Báo cáo khoa học: Ascorbic acid induces a marked conformational change in long duplex DNA doc

We examined the effect of ascorbic acid on the higher-order structure of DNA through real-time observa-tion by fluorescence microscopy.. We found that ascorbic acid generates a pearling st

Ascorbic acid induces a marked conformational change in long

duplex DNA

Yuko Yoshikawa1,5, Mari Suzuki1, Ning Chen2, Anatoly A Zinchenko2, Shizuaki Murata2,5,

Toshio Kanbe3, Tonau Nakai4, Hidehiro Oana4,5and Kenichi Yoshikawa4,5

1

Department of Food and Nutrition, Nagoya Bunri College, Japan;2Graduate School of Environmental Studies, Nagoya University, Japan;3Laboratory of Medical Mycology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Japan;4Department of Physics, Kyoto University, Japan;5CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation

Ascorbic acid is often regarded as an antioxidant in vivo,

where it protects against cancer by scavenging

DNA-dam-aging reactive oxygen species However, the detailed

mech-anism of the action of ascorbic acid on genetic DNA is still

unclear We examined the effect of ascorbic acid on the

higher-order structure of DNA through real-time

observa-tion by fluorescence microscopy We found that ascorbic

acid generates a pearling structure in single giant DNA

molecules, with elongated and compact regions coexisting

along a molecular chain Results from electron microscopy and atomic force microscopy indicate that the compact regions assume a loosely packed conformation A possible mechanism for the induction of this conformational change

is discussed in relation to the interplay between the higher-order and second-higher-order structures of DNA

Keywords: ascorbic acid; higher order structure of DNA; pearling structure; single molecular observation

Vitamin C (ascorbic acid) is ubiquitous and fundamental in

living cells, where it acts as a water-soluble antioxidant and

an essential cofactor for many enzymes involved in diverse

metabolic pathways Several epidemiological and

experi-mental studies have shown that the consumption of foods

rich in vitamin C is associated with a decreased risk of

several chronic diseases, including cardiovascular disease

and cancer [1–4] However, the extent to which vitamin C

contributes to these effects is still unclear [5] There is

evidence that vitamin C inhibits oxidative DNA damage

in isolated and cultured cells exposed to reactive oxygen

species and UV/visible light [5–9] On the other hand,

several studies have shown that vitamin C sometimes

increases DNA damage in humans [5,10,11] These studies

suggest that vitamin C may have anti-oxidative or

pro-oxidative properties depending on the conditions in the cell

Thus, it may be useful to clarify the mechanism of the action

of vitamin C on DNA [12,13]

It is well known that genomic DNA molecules are very

long, e.g of the order of 1 cm in human cells Recently,

it has become clear, from single-chain observation using

fluorescence microscopy together with electron microscopy,

that long DNA exhibits unique responses to different

condensing chemicals [14] Polyamines, metal cations,

neutral polymers, polypeptides and basic proteins have all

been found to be efficient condensing agents [15] A variety

of higher-order structures can be generated from the same long DNA molecule: e.g toroid, rod, spherical, spool and intrachain segregated structures [14]

We performed a single-molecule observation of giant DNA molecules to examine the effect of ascorbic acid at physiological pH Surprisingly, we found that ascorbic acid generates a pearling structure in a giant DNA molecule, in which elongated and compact parts coexist along a single molecular chain A possible mechanism is discussed in relation to the interplay between the higher-order and second-order structures of DNA

Experimental procedures

Materials T4 phage DNA, 166 kbp with a contour length of 57 lm, was purchased from Nippon Gene (Toyama, Japan) The fluorescent dye YOYO-1 was obtained from Molecular Probes, Inc (Portland, Oregon, USA) An antioxidant, 2-mercaptoethanol, and L-ascorbic acid were purchased from Wako Pure Chemical Industries (Osaka, Japan) Fluorescence microscopic observations

T4 phage DNA was dissolved in 10 mMTris/HCl buffer with 0.1 lMYOYO-1 (nucleic acid staining) and 4% (v/v) 2-mercaptoethanol at pH7.4 Various concentrations of

L-ascorbic acid (50 lM to 10 mM) were added To avoid intermolecular DNA aggregation, measurements were con-ducted at a low DNA concentration, 0.3 lMin nucleotide units Fluorescent DNA images were obtained using a microscope (Axiovert 135 TV; Carl Zeiss, Jena, Germany) equipped with a 100· oil-immersion objective lens and a

Correspondence toY Yoshikawa, Department of Food and Nutrition,

Nagoya Bunri College, Nagoya, 451-0077, Japan.

Fax: + 81 52 521 2259, Tel.: + 81 52 521 2259,

E-mail: yuko@chem.scphys.kyoto-u.ac.jp

Abbreviations: AFM, atomic force microscopy; TEM, transmission

electron microscopy.

(Received 18 March 2003, revised 4 May 2003,

accepted 2 June 2003)

highly sensitive Hamamatsu SIT TV camera, which allowed

recording of images on video tapes The video image was

analyzed with an image processor (Argus 20; Hamamatsu

Photonics, Hamamatsu, Japan)

Imaging by atomic force microscopy (AFM)

A DNA solution containing ascorbic acid was prepared

as described above, and 5 lL was adsorbed on to freshly

cleaved mica for 1 min The mica surface was washed with

Milli-Q-purified water, and dried in N2gas An NVB 100

(Olympus, Tokyo, Japan) operated in trapping mode was

used Images were displayed without modification except

for flattening to remove the background curvature of the

mica surface

Electron microscopic observations

Samples used for electron microscopy were mounted on

carbon-coated copper grids (No 200), negative-stained

with 1% uranyl acetate, and observed with a transmission

electron microscope (Jeol 1200EX, Tokyo, Japan) at

100 kV

CD spectroscopic measurements

Measurements were performed at a T4 phage DNA

concentration of 30 lMin 20 mMTris/HCl, pH 7.4

Vari-ous concentrations of L-ascorbic acid (10 lM to 150 lM)

were added to the DNA solution CD spectra were recorded

on a Jasco J-720 spectropolarimeter in a 1.0· 1.0 · 5.0 cm

quartz cell at room temperature

Results

Figure 1 shows fluorescence microscopic images of

individ-ual T4 DNA molecules in aqueous solution at pH7.4 in the

absence and presence of ascorbic acid Depending on the

concentration of ascorbic acid, DNA molecules exhibit

intrachain and translational Brownian motion with

differ-ent conformations Without ascorbic acid (Fig 1A), DNA

molecules assume an elongated coil conformation At

200 lM ascorbic acid, DNA remains in a coiled state

(Fig 1B) At 5 mM, folded compact and elongated regions

coexist along a single molecular chain, i.e intrachain

segregation is observed (Fig 1C) This segregated

confor-mation appears at concentrations of ascorbic acid of 1 mM

and above At 1 mM, about 10% of the DNA molecules

show segregated structures, the majority exhibiting the

elongated coil conformation Above 3 mM, most of the

DNA molecules (>80%) are in the segregated state

To examine the segregated conformation, we used

fluorescence microscopic observation of fixed DNA

mole-cules on a glass surface Figure 2 shows the fluorescence

images of T4 DNA molecules on a glass slide in 5 mM

ascorbic acid solution The DNA molecule was extended on

a glass slide by introducing shear to the solution with a

cover slide Mini-globules are observed along an extended

DNA molecule on a 2D glass plate On the other hand,

under the same conditions, the segregated DNA molecules

in the bulk solution show only a few compact regions

(Fig 1C) As the effective resolution becomes higher on the

fixed DNA, the small mini-globules became visible From these observations and the following results obtained by AFM, it is expected that such small mini-globules observed

A

B

C

5 µm Fig 1 Fluorescence microscopic images of T4 DNA moving freely in aqueous solution at different ascorbic acid concentrations (A) Buffer solution; (B) 200 l M ascorbic acid; (C) 5 m M ascorbic acid.

Fig 2 Fluorescence microscopic images of T4 DNA fixed on a glass surface in the presence of 5 m M ascorbic acid.

on the fixed DNA may also exist on the segregated DNA in

bulk solution

Figure 3A,B shows the detailed morphological features

of DNA molecules observed by AFM in the presence of

3 mM ascorbic acid The intrachain segregated state is

observed with much higher resolution than those observed

by fluorescence microscopy It is clear that the mini-globule

part assumes a loosely packed conformation This AFM picture corresponds well to the segregated structure observed by fluorescence microscopy with a lower resolu-tion (Figs 1 and 2) Figure 3B shows a magnified view of a condensed part from Fig 3A Interestingly, the condensed part shows the irregular packing of DNA segments Figure 3C shows the transmission electron microscopy

0.2 µm

0.1 µm

C

Fig 3 AFM and TEM images of T4 DNA molecules in the presence of ascorbic acid (A) and (B) AFM images of T4 DNA molecules in the presence

of 3 m ascorbic acid, where (B) is a magnified view of (A) (C) TEM image of T4 DNA in the presence of 5 m ascorbic acid.

(TEM) image of T4 DNA in the presence of 5 mMascorbic

acid, and indicates that a loosely packed structure is formed

The morphological features of the condensate observed by

TEM are similar to those observed by AFM, although with

TEM it was difficult to judge whether an elongated coil

region existed in the DNA chain

It has been well established that single DNA molecules

are packed into a compact toroidal structure with a

diameter of 50–100 nm on the addition of condensing

agents such as polyamines or tervalent metal cations [16,17]

Compared with such a tightly packed state, the folded state

of DNA induced by ascorbic acid is rather swollen

Next, we measured the changes in the CD spectra of

DNA Figure 4 shows that the positive Cotton sign at the

280 nm band almost disappears when the ascorbic acid

concentration is above 100 lM The change in the negative

band at 246 nm to a positive value is mostly attributable to

the contribution from the added ascorbic acid (see

Fig 4B) Figure 4C shows the differential CD spectrum

[(30 lMDNA + 100 lM ascorbic acid)) (100 lM

ascor-bic acid)] A decrease in the plus Cotton band is observed

at 280 nm, whereas the minus band at 246 nm remains

essentially constant Interestingly, the band shape in the

difference spectrum resembles that of the C-form of DNA

[18,19]

Discussion

Ascorbic acid induces substantial changes both in the higher-order and second-order structures of DNA

It is clear that ascorbic acid induces a significant change in the higher-order structure of DNA We confirmed the generation of a segregated structure with multiple mini-globules using different experimental tools: fluorescence microscopy and AFM The mini-globule shows irregular packing and is very different from previously observed regular conformations, such as toroid and rod [14,15] A similar pearling structure was generated from a long DNA molecule complexed with histone H1 [20] However, the action of ascorbic acid on the pearling structure is thought

to be very different from that of histone H1, as the former is anionic and the latter is cationic As ascorbic acid is negatively charged, it cannot neutralize the charges on the negative phosphate groups of DNA Instead, it may interact with the bases inside the double-stranded structure Such an interaction may cause distortion in the double-stranded structure Neault et al [12] performed a Raman and infrared spectroscopic study on the effect of ascorbic acid

on DNA They suggested that the OHand C-O groups of ascorbic acid interact directly with DNA bases

We shall now discuss the mechanism of the large conformational change in DNA induced by ascorbic acid,

in relation to the change in second-order structure Figure 3C shows the presence of a gnarled conformation

in the condensed part of the chain This result suggests that torsional stress is generated along the double-stranded DNA It is known that the C-form-like structure of DNA

is found on nucleosome particles [18,21–23], where DNA is wound around the histone core proteins with high curvature; the radius is
5 nm The similarity of the CD spectrum to the C-form in Fig 4C may be explained by the formation of such an over-wound double-stranded con-formation, as in a nucleosome To interpret the CD spectra, we need to consider the possible effect of aggregates [24] It is known that aggregates induce distortion of the absorption spectrum, as well as the CD spectrum, through scattering of light We have confirmed that, when the concentration of ascorbic acid is increased, the UV spectra remain on the null level for the region above 310 nm where both DNA and ascorbic acid exhibit

no light absorption This indicates that there will be negligible contribution from aggregates on the CD band The above consideration suggests that the unique features observed by AFM and TEM are closely related to the change in the secondary structure of DNA

Theoretical consideration of the stability

of the segregated structure

We now consider the stability of the intrachain segregated structure induced by ascorbic acid In general, the free energy of a condensed object is the result of two different contributions: bulk and surface energies For the conden-sation of DNA induced by ascorbic acid, we have to take into account the effect of the surviving negative charge, because negatively charged ascorbate cannot neutralize the negative charge of DNA

0 2

220 240 260 280 300 320

220 240 260 280 300 320

0

-4

2

-2

4

6

-2

-4

220 240 260 280 300 320

0

4

8

Wave length[nm]

Wave length[nm]

Wave length[nm]

A

0 µM

20

40

60

100

150

Control

Difference

Fig 4 CD spectra (A) 30 l M T4 DNA in the presence of different

concentrations of ascorbic acid (B) 100 l M ascorbic acid (C) Solid

line, difference spectrum [(30 l M T4 DNA + 100 l M ascorbic acid)

solution ) (100 l M ascorbic acid) solution] Broken line, 30 l M T4

DNA in the absence of ascorbic acid.

It is well established that, when DNA is folded into a

tightly packed structure accompanied by parallel ordering

of the segments, the negative charge on the DNA molecule

almost completely disappears This is similar to the

com-paction by spermidine(3+) at low salt concentrations

[25] On the other hand, when DNA is compacted with

spermidine(3+) at high salt concentration, its volume

becomes one order larger than that of the tightly packed

and ordered state, suggesting the survival of negative charge

in the volume part of compact DNA [26] Thus, the

remaining negative charge would make a significant

contri-bution to the stability of the compact state when the packing

is less dense, as in the case of DNA complexed with ascorbic

acid

In the situation of less dense compaction, the free energy

of a single giant DNA in the compact globular state with

respect to the elongated state is given as

where N is the number of Kuhn segments on the DNA It is

well established that a single Kuhn segment in

double-stranded DNA is composed of 300 base pairs [14,27,28] In

eqn (1), the first and second terms correspond to the volume

and surface energy of a globular compact state, respectively

The third term represents the instability due to the

remaining charge in the globule Q and R are the remaining

electronic charge and radius of the condensed particle The

constants a, b, and c depend on the manner of compaction

or condensation, and are all positive It is reasonable to

expect that QN and RN1/3 In such a framework, the

following relationship is deduced [14,27,28], where c1is a

constant

F¼ aN þ bN2=3þ c1N5=3 ð2Þ

Equation (2) implies that a condensate with a surviving

electronic charge in the bulk should be destabilized above a

critical number Ncof segments When N is larger than Nc,

the single-globule conformation is destabilized and, as a

result, multiple mini-globules are formed Thus, the fully

compact state becomes unstable when the residual charge

becomes large, which may correspond to the present case

It has been found that polycations, such as histone H1

[20] and aminated poly(ethylene glycol) [29], induce a similar

intrachain segregated structure in giant DNA molecules

The stability of a segregated structure induced by

poly-cations has been interpreted in terms of the incomplete

charge neutralization [28,29] Although the mechanism of

the compaction induced by ascorbic acid is quite different

from that caused by polycations, the contribution of the

surviving negative charge to the stability of the segregated

state will be the same

It has been reported that giant DNA molecules are folded

into a compact state by the addition of a negatively charged

polymer, polyglutamic acid [30] In this case, DNA

com-paction is induced by the Ôcrowding effectÕ, similar to the

mechanism of compaction induced by neutral hydrophilic

polymers such as poly(ethylene glycol) [15] The

concentra-tion of polyglutamic acid required in monomer units to

induce compaction is very high, of the order of 1M Thus,

ascorbic acid induces compaction of DNA in a very

different way from the negatively charged polymer

Biological significance of the action of ascorbic acid

on DNA Ascorbic acid is present in human blood at a concentration

of
50 lM[31,32] Moreover, the concentration of ascorbic acid in human cells and tissues can exceed that in the blood

by one order of magnitude [31–33] In particular, human circulating immune cells, such as neutrophils, monocytes and lymphocytes, accumulate ascorbic acid in millimolar concentrations [32,33] Therefore, the ascorbic acid concen-tration that induced the large conformational change in DNA in this study may be of physiological significance

It is thought that the folded compact state of DNA is resistant to external stimuli, such as reactive oxygen species and restriction enzymes For example, it has been reported that the tight and ordered DNA packing in the bacterium Deinococcus radiodurans promotes resistance to environ-mental stress [34] It has also been shown that compacted DNA is highly resistant to the action of a restriction enzyme [35] It is possible that ascorbic acid may reduce oxidative damage by changing the higher-order structure of DNA To our knowledge, this possible biological effect of ascorbic acid has not previously been considered

In this study, it has become clear that ascorbic acid has a dramatic effect on the conformation of giant DNA mole-cules At present, the physicochemical mechanism of the conformational transition of DNA remains an open ques-tion Presumably, direct interaction of ascorbic acid with DNA bases and distortion of the double-stranded structure may explain the large change in the higher order structure It may be of importance to clarify the biological significance of the action of ascorbic acid, in relation to its effects on both the higher-order and second-order structures of DNA

Acknowledgements

This work was supported in part by a Grant-in-Aid from the Ministry

of Education, Science, Sports, and Culture of Japan.

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