 2). The increase in FRET time trajectory A
representative
dsDNA substrate was immobilized on a quartz surface. When ExoIII with a reaction buffer was showing how the degradation time is
measured. The total intensity is the sum of
added to the DNA substrate, the enzyme degraded only the 3' hydrolyzed strand. The the donor and acceptor intensities (black,
degradation of the 3' strand leaves 5' single-stranded (ss)DNA, which undergoes ssDNA top). Green and red curves represent the
shrinkage and show increased FRET due to decreased distance between Cy3 (green line) and donor and acceptor intensities,
respectively (middle), and the blue curve
Cy5 (red line) (right in Fig. 2). represents the calculated FRET efficiency
(bottom) (right). a.u., arbitrary units.
RESULTS
ExoIII processively degrades DNA substrates containing an AP site, whereas it performs Coordinated gap processing and gap filling by ExoIII
distributive degradation on dsDNA substrates lacking an AP site. and pol I.
A A B A B C
C
B C D

E
Fig. 3. Schematics of different substrates(A). (B) Average degradation time per
nucleotide for various DNA substrates with SEM. Degradation times were Fig. 5. Coordinated gap processing and gap filling by ExoIII and pol I. (A) Representative fluorescence
determined during which FRET increases from the minimum to the maximum intensity (top, green for donor and red for acceptor) and FRET efficiency time trajectories (blue) with the
characteristic features of degradation (orange region), polymerase binding (violet region), and
values. (C) Fraction degraded versus NaCl concentration for the three different
substrates (AP-DNA, nicked AP-DNA, and nicked DNA), showing a strong ExoIII polymerization (green region). The experiments were performed by adding ExoIII and pol I to AP-DNA in
affinity for the AP site even at high NaCl concentrations. a solution containing Mg2+ and dNTPs. (B) Histograms (top) before (black) and after (gray) degradation.
The FRET efficiency shift caused by degradation in the presence of pol I indicates that the gap size is
approximately 5 to 6 nt long. Calibration curve of FRET efficiency and gap size measured at various salt
To investigate the effect of AP sites on enzymatic activity, concentrations with 10 mM Mg 2+ (bottom). (C) Degradation rate and polymerization rate as a function of
we compared the degradation activity of different DNAs Fig. 4. The AP site provides a strong affinity to the DNA substrate and processive dNTP concentration. The degradation rate does not change, whereas the polymerization rate is strongly
(Fig. 3A). At 5 nM [ExoIII], the degradation time of the degradation in the presence of Mg2+, directly monitored by Cy3-labeled WT ExoIII and dependent on the dNTP concentration.
DNAs w/ the AP site (AP-DNA and nicked AP-DNA) were Cy5-labeled AP-DNA. (A) A WT ExoIII is labeled with a Cy3 fluorophore (green star) at We performed experiments for gap processing by ExoIII and gap
shorter than DNAs w/o the AP site (blunt-ended DNA and the N terminus via site-specific labeling by sortase. (B) Experimental setup for filling by pol I consecutively and more than ~52% (n = 145) of all
monitoring direct protein binding, where Cy3-labeled ExoIII was added to a Cy5-labeled
nicked-DNA) (Fig. 3B). The DNA w/ the AP site (i.e., AP- AP-DNA substrate immobilized on a fluorescence imaging surface. (C) Representative traces displayed a unique pattern: an increase in FRET (the
fluorescence intensity (top, green for donor and red for acceptor) and FRET efficiency
DNA & nicked AP-DNA) showed robust degradation (second and below) time trajectories show a gradual FRET increase during gap degradation by ExoIII-orange region) followed by a decrease in FRET
regardless of salt concentration, whereas nicked DNA creation. (D) FRET evolutionary tendency calibrated as a function of gap size (the polymerization activity of pol I-green region) and a sharp increase
generated during gap processing (x stands for the gap size in number of nucleotides).
lacking the AP site displayed a strong salt-dependent (E) Histogram of total degradation (left) and binding (right) times. in fluorescence (the binding of pol I-violet region) between them
inhibition of degradation, with the lowest activity at 100 We monitored the degradation reaction by FRET between (Fig. 5A, arrow). The histogram obtained from the degradation (~443
mM NaCl (Fig. 3C). These results demonstrate that the Cy3-labeled WT ExoIII and Cy5-labeled AP-DNA (Fig. 4A molecules) revealed a FRET peak shift from ~0.36 to ~0.54,
enzyme efficiently digests AP-containing substrates even and 4B) in the presence of Mg 2+ during gap creation. The corresponding to an average gap of ~5 nt, based on the calibration
at higher [NaCl] but less efficiently digests AP-lacking FRET evolution of time traces (Fig. 4C) did not show any curve (Fig. 5B). The degradation rate obtained from the FRET
nicked DNA in a distributive manner, confirming that disappearance of the Cy3 signal until the FRET reached increase was ~11.8 nt/s, whereas the polymerization rate obtained
ExoIII binds tightly to the AP site. Both the faster the maximum value (Fig. 4E) and was consistent with the from the FRET decrease varied and fit Michaelis-Menten kinetics with
degradation (Fig. 3B) and the robust affinity (Fig. 3C) in degradation mimic tendency with parabolic FRET growth a Km value of ~531 nM and a maximum velocity of ~12.5 nt/s, which
the presence of AP sites imply that strong AP site (Fig. 4D), indicating that gradual exo-nucleolytic was consistent with previous data (Fig. 5C). This suggests that when
recognition allows the enzyme to processively degrade a degradation took place while FRET increased and the gap ExoIII first releases the 3′ end, pol I quickly binds and synthesizes
series of nucleotides without dissociation. creation is processive in the presence of AP sites. DNA from it.
CONCLUSIONS REFERENCES
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