AP sites and AP endonucleases

BIOCH1MIE, 1982, 64, 577-580.

CNRS symposium, May 1982 - TOULOUSE Inducible responses to DNA damages

AP sites and AP endonucleases. Lawrence GROSSMAN and Robert GRAFSTROM (*).

Department of Biochemistry, The Johns Hopkins University, School of Hygiene and Public Health, 615 North Wolfe Street, Baltimore. Maryland 21205, USA.

Rdsumd.

Summary.

Nous ddcrivons la sensibilitd des sites apuriniques/apyrimidiniques (AP) internes et terminaux d l'hydrolyse alcaline et ft la bdta-~limination. Nous comparons les modes symdtriques et asym~triques de l'hydrolyse endonucl~olytique par des endonucldases A P sp6ciJiques et nous discutons de la discrimination entre leurs mdcanismes non-enzymatique et catalytique.

The sensitivity of internal and terminal apurinic/ apyrimidinic (AP) sites to alkaline hydrolysis and p-elimination is described. The symmetric and asymmetric modes oJ endonucleolytic hydrolysis by specific A P endonucleases are compared and the discrimination between their non-enzymatic and catalytic mechanism is discussed.

Mots-cl~s : sites apuriniques / sites apyrimidiniques / incision / ~-~limination / hydrolyse alcaline.

Key-words: apurinic / apyrimidinie / incision / R-elimination / alkaline hydrolysis.

Since AP endonucleases act specifically at depurinated and/or depyrimidinated sites it is valuable to examine the nature of the strbstrate for such enzymes. Obviously not all AP sites are the same. From a casual perception these sites with potential aldehydes at their C-1 positions appear to be rather uncomplicated structures. In reality though an apurinic site is distinct from an apyrimidinic site because of the nature of the complementary nucleotide which retains template information after excision of the AP nucleotides. Hence the specificity of AP endonucleases may be influenced by the nature of the complementary nucleotide. For instance, the 3' apyrimidinic endonuclease associated with the Micrococcus luteus dimer DNA glycosylase [1, 2], prefers apurinic sites over apyrimidinic sites. In addition to interstrand specificity AP endonucleases may also be able to discriminate

AP sites according to the nature of its flanking sequences. Do some AP endonucleases exhibit Km differences for AP sites if there are specific purines, pyrimidines or combinations 3' or 5' to such st~bstrates ? The 3' aprymidinic endonuclease of M. luteus associated with the dimer DNA glycosylase, for example, shows a marked specificity for a 5' apyrimidinic site which is adjacent to a pyrimidine : pyrimidine nucleotide cyclobutane dimer [3] over any other type of apurinic or apyrimidinicite. Removal of the free pyrimidine moiety of the dimer by yeast photolyase leads to a marked reduction in the initial velocity of the incision reaction.

* Present address : Cancer Biology Program, NC1 Frederick Cancer Research Facility, Frederick, Maryland 21701. To whom all correspondence should be addressed.

Due to the availability of enzymatically synthesized polydeoxynucleotides that contain uracil either in place of thymine or through the deamination of cytosine, it is feasible to prepare a variety of substrates with potentially interesting sequences as neighbors to AP sites throt~gh the action of uracil DNA glycosylases [4, 5]. Furthermore, the chemical reduction of AP sites with NaBH4, [6, 7] permits a comparison of the substrate specificity of a variety of bacterial and mammalian AP endormcleases as well as elucidation of possible reaction mechanisms.

578

L. Grossman and R. GraJstrom. well as D-elimination since the C-4, hydroxyl is cis to the C-5 phosphodiester bond with potential for forming a C5, C4 inner cyclic phosphoanhydride. In this pathway C5 or C4 phosphorylated sugar derivatives are liberated generating, a 3' hydroxyl terminatect DNA fragment. Given this range of chemical reactivities the sensitivity of AP termini to certai~ chemical conditions is predictable. A 5' terminal AP site is specifically sensitive to D-elimination whereas a 3' ter-

Chemical sensitivity of AP sites (*). One of the interesting aspects of the chemistry of AP sites is the variety of chemical reactions which they can undergo. Not only are these sites subject to potential D-elimination reactions by a variety of cellular nucleophiles such as thiols and by primary and secondary amines [8], but they are also amenable to alkaline hydrolysis because of a cis-glycol at the positions C4 and C5. The ~-elimination reaction occurs when there is a good leaving

_~-Elimination at Internal AP Sites

. o.

,

/M,...,,~H

R'O ..0 . /

R. n

H"l

~

~P'~_ ~ H.J O o o . . . . ~_.~-~"'~o-R k,n2 n I~ n I ""R O_

--

",#

~

~

-"IP"o O -

H'~.'B"

~'l

-,

_ ~

"CH2..~ HO~I.. ^ iOI ":,:I>" u-r?., I 0 H

0

o

l~"O./t

r'O._..

O_

H.[,O, 0

GH2~H,O,,.~-6 II "i" - P.'o u

"R

~

n

O_

"~R

I I~ Phosphote Elimination

H O

, R'o..O

.

" 0 ' ' 0..

"5 , - -

0,,

oo'11 O_

HaC

H OH

2 ~ Phosphate Elimination

Fie.

1. - -

~-elimination reactions at internal A P sites.

group D to an unsaturated C atom and in this case involves C3 phosphate-elimination generatillg a 3'-terminal AP site and a 5'-phosphorylated terminus (figure 1). 5-phosphate elimination occurs in the presence of excess D-eliminating agent generating a C3-phosphorylatect terminus and an unsaturated sugar derivative. This sequence of reactions has been exploited for the strancL breakage in the chemical DNA sequencing techniques developed by Maxam and Gilbert [9]. When the initial D-elimination is catalyzed by alkali, the 3' terminal AP site is also sensitive to alkaline hydrolysis as (~) The numbering system for nucleotides requires a (') prime to distinguish between sites on sugars from the numbering o/ purines and pyrimidines. Since an A P site lacks such bases the sites on the sugar are unprimed except when directionality is emphasized. BIOCHIMIE,

1982,

64, n ° 8 - 9 .

Hf:B-

minal AP site (with a 3' OH group) is refractory to ~-elimination, but is sensitive to alkaline hydrolysis. ~-elimination appears to be the preferred mechanism under mild alkaline conditions (pH 11.7, 37°C, 1.5 hr (1,8). Since reduction of the potential aldehyde with NaBH4 interferes with ~-elimination [10], then, this resistance can be used to discriminate between reactions which follow a ~-elimination mechanism from those which are sensitive to alkaline hydrolysis. Nature oJ the enzymes which hydrolize A P sites. There are two types of AP endonucleases : the complex bacterial enzymes in which AP endonucleolytic activity is only one of many enzyme ftmctions and the simple AP endonucleases whose only known catalytic function is the endonucleolytic scission at AP sites. E. call and H. influenzae exo-

DNA

nuclease III [11-14] are single polypeptides of molecular weight in the range of 25,000 and 30,000 respectively which possess four distinct catalytic activities: (i) a 3' to 5 ~ exonucleolytic activity prefering duplex DNA (ii) an AP endonu~ cleolytic activity, (iii) a DNA 3'-phosphomonesterase activity and (iv) an RNase H activity. The associated AP endormcleolytic activity hydrolyzes 5' to the AP site generating a C3 hydroxyl group and a C5 phosphomonoester linked to the terminal AP site. Since exonuclease III hydrolyzes on the 5' side of the AP site this enzyme has been further categorized as a class II AP endonuclease [15]. N

N

N

T~T

N

N

579

repair e n z y m e mechanisms.

pyrimidine: pyrimidine nucleotide cyclobutane dimer [3] (fig. 2). AP endonucleolytic activity appears at this juncture of our knowledge to be the sole catalytic capability of the simple AP endonueleases. These NI

3'-Incision ~

NI

NZH 0

Na N4

N~

N2

Hv
,

°

H 0 N3 N4

H~
Nn

N4

o

:oroc.,

o'

o_

DNA-glycosylase ,,x N O"c'H l~]), N

N = N2

*elim{nation

Pyrimidine dimer

N

5'-Incision

N

5'-~P-~P-~P.~P~P.~P.~P..~ 3' N

N2H 0 N3 N4

NI

N

Nz

H.,,~.O H

Hv'O

H

N3 N4

N

FIG. 3. - - Chemistry o] terminal AP site release. AP Endonuclease

N

N

N O"c'H ~,,x. N

N

N

H FIG. 2. - - Mechanism oJ the M. luteus pyrimidine dimer DNA glycosylase : 3" AP endonuclease.

The M . luteus and bacteriophage T4-V gene encoded enzymes [2, 16] which incise pyrimidine dimer containing DNAs, are small single polypeptides (Mr 18,000) which possess two distinct catalytic activities: (i) a h emi-DNA glycosylase specific for the 5' pyrimidine N-glycosyl bond of a cyclobutane dimer and (ii) an apyrimidinic endonucleolytic activity. The AP endonuclease cleaves 3' to the AP site ger~erating a 5' PO4 termini and is thus a Class I endonuclease. This AP endonuclease shows a preference for those apyrimidinic sites which are adjacent to a pyrimidine : pyrimidine nucleotide cyclobutane dimer [2, 3]. Incision of the apyrimidinic site as a consequence of enzymarie hydrolysis generates a 3' terminal AP site and a 5' phosphoryl group associated with the BIOCH1MIE, 1982, 64, n ° 8-9.

enzymes are ubiquitous having been purified from bacterial [17, 18] and mammalian tissues [19] in which their catalytic specificities are seemingly limited to whether they hydrolyse 3' or 5' to an AP site. In analyzing the specificity of the simple AP endonuclease purified to homogeneity from human placenta [5, 7] it was observed that hydrolysis 5' to an AP site did not proceed to completion, but instead reached a plateau at 60 per cent of stoichiometry. This finding prompted an analysis of 3'-AP site hydrolysis by this homogeneous enzyme. The results were surprising in that this AP e n d o n ~ clease also hydrolyzed 3' to AP sites to the extent of 40 per cent of stoichiometry. If these AP sites were reduced with NaBH4 the same symmetry in the distribution of hydrolysis was observed clearly ruling out ~-elimination as the mechanism for 3'-AP site hydrolysis. In reevaluating the results of similar experiments with other AP endonucleases many of the hydrolytic reactions with specifically labelled AP polynucleotides were only investigated with reference to one site of cutting and do not appear to reach completion. It is, therefore, not possible to unequivocally assign a classification to AP endonucleases based orL the sidedness of

580

L. G r o s s m a n and R . Grafstrom.

cleavage in the absence of such necessary information. Because the htmaan placental A P endonuclease hydrolyzed A P sites in almost a symmetric m a n n e r it was of apparent interest to determine whether the terminal A P sites which were initially generated were also substrates for the same enzyme. Sugar nucleotide release was only observed f r o m 5'-terminal A P sites and when that same site was reduced to the corresponding alcohol no sugar release could be detected. Since in this case [3-elimination occurs when a phosphodiester group is [3 to a carbonyl oxygen [10] it was suspected that the observed release of the 5' terminal A P site was non-enzymatically stimulated. This proved to be the case since both denatured enzyme and enzyme inhibited by E D T A released 5'-terminal A P sites. However, the rate of 5' terminal A P site release was two orders of magnitude slower than internal A P site hydrolysis by the enzyme. T h e p r o d u c t which was released did not behave chromatographically as deoxyribose-5-phosphate and is thought to be an a, [3 unsaturated deoxyribose-5-PO4 intermediate in the [3-elimination reaction as suggested in figures 1 and 3. I n conclusion the features of the chemi'stry of apurinic and apyrimidinic sites in D N A have been discussed as well as the specificity of the h u m a n A P endonuclease which can act symmetrically as a phosphodiesterase to generate A P terminated D N A fragments. As a result of these observations it is critical when performing terminal A P site release experiments to minimize those conditions favoring [3-elimination reactions. The presence of thiols and those primary and secondary amines in buffers [8, 20] and associated with the protein itself must be carefully considered, therefore, when studying D N A s containing A P sites.

BIOCH1MIE, 1982, 64, n ° 8-9.

Acknowledgements. The research described in this article was supported by grants from the National Institutes of Health (CP-85624 ; GM22846), and the Department of Energy (EY-76-5-0278-14).

REFERENCES. 1. Haseltine, W. A., Gordon, L. K., Lindan, C. P., Grafstrom, R. H., Shaper, N. C. ~ Grossman, L. (1980) Nature, 634-641. 2. Grafstrom, R. H., Shaper, N. L., Grossman, L. & Hasettine, W. A. (1980) in <~Chromosome Damage and D N A Repair ~, eds. Kieppe, K., Seberg, E. Plenu~m Press, in press. 3. Grafstrom, R. H., Park, L. & Grossman, L. (1982) J. Biol. Chem., st~bmitted. 4. Clements, J. E., Rogers, S. G. & Weiss, B. (1978) J. Biol. Chem., 253, 2990-2999. 5. Grafstrom, R. H., Shaper, N. L. ~ Grossman, L. (1982) J. Biol. Chem., submitted. 6. Hadi, S. & Goldthwait, D. A. (1971) Biochemistry, 26, 4986-4994. 7. Shaper, N. L., Grafstrom, R. H. & Grossman, L. (1982) J. Biol. Chem., submitted. 8. Lindahl, T. & Anderson, A. (1972) Biochemistry, 11, 3618-3622. 9. Maxam, A. & Gil~bert, W. (1977) Proc. Natl. Acad. Sci. USA, 74, 560-564. 10. Jones, A. S., Mian, A. M. & Walter, R. T. (1981) J. Chem. Soc. (c), 1968, 2042-2044. 11. Gossard, F. & Verly, W. G. (1978) Eur. J. Biochem., 82, 321-322. 12. Verly, W. G. (1978) in <> (Hanawalt, P. C., Friedbexg, E. C. 8, Fox, C. F. eds.), pp. 187-190 Academic Press, New York, N.Y. 13. Mil¢arek, C. ~ Weiss, B. (1972) J. Mol. Biol., 14, 1-7. 14. Weiss, B. (1981) in ¢ The Enzymes~ (Boyer P. D. ed.), vol. 14, pp. 251-279, Academic Press, N.Y. 15. Mosbaugh, D. W. & Lima, S. (1981) J. Biol. Chem., 255, 11743-11752. 16. Radany, E. H. & Friedberg, E. C. (1980) Nature, (London), 286, 182-185. 17. Pierre, J. ~ Laval, J. (1~80) Biochemistry, 19, 50185024. 18. Pierre, J. & Laval, J. (1980) Biochemistry, 19, 50245029. 19. Lindahl, T. (1979) Prog. Nuc. Acid Res. Mol. Biol., 22, 135-192. 20. Behmoares, T., Toulm6, J. J. & H61~ne, C. (1981) Proc. Natl. Acad. Sci., USA, 78, 926-930.