The accuracy of DNA replication

BIOCHIMIE, 1978, 60, 1083-1095.

The accuracy of DNA replication. Fran~oise BERNARDI (*) and Jacques NI.NIO.

ERROR RATES. A c c o r d i n g to Drake's rule of t h u m b [1], spontaneous m u t a t i o n s occur at a rate of about 10 -a per genome per generation i n procaryotes. Thus, w h e n the E. coU c h r o m o s o m e - c o n t a i n i n g 5 X 106 base-pairs - is replicat.ed, the f r e q u e n c y of errors per base-pair, is a r o u n d 10-3/(5 × 106) = 2 X 10 -10. T a k i n g a cistron of 300 base-pairs, the mutation rate for this locus w o u l d be 500 × (2 X 10 -10) = 10 ~7. Indeed, spontaneous m u t a t i o n s i n m i c r o b e s generally occur at frequencies of 10 -7 to 10 -6 per gene per g e n e r a t i o n [2]. Similarly, Drake gives a total m u t a t i o n rate of 1 to 3 × 10 -3 for the phages )~ a n d T4. The c o r r e s p o n d i n g error-rate per base-pair r e p l i c a t i o n is then about 2 × 10 -s. However, the e r r o r - r a t e in the r e p l i c a t i o n of RNA phages a n d viruses m a y be m u c h higher. Direct s e q u e n c i n g studies on phage ( ~ RNA show [3] that the RNA p o p u l a t i o n s are extremely heterogeneous. The Q~ genome contains about 4,500 base pairs. The m u t a t i o n frequency, d e t e r m i n e d at one specific nucleotide position was estimated to be about 10 -4 [4]. Let us, for the sake of the argument, apply the same figure of 10 -3 m u t a t i o n s per genome replication to a h i g h e r eucaryote, drosophila. Since, on the average, 40 cell generations separate the fertilized egg from the p r o g e n y gametes, this w0u)d give 4 × 10 -2 m u t a t i o n s per sexual generation. The actual figure, given by Drake [1] is 20 times higher : 0.8 m u t a t i o n / g e n e r a t i o n . Since the haploid genome of drosophila c o n t a i n s about 2 × 10 s base pairs, the c o r r e s p o n d i n g e r r o r rate per base-pair r e p l i c a t i o n is about 0.8/(40 × 4 × 10 s) = 5 × 10'-lz. Thus, DNA r e p l i c a t i o n w o u l d be about 10 times more accurate in d r o s o p h i l a t h a n i n E. colt. However, the m u t a t i o n rate per gene and per sexual g e n e r a t i o n is higher in drosophila. According to the above figures, it should be about

(% Adresse prdsente : Laboratoire d'Enzgmologie du CNRS, 91190 Gif sur Yvette. Lecture delivered at the <>,May 31 June 2, 1978 on ¢ Mechanisms of DNA repair , .

I n s t i t u t de R e c h e r c h e en B i o l o g i e Mol~culaire, 2 P l a c e J u s s i e u , 75221 P a r i s C e d e x 05.

500 X 40 X 5 × 1~)- n = 1'{)-e. The s t u d y of natural p o p u l a t i o n s both i n d r o s o p h i l a a n d m a n give m u t a t i o n f r e q u e n c i e s per gene a n d per sexual g e n e r a t i o n of about 10 -5 ; but since the studies are c a r r i e d on the <>. A c c o r d i n g to the n e u t r a l i s t school in p o p u l a t i o n genetics, most m u t a t i o n s at the gene level should lead to proteins w h i c h do not differ d r a m m a t i c a l l y from w i l d t y p e p r o t e i n s [12]. Stu-

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dies of h e m o g l o b i n mRNA sequences i n r a b b i t a n d m a n show that about 2/3 or 3/4 of the gene m u t a t i o n s that were r e t a i n e d i n evolution were silent -they did not change the n a t u r e of the coded a m i n o acid [13]. This implies that n o n - s i l e n t gene m u t a t i o n s are in general counter-selected. F i n a l l y , m u t a b i l i t y m a y vary w i t h i n a n y species. Mutation rates are u n d e r genetic control. Mutator a n d a n t i m u t a t o r v a r i a n t s will be discussed in detail later.

lity for the cell w o u l d be to use polymerases with high~ly a s y m m e t r i c a l patterns of mistakes. F o r instance, if G.C. ---> G.T is m u c h more p r o b a b l e t h a n A.T ~ G.T, then a G.T m i s m a t c h should be corrected as G.C. Aetua.l'ly, t~e real t r i c k seems to be different. W a g n e r a n d Meselson [15] suggest that since the n e w l y synthesized c h a i n of DNA has not h a d the time to become fully inethylated, it w o u l d be d i s t i n g u i s h e d from the other c h a i n on the basis of its u n d e r m e t h y l a t i o n .

Error-rates m e a s u r e d in vitro for the r e p l i c a t i o n of n u c l e i c acids w i t h purified DNA or RNA polymerases are extremely v a r i a b l e (table IlL U s i n g a <> DNA (from phage ¢X 174, h a v i n g an a m b e r m u t a t i o n ) , W e y m o u t h a n d Loeb [14] did not detect a n y r e v e r t a n t at the a m b e r site after in vitro r e p l i c a t i o n w i t h E. colt DNA polymerase 1, the sensitivity of the method allowing the detection of about one e r r o r in 10,0,00. W h e n substituting m a g n e s i u m w i t h manganese, the level of errors became m e a s u r a b l e [14]. T a k i n g a value of 10-5 to 10 -6 for the in vitro error-rate w i t h DNA polymerases, we w o u l d have a gap of 10~ b e t w e e n in vitro a n d in vivo error rates in DNA replication. How is the h i g h e r a c c u r a c y level achieved in vivo ? T h e r e are two possibilities. A first one is to invoke the activity of r e p a i r enzymes. The cell possesses a w h o l e set of r e p a i r enzymes that r e s p o n d to various k i n d s of dammages ( p y r i m i d i n e dimers due to U-V lesions, accid,ental d e p u r i n a t i o n , m i s i n c o r p o r a t i o n of uracil, etc.). E n z y m e systems s p e c i a l i z e d i n the detection a n d r e p a i r of i n t e r n a l mismatches betw e e n s t a n d a r d bases (for instance, G. T pairs) have been studied [15, 161 but not to the p o i n t of detailed b i o c h e m i c a l characterization. Here comes a mystery. If a n i n t e r n a l G.T. p a i r is to be repaired, h o w is the enzyme system able to d e t e r m i n e w h e t h e r it must be corrected into G.C or into A.T? One proposal, i n s p i r e d by yon N e n m a n n ' s w o r k on automata, b u t not m u c h rooted on b i o c h e m i c a l evidence, states that at least three p a r t i a l copies are made of a n y stretch of DNA. An enzyme w o u l d compare the three copies and homogenize the output a c c o r d i n g to the m a j o r i t y rule [17]. Actually, two copies are sufficient in the case of DNA replication. If the two daughter molecules c o n t a i n at a given position G.T a n d G.C, then there is all l i k e l i h o o d that G.C is the correct antecessor of G.T. Geneticists have been c o n v i n c e d for a long time that r e c o m b i n a t i o n - - a process w h i c h requires the m a t c h i n g of homologous DNA stretches - - is s o m e w h a t l i n k e d to r e p a i r [18-20]. However, the k i n d of r e p a i r system w h i c h is involved i n gene c o n v e r s i o n does not seem to w o r k as a very efficient homogenizer. A n o t h e r possibi-

Still, it is not c e r t a i n that r e p a i r enzymes are sufficient to a c c o u n t for the r e d u c t i o n i n errorrate from in vitro to in vivo replication. It m a y well be that DNA polymerases are i n t r i n s i c a l l y more accurate t h a n appears from in vitro experiments, for reasons that wil,1 be clarified later. We m a y just note at the m o m e n t that biochemists accept n o w the view that r e p l i c a t i o n is p e r f o r m e d in vivo by a h i g h l y .complex m u l t i - e n z y m e system [21]. Yet, a c c u r a c y is generally studied w i t h pure or s u p p o s e d l y pure DNA polymerases. The accur a c y of r e p l i c a t i o n may d e p e n d u p o n the interaction of the polymerase w i t h other proteins. Mutations i n the gene 32 of phage T4, w h i c h codes for a DNA b i n d i n g p r o t e i n do influen,ce the rates of s p o n t a n e o u s or i n d u c e d m u t a t i o n s [22, 23]. The p r o d u c t of gene 32 is though to w o r k in c o n j u n c t i o n w i t h the DNA polymerase. I n its presence, DNA r e p l i c a t i o n is both more efficient [24, 25] a n d more accurate [26].

BIOCHIMIE, 1978, 60, n* 10.

ERROR PATTERNS. What are the m a j o r mistakes that are made dur i n g DNA r e p l i c a t i o n ? Since each of the four bases A, T, G, C can be replaced by mistake b y a n y of the three others, there are twelve possib~le types of substitutions. Ideally, we w o u l d like to give estimates of the relative frequencies of each of these, a n d make some c o m m e n t s on the p a t t e r n s of errors. There is an e n o r m o u s a m o u n t of k n o w ledge w h i c h may, in p r i n c i p l e , be exploited to give i n d i c a t i o n s or clues as to the frequencies of the various mistakes. Genetic studies on the n a t u r e of s p o n t a n e o u s m u t a t i o n s (mostly in procaryotes) generally give i n c o m p l e t e i n d i c a t i o n s . By s t u d y i n g h o w easily various mutagens reverse the spontaneous mutation, one infers the target base-pair (A.T or G.C) a n d the s u b s t i t u a n t one. By this c r i t e r i u m the various mistakes are grouped two by two into six classes (A.T ~ T.A, A.T ---> G.C, etc). T y p i c a l frequencies observed in E. colt [27] are s h o w n in table I. Any of the base-pair substitutions may

Accuracy of DNA replication. a priori occur via one of two pathways. F o r instance, the t r a n s v e r s i o n A.T --> T.A m a y be due to a T.T or an A.A m i s p a i r : J A.A "--> T . A A . T .., T.T --> T.A

1085

t h a n p u r i n e - p y r i m i d i n e or p y r i m i d i n e - p u r i n e substitutions. It seems that no single generalization w i l l hold for all polymerases simultaneously. Three or four generations of evolutionnists have c o n s i d e r e d that the development of clever devices

T A B ~ I.

Some mutation frequencies, in the TrpA gene of E. colt [[rom Fowler, Degnen and Cox, re[. 27]. TrpA allele A A A A A

58 3 78 88 446

A 11 A 23

B:~se-pair substitution A.T A.T A.T G.C

--~ ---> --> id. ~

G.C T.A C.G T.A

G.C ---> C.G G.C ---> T.A

There are i n p r i n c i p l e , methods for d e t e r m i n i n g the p a t h w a y of given substitutions [reviewed i n 28] b u t these have not been a p p l i e d systematically. Most reviews or textbooks on mutagenesis say little on the p a t t e r n s of s p o n t a n e o u s m u t a t i o n s [5, 28-30] a n d ~ve shall not, i n this domain, propose generalizations of our own. As was p o i n t e d out b y Orgel, if there is a n y good physico-chemica'l reason w h y a c e r t a i n m u t a t i o n o r lesion should be p a r t i c u l a r l y frequent, t h e n it is a good bet to p r e d i c t that the cell has developped a counter-measure to keep this class of m u t a t i o n s or lesions down. This a r g u m e n t gives little hope of d e r i v i n g the p a t t e r n s of mutations from any straight p h y s i c o - c h e m i c a l p r i n c i p l e . One w o u l d r a t h e r predict, i n d e p e n d e n t l y of a n y detailed consideration, that there should be a t r e n d t o w a r d s the equalization of the various e r r o r - f r e q u e n c i e s [31]. The ,large r e p e r t o i r e of d e t e r m i n e d p r o t e i n sequences a n d c o m i n g RNA and DNA sequences m a y be exploited, i n comparative studies, to det e r m i n e ¢ m u t a t i o n a l t r e n d s >> [32-~5~. W h e t h e r the observed l i m i t e d G --> A t r e n d reflects mutational pressure, or is due to n a t u r a l selection acting at the n u c l e i c acid or the p r o t e i n level, r e m a i n s unclear. A c o m p i l a t i o n of the well-characterized m i s i n c o r p o r a t i o n s that have been observed in vitro is s h o w n i n table II. Base substitutions l e a d i n g to t r a n s i t i o n s are often b u t not always more frequent

BIOCHIMIE, 1 9 7 8 ,

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lteversion trequency in wildtype 4.3 3.9 2.0 6.1 3.1

X X X X X

1 0 -~° 1 0 -9 1 0 -:* 1 0 -j° 1 0 "I°

8.6 X 10-4° 3.1 X 10 -9

ReversionIrequency ia mutator mut D5 1.4 2.3 3.I 4.4 1.9

X X X X X

1 0 -~ 1 0 -6 1 0 -: 1 0 -8 1 0 -:~

2.9 X 10-6 1.9

X

1 0 -6

that p e r m i t t e d the genetic m a t e r i a l to mutate was one of the most b r i l l i a n t i n v e n t i o n s of evolution. I n line w i t h this c o n c e p t i o n - - but not necessarily l i n k e d to it - - m a n y attempts were made to e x p l a i n how m u t a t i o n s could, after all, occur. Most theories [reviewed i n 5] i n v o k e d some specific d e v i a t i o n of a nucleotide from its n a t u r a l state : t a u t o m e r i c shifts, ionization, flipping of bases a r o u n d the base to sugar bond. The idea that a s u b s t a n t i a l p r o p o r t i o n of the m i s i n c o r p o r a t i o n s m a y occur via m i s p a i r s b e t w e e n nucleotides in their most n a t u r a l states does not seem to have received m u c h attention. Yet, c o n s i d e r i n g some extreme cases of m i s i n c o r p o r a t i o n s , one m a y doubt that a b n o r m a l states need to be involved. The repeating p o l y m e r Poly(dC-dC-dT) n c a n be t r a n s c r i b e d e f f c i e n t l y as Poly (rG) b y E. colt RNA p~lymerase [36]. The structure of DNA c a n n o t b y itself e x p l a i n error-levels as tow as 10~8 or 10-lo. Attempts to replicate n u c l e i c acids t h r o u g h n o n - e n z y m a t i c catalysis lead to estimates of the <> level of errors, at best i n the 10 -1 to 10-2 range [37, ~ ] . A parallel can be d r a w n here b e t w e e n the concepts that p r e v a i l for r e p l i c a t i o n a n d for translation. At the time the w o b b l e hypothesis was proposed [38], most w o r k e r s in the field of the genetic code c o n s i d e r e d that a special hypothesis was needed in order to explain h o w G.U. pairs could occur i n the t h i r d position of the codon-anticodon association. Special configurations [38, 39],

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F. Bernardi

a n d J. N i n i o .

TABLE II. Misincorporations Template Poly(dC). Oligo(dG)

Poly(dC) . Poly(dC0

Poly(rC) . Oligo(dG) Poly(dI-dC)

Poly(dI) . Poly(dC) Poly(rC) . Poly(rI) P oly(dA-dT)

observed with some DNA and RNA

Polymerase T4, wildtype T4, m u t a t o r L56 T4, wildtype T4, m u t a t o r L56 Spleen necrosis v i r u s DNA polymerase

P o l y ( r A ) . Oligo(dT)

T

G

Yeast, m i t o c h o n d r i a l DNA p o l y m e r a s e Calf t h y m u s DNA polymerase ~

G

AM,V DNA p o l y m e r a s e E. coli DNA p o l y m e r a s e 1 E. eoli RN,A polymerasc AM'V DNA polymerase SNV DNA p o l y m e r a s e AMV DNA p o l y m e r a s e H u m a n DN~A p o l y m e r a s e c~ f r a c t i o n I f r a c t i o n II H u m a n DI~A p o l y m e r a s e u, f r a c t i o n I A ~ V DNA p o l y m e r a s e AM~V DNA p o l y m e r a s e

H u m a n I)N,A p o l y m e r a s e c~ f r a c t i o n I H u m a n DN~A p o l y m e r a s e a, f r a c t i o n II H u m a n c~ f r a c t i o n I fraction H SNV DNA p o l y m e r a s e AMV DN~A p o l y m e r a s e

Poly(dA) . Oligo(dT)

G

G

Bacillus S t e a r o t h e r m o p h i l u s DNA p o l y m e r a s e

P o l y ( r A ) . Poly(dT)

Incorrect nucleotide

E. coli DNA polymerase 1

E. coli RNA ~polymerase Bacillus ~ L i c h e n i f o r m i s DNA polymerase

P o l y ( d A ) . Poly(dT)

Correct nueleotide

E. coli RN~A p o l y m e r a s e E. coli DNA p o l y m e r a s e 1 Calf t h y m u s DNA p o l y m e r a s e

G

G G, C G G G G, C

G, C G A, T

T C A T C A T T C A T C A A A T A A T A A T A T

polgmerases. Discrimination Rele(1) Conditions rence 14,800 660 2,9'00 167 0.8 ! 37 37 44 490 240 22,600 8,000 4,0'00 1,40,0 (2) 300,000 2,000 400 67'0 130 940 9,40'0 740 0.006 ! 2,860 986 1,760 160 880

A, T

A C G C G C G C G C G C G

250 2,950 3,100 1,490 180 410 210 420 220 720 240 840 370

A, T

G

145

A, T

G

T

G

T

C rG C G C G

2~30 150 3,390 0.8 ! 750 8,2~)0 ~,000 895 375 925

A, T A, T

A, T

T T, A T

Mg, 30°C

[69]

~ n , 30°C Mn,37°C

[92]

Mn,37°C

[92]

Mg or Mn, [90] 35°C M,g, 35°C [87] Mn, 35°4

Mg, 37°C [77] Mn, 37°C [93] Mg, 37°C Mg, 37°C Mn, 37°C Mg, 37°C Mn, 37°C

[88] [77] [92] [77] [91]

Mn, 37°C [91] Mg, 37°C [77] Mg, 37°C [77] Mg, 37°C Mg, 37°C

[88] [94]

Mg, 55°4 Mg, 37°C

[94]

Mg, 55°C Mg or Mn, [91] 37,°C Mn, 37°C [91] Mn, 37°C [91] Mg, 37°C Mn, 37°C ~/~g, 3r7°C ~Ig, 37~°C Mg, 37°C Mn, 37°C

[92] [77] [89] [88] [93]

Mn, 35°C [87]

(1) D i s c r i m i n a t i o n is defined b y Eq. [9] : correct over incorrect i n c o r p o r a t i o n divided b y correct over incorrect s u b s t r a t e concentrations. (2) A s s u m i n g equal c o n c e n t r a t i o n s of correct a n d incorrect substrates. Most of the articles quoted here c o n t a i n a d d i t i o n a l d a t a o b t a i n e d u n d e r o t h e r conditions (for instan.ce, w i t h different metals). T h i s t a b l e is s i m p l y m e a n t to b e a first guide to a growing b o d y of data. BIOCHIMIE, 1978, 60, n ° 10.

Accuracy of DNA replication. tautomeries [40] or f l i p p i n g a r o u n d of bases [41] were invoked. The idea that the G.U. pair was more <> t h a n p r e v i o u s l y realized, a n d that one needed to explain, not be occurence of G.U. p a i r s at the t h i r d position of the c o d o n - a n t i codon association, but its p r a c t i c a l avoidance in the first two positions [42, 43] was not, u n t i l recently, seriously taken into c o n s i d e r a t i o n .

PROOFREADI,NG. I n the cell, a c c u r a c y is not achieved at once : errors are made, then corrected. Sometimes the s y n t h e t i c a n d the corrective activities are exerted by the same enzyme. F o r instance, the isoleucyltRNA ligase is able to acylate the tRNA TM w i t h valine but then hydrolyzes the m i s t a k e n p r o d u c t quite efficiently [44, 45]. Similarly, the DNA poly. merases from phage [46-48], b a c t e r i a [49-54], some lower eucaryotes [55-57], a n d some DNA polymerases from m a m m a l s [58] are able to excise from the g r o w i n g c h a i n the last nucleotide that h a d been i n c o r p o r a t e d . E x c i s i o n is in general very efficient t o w a r d s a m i s m a t c h e d t e r m i n a l nucleotide, less so w h e n the last n u c l e o t i d e is comp l e m e n t a r y to the opposite base on the template [47, 48]. Thus, E. coli DNA polymerase 1 will effic i e n t l y remove the labelled residue in : .... AAAAAAAAAAAAAAA ..... .... TTTT* C AAAAAAAAAAAAAAA TTTT* T

.....

A n a i v e view of the f u n c t i o n n i n g of such enzymes w o u l d be that the DNA polymerases execute an o r d e r e d p r o g r a m : 1 - - i n c o r p o r a t e a nucleotide 2 - - check w h e t h e r the match w i t h the opposite base is good or not. 3 - - if the m a t c h is bad, excise the nucleotide and go back to the previous position. 4 - - if the m a t c h is good, move f o r w a r d on the template a n d i n c o r p o r a t e the next nucleotide. This is p r o b a b l y not the good w a y to t h i n k about DNA polymerases. The above scheme, w h i c h looks simple on paper, requires extremely complex enzyme kinetics. W e w i l l stick to anot h e r view, w h i c h m a y seem at first sight more complicated, but is a c t u a l l y m u c h simpler in terms of f u n d a m e n t a l enzyme kinetics. It also has the virtue of b e i n g compatible w i t h all the avai-

BIOCHIMIE, 1 9 7 8 ,

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lable evidence. I n this model, first outlined by Goodman et al. [59] a n d s u b s e q u e n t l y analyzed m a t h e m a t i c a l l y by us .(see ref. [60]; w e will p u b l i s h elsewhere the detailed m a t h e m a t i c s of the p o l y m e r i z a t i o n / e x c i s i o n kinetics) a n d b y Branscomb a n d Galas ( p e r s o n n a l c o m m u n i c a t i o n and [61]), the p o l y m e r i z a t i o n a n d the excision activities are s w i t c h e d on p e r m a n e n t l y . W h i l e the polymerase is at position n on the template, there is a k i n d of race b e t w e e n the two activities : - - i n c o r p o r a t i o n of a nucleotide w i t h a subseq u e n t f o r w a r d m o t i o n of the polymerase to position n + 1 (or, if one considers a d i s t r i b u t i v e m e c h a n i s m , the polymerase leaves the template, a n d a n o t h e r enzyme molecule eventually b i n d s to position n + 1) - - excision of the last nucleotide, w i t h a subsequent ~backward motion of the p o l y m e r a s e to position n - - 1 (or, eventually, d e p a r t u r e of the enzyme a n d b i n d i n g of a n o t h e r polymerase to position n - - 1).

The outcome of the race b e t w e e n the two activities is probabilistic. Take the simplest case in w h i c h a h o m o p o l y m e r , Poly (dA) is replicated in the presence of a single c o m p l e m e n t a r y nucleotide, dTTP. T h e r e are two r a t e - c o n s t a n t s : a kinetic c o n s t a n t k 1 for the i n c o r p o r a t i o n of a nucleotide, a n d a c o n s t a n t k=, for the excision of a T residue: k.2 g

n--1

but not the labelled T i n : .... ....

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k 1 [S]

---0

n

~" -0 n+

(,1) 1

where [S] is the c o n c e n t r a t i o n of dTTP. The prob a b i l i t y of going f o r w a r d is : k I [S] P ~ = (2) k t [S] + k~ Similarly, the p r o b a b i l i t y of excision is : ke P <-- = k I [SJ + k~

(3)

The growth of the p o l y m e r i z e d c h a i n has the character of a r a n d o m walk, w i t h a succession of f o r w a r d a n d b a c k w a r d steps, i n a r a n d o m fashion, a c c o r d i n g to their respective probabilities. It is f u n d a m e n t a l to realize that the respective p r o b a b i l i t i e s of going f o r w a r d and b a c k w a r d dep e n d u p o n the c o n c e n t r a t i o n of substrate. W h e n there is no d T T P in the r e a c t i o n mixture, [$1 = 0, the p r o b a b i l i t y of going f o r w a r d is nil, a n d the p o l y m e r a s e behaves as a pure exonuclease. On the other hand, at very high c o n c e n t r a t i o n s of substrate, the p r o b a b i l i t y of going f o r w a r d becomes close to one, and there is little excision.

F. B e r n a r d i a n d J. N i n i o .

1088

ginning, the polymerization steps are preponder a n t . S m a l l b u t m e a s u r a b l e a m o u n t s of d ' [ ~ I P a r e p r o d u c e d . As t h e s u b s t r a t e d T T P is c o n s u m e d (IS] goes down), excision becomes more and m o r e f m p o r t a n t w i t h r e s p e c t to i n c o r p o r a t i o n . Finally, excision takes over, and the overall trend r e v e r s e s . If y(t) is t h e a m o u n t of p o l y m e r i z e d m a t e r i a l at t i m e t, a n d x(t) t h e a m o u n t of n u c l e o side monophosphate that has accumulated, we

T h i s s i m p l i f i e d m o d e l p e r m i t s to u n d e r s t a n d , as 'we s h a l l see, t h e m o s t p u z z l i n g o b s e r v a t i o n s o n t h e a c c u r a c y of D N A p o l y m e r a s e s . M o r e c o m p l e x schemes are discussed later, but bring little imp r o v e m e n t to t h e u n d e r s t a n d i n g of t h e b a s i c effects. P y r o p h o s p h o r o l y s i s b r i n g s a b o u t a p r a c tical complication. A residue may be removed, n o t o n l y b y e x c i s i o n , b u t also b y p y r o p h o s p h o r o _ lysis. L e t k 3 b e t h e r a t e - c o n s t a n t f o r p y r o p h o s -

(b)

(a)

1.0

.8(

0,.3

Tinc (y)

.00

.40 0.5

9.2

.20 ~.1 X [

I

0.05 V-_

I 15

I 30

dy/dx

21 15 10 5 4*.'~

~ _ 0.1xlo-4M

I 0.2

I 0.3

I 0.4

p h o r o l y s i s , a n d let u s a s s u m e t h a t e v e r y i n c o r p o r a t i o n of a d T T P m o l e c u l e l i b e r a t e s a p y r o p h o s phate. In the experiment shown in figure 1 we replic a t e t h e h o m o p o l y m e r P o l y (dA) w i t h d T T P at a n i n i t i a l c o n c e n t r a t i o n [S(0)] : 1~-~ M. At t h e b e -

BIOCHIMIE, 1978, 60, n ,° 10.

0.15

I

•

0.20

Fro. 1. - - Replication of the homopolymer Poly (dA) : incorporation and excision of T, the complementary nucleotide. Fig. la shows the i n c o r p o r a t i o n of T into p o l y m e r

I 45 m n

Time

_

I

0.1

(curv.e y) a n d t h e a c c u m u l a t i o n of dTMIP (curve x), reflecting excision. T h e curve y gives the a m o u n t of T t h a t h a s escaped from excision, a f t e r incorporation. As the s u b s t r a t e dTTP is consumed, i n c o r p o r a t i o n declines a n d excision takes over. The r e l a t i o n s h i p between polym e r i z a t i o n a n d excision is well described b y Eq. (4). I n t e r p r e t e d in t e r m s of t h i s equation, the e x p e r i m e n t gives the following r a t i o s for the kinetic c o n s t a n t s : k3/k2 = 3.21 × 104 ; kl/k~ = 49.3 × 104 ; k~/k3 ---- 15.4. Thus, at equal c o n c e n t r a t i o n s of dTTP and pyrophosphate, there is o n l y a 15 fold preference for incorporation. At 10-4M p y r o p h o s p h a t c , the r e m o v a l of a T residue by p y r o p h o s p h o r o l y s i s would be t h r e e times less f r e q u e n t t h a n its r e m o v a l b y excision. The data are given as fractions of the initial a m o u n t of s u b s t r a t e f o u n d in the dTTP, dTMP, or polymerized form. The t e m p l a t e was Poly (dA). dTl~_a8 (50~ residues of A for 1 T), at 0.5 o.d.u./ml. The enzyme (E. coli DNA polymerase 1) was a generous gift of Dr Gilbert Brun. The i n c u b a t i o n m i x t u r e c o n t a i n e d 10,-4M ~nCl.,, 10-1M KG1, 7.5 × 10-SM dTTP, 2.5 X 10-SM dATP and 0.8 unit of e n z y m e / m l ; the buffer was Tris-HC1, 50 raM, pH 7.4.

should observe d y / d x a n d E S] dy

ax

-

a k:~

,(1 + - -

~

linear

dependency

[S,(O)])

+

k1 + k3

( t h e d e r i v a t i o n s of all t h e e q u a t i o n s here will be published elsewhere).

between

ES~

(4)

presented

Accuracy of DNA replication. As s h o w n dependency t h e n assign tants k~, k2,

in figure l c , t h e r e is i n d e e d a l i n e a r b e t w e e n d y / d x and [S]. W e can relative values to the t h r e e rate-consk 3 (see the legend to figure 1).

Actually, the same l i n e a r d e p e n d e n c y b e t w e e n d y / d x and IS] should be o b s e r v e d w i t h a g e n e r a l class of m o d e l s first p r o p o s e d b y Brutlag a n d K o r n b e r g [49], a n d f u r t h e r dewelopped b y Branscomb a n d Galas [61 and m a n u s c r i p t p r i v a t e l y circ u l a t e d ] . The i d e a in this class of models is t h a t although the p o l y m e r i z a t i o n a n d e x c i s i o n activities of the p o l y m e r a s e are s w i t c h e d on p e r m a n e n tly, the c o n f o r m a t i o n a l state of the p r i m e r t e r m i nus d e t e r m i n e s w h i c h a c t i v i t y m a y p r a c t i c a l l y be exerted. If the t e r m i n u s is b a s e - p a i r e d to the opposite base on the template, p o l y m e r i z a t i o n m a y occur, but e x c i s i o n is impossible. If the term i n u s is u n p a i r e d , e x c i s i o n becomes possible b u t . p o l y m e r i z a t i o n c a n n o t occur. Such m o d e l s l e a d also to the above equation, w h e r e k I and k 2 become p h e n o m e n o l o g i c a l constants. Note also that the equation m a y be c o n s i d e r e d as a l i m i t i n g case, v a l i d for most k i n e t i c schemes, w h e n the c o n c e n t r a t i o n of s u b s t r a t e is low.

~UTATORS

AND ANTIMUTATORS.

The f r e q u e n c y of s p o n t a n e o u s m u t a t i o n s observed in each species achieves some k i n d of comp r o m i s e for the species b e t w e e n the a d v a n t a g e s a n d d i s a d v a n t a g e s of v a r i a b i l i t y (potential increases in fitness versus i n c r e a s e s in genetic load). The rate of s p o n t a n e o u s m u t a t i o n s is not i m m u t a b l e . One sihgle m u t a t i o n a l step at an a p p r o p r i a t e locus i s . s u f f i c i e n t to c h a n g e the freq u e n c y of m u t a t i o n s i n the o r g a n i s m and its descent. Varieties i n w h i c h t h e m u t a t i o n rate is h i g h e r t h a n it is i il w i l d t y p e h a v e been o b s e r v e d both in procary()fes and e u c a r y o t e s [23, 62-64], Competitions. i,~bet~veen w i l d t y p e a n d m u t a t o r p o p l j l a t i o n s h f i y e b e e n s t u d i e d in a few cases [65, 66]. Mutators s e e m to have the edge in a r a p i d l y c h a n g i n g envfi:on.ment. S t a b i l i t y in the e x t e r n a l c o n d i t i o n s seem to give the a d v a n t a g e to w i l d type. A n t i m u t a t o r s - i.e., v a r i e t i e s w i t h a r e d u c e d f r e q u e n c y of s p o n t a n e o u s m u t a t i o n s are also k n o w n , m a i n l y i n p h a g e T4. Genetic [23, 67, 68, 73] and bioch:emical ,~46, 47] studies on m u t a t o r s a n d antimuta!obS, of 'pliage T4 have s h o w n in most cases that a l t e r e d DN.A p o l y m e r a s e s are r e s p o n sible for the c h a n g e : i n ? n u t a t i o n frequency. The b i o c h e m i c a l sl~dies 0rr .mutant DNA p o l y m e r a s e s from p h a g e T4"~hhve p r o v i d e d a c r o p of results [46,. 47, 69-741 t h a t c o n t r i b u t e d to change our con-

BIOCHIMIE, 1978, 60, n,P 10.

1089

c e p t i o n s on the r a t i o n a l e for a c c u r a c y in e n z y m a tic d i s c r i m i n a t i o n s . Hershfiel.d's results [47] w e r e still c o m p a t i b l e w i t h t r a d i t i o n a l views. He f o u n d that the m u t a t o r DNA p o l y m e r a s e LS88 w a s less selective than w i l d t y p e in the choice b e t w e e n c o r r e c t a n d i n c o r r e c t s u b s t r a t e at the i n c o r p o r a tion revel. On t h e o t h e r h a n d , Muzyczka et al. [46] f o u n d that the difference in a c c u r a c y betw e e n w H d t y p e , m u t a t o r and a n t i m u t a t o r DNA p o l y m e r a s e s could not be a c c o u n t e d for b y changes in the s e l e c i i v i t y of i n c o r p o r a t i o n , or changes in the s e l e c t i v i t y of excision. The best c o r r e l a t e of the genetic p h e n o t y p e was w i t h the general b a l a n c e b e t w e e n i n c o r p o r a t i o n a n d excision. W i t h r e s p e c t to w i l d t y p e , a m u t a t o r enzyme has an i n c r e a s e d t e n d a n c y of m o v i n g f o r w a r d , w h i l e an a n t i m u t a t o r p o l y m e r a s e w o u l d on the average go t h r e e s t e p s b a c k w a r d for e v e r y four f o r w a r d steps. An e n e r g e t i c p r i c e is p a i d for the i n c r e a s e d a c c u r a c y , s i n c e for e v e r y dNMP t h a t is f i n a l l y i n c o r p o r a t e d , 4 d N T P ' s are on the average, c o n v e r t e d into dNMP. Can w e u n d e r s t a n d w h y , b y i n c r e a s i n g in a g e n e r a l m a n n e r the p o l y m e r i z a t i o n / e x c i s i o n ratio, the e r r o r - r a t e i n c r e a s e s ? W e shall give the explan a t i o n b y d i s c u s s i n g a c o n c r e t e example, t a k e n f r o m Bessman et al. [71]. Bessman a n d co-work e r s r e p l i c a t e d a n a t u r a l DNA in the p r e s e n c e of the f o u r usual nucleotides, plus the base-analog 2-amino p u r i n e in the d e o x y n u c l e o s i d e t r i p h o s p h a t e f o r m : (2AP)TP. T h e y m e a s u r e d the i n c o r p o r a t i o n a n d e x c i s i o n of A and 2AP w i t h w i l d t y p e a n d several m u t a t o r a n d a n t i m u t a t o r DNA p o l y m e r a s e s . Tile f o l l o w i n g results (here in arbit r a r y units) w e r e o b t a i n e d w i t h the w i l d t y p e polymerase : y(A) = r e s i d u e s o f A f i n a l l y p o l y m e r i z e d : 3.29 x(A) = r e s i d u e s of A f o u n d in the f o r m of dAMP ( i n c o r p o r a t e d , then excised) : 0.42 y(2AP) : 0.27 x(2AP) : 0.19 On the average, w h e n 371 r e s i d u e s of A are init i a l l y i n c o r p o r a t e d into p o l y m e r (371 = 329 + 42), 42 r e s i d u e s are s u b s e q u e n t l y excised a n d 329 finally r e m a i n in p o l y m e r i z e d form. W h e n 46 residues of 2AP are i n i t i a l l y i n c o r p o r a t e d , 19 on the average are e x c i s e d a n d 27 r e m a i n in the polymer. The initial p r o b a b i l i t y of 2AP i n c o r p o r a t i o n b y the w i l d t y p e enzyme is thus 4.6/(46 + 371) = 0.11. The p r o b a b i l i t y of e x c i s i o n of an A after i n c o r p o r a t i o n is : 42'/371 = 0.113, and the p r o b a b i l i t y of excision of 2AP is : 19/46 = 0.413. One m a y i n t r o d u c e a coefficient Q w h i c h d e s c r i b e s the relative efficiencies of the excision activities

F. Bernardi and J. Ninio.

109'0

t o w a r d s the i n c o r r e c t (2AP) a n d the correct (A) base : Q = 0.43/0.113 = 3.65. The global errorrate is : y(2AP)/[y,(A) + y(2AP)] = 7.6 per cent. The results o b t a i n e d with the a n t i m u t a t o r L141 were the f o l l o w i n g : y(A) x~(A) y(2AP) x(2AP)

: 1.17 : 0.78 : 0.03 : 0'.32

The overall error-rate w i t h the a n t i m u t a t o r enzyme is 0..03/(1.17 + 0.03) = 2.5 per cent, it is three times lower t h a n the error-rate achieved w i t h the w i l d t y p e enzyme. Yet, the specificity at the i n c o r p o r a t i o n level is not improved. We f i n d a p r o b a b i l i t y of i n i t i a l i n c o r p o r a t i o n of 2AP of 0.17 - i.e., from this p o i n t of vie~v, the situation is even worse t h a n w i t h the w i l d t y p e enzyme. If we c o m p u t e the coefficient Q giving the selectivity of excision, we f i n d 2.3 w h i c h m e a n s again that the a n t i m u t a t o r is less specific at the level of excision t h a n wildtype. Being less specific both at the i n c o r p o r a t i o n level a n d at the excision level, h o w can it be, on the whole, more accurate ? The e x p l a n a t i o n is i n fact r a t h e r simple. The ratios that we i n t r o d u c e d to compute the specificity factor Q are not those that really matter. Here comes the c r u c i a l p o i n t that was not discussed in ref. [59]. Let us c o n s i d e r again the simplified k i n e t i c scheme of the p r e c e d i n g section a n d study the fate of a 2~AP that has just been i n c o r p o r a t e d . A 2AP w h i c h is finally i n c o r p o r a t e d is a 2AP that has escaped from excision. The e l e m e n t a r y p r o b a b i l i t y of excision is kgAP/(k~AP + hi). I n the f o r w a r d constant 2 2 h I we are l u m p i n g the two possible ways of i n c o r p o r a t i n g a b a s e : h 1 = k~ [A] + klSAP

[2AP]. The p r o b a b i l i t y of escape from excision i s : sAP = h i / ( h i + k ssAP ) p____~ (5) The p r o b a b i l i t y of escape from excision is, for the correct base : p____>A= hl/,(h 1 + k ~ ) (6) Let us compare the two p r o b a b i l i t i e s of escape : -----> psAP A

P----~

h I + k~

(7)

2AP

hi + ks

Let us assume that excision is more efficient t o w a r d s the i n c o r r e c t than t o w a r d s the correct base (k~ AP > k#). W h e n h 1 is very large compared to the k2's ( w h i c h m a y occur for i n s t a n c e if the c o n c e n t r a t i o n of dNTP is high), the p r o b a b i lity of escape is close to one for both the correct an dthe i n c o r r e c t base. The fact that the k i n e t i c constants for excision k~ a n d k~^P are different

BIOCHIMIE, 1978, 60, n ° 10.

is of no c o n s e q u e n c e . The p o t e n t i a l specificity of the e x c i s i o n activity is not exploited. The smaller the value of h i ( c o m p a r e d to the k.2's), the more the p r o b a b i l i t i e s of escape from excision become different, a n d t h e i r ratio approaches the ratio of the kz's. Thus, b y c h a n g i n g i n a non-specific w a y the ratio of the i n c o r p o r a t i o n activity (reflected i n h 1) to the excision activity (reflected i n the k2's) , the f r e q u e n c y of errors must vary. The h i g h e r the p o l y m e r i z a t i o n / e x c i s i o n ratio, the h i g h e r the error-rate. F o r a more rigorous treatment, see [60]. A p r e d i c t i o n of the model is that the m u t a t o r a n d the a n t i m u t a t o r effects m a y be m i m i c k e d b y s i m p l y c h a n g i n g the overall c o n c e n trations of substrates a n d k e e p i n g the same enzyme. Consider the r e p l i c a t i o n of a heteropolymer : ....... ATTGCGCTATTCACT ...... ....... TAACA The p r o b a b i l i t y of excision of the last residue will d e p e n d u p o n the rate-constant for the excision of A w h e n it is opposite to a C, and u p o n the rate-constant for the i n c o r p o r a t i o n of the next nucleotide, a C. Thus, a c c u r a c y at one position depends u p o n the rate of i n c o r p o r a t i o n of the nucleotide w h i c h comes next. This gives one possible source for position effects in mutagenesis. The p r e c e d i n g discussion of the specificity of the p o l y m e r i z a t i o n / e x c i s i o n process follows a line that is very s i m i l a r to the earlier t r e a t m e n t of the specifi.city of m u t a n t ribosomes that make m o t or less mistakes [75]. An almost perfect illust r a t i o n of the effect of substrate c o n c e n t r a t i o n u p o n t the error-rate at a given position is provided i n figure 2. Here, we replicated Poly (dC) w i t h the substrate dGTP, and small a m o u n t of dATP. The efficiency of the .excision activity t o w a r d s G residues is rather low. Calling y(A) the a m o u n t of A finally i n c o r p o r a t e d i n the Poly (dG), a n d x(A) the a m o u n t s of A t u r n e d into nucleoside m o n o p h o s p h a t e , the simplest model p r e d i c t s : dy k [ dGTP] (8) dx k W h i l e G residues n e a r l y always escape from excision, the p r o b a b i l i t y for an A of not b e i n g excised decreases as the c o n c e n t r a t i o n of G decreases.

SPECIAL KINETIC EFFECTS. SUBSTRATE CONCENTRATION. In a previous paper, we d e f i n e d a <> enzyme as an enzyme w h i c h has only one

A c c u r a c y o[ D N A r e p l i c a t i o n . conformational state for picking up the substrate, and which transforms th~ substrate into product under conditions where the back-reaction is

O)

1091

w h a t e v e r t h e c o n c e n t r a t i o n s of A a n d B. A g o o d e x a m p l e of t h e c o n s t a n c y of t h e a b o v e r a t i o is g i v e n i n t h e w o r k of B a t t u l a a n d L o e b o n A M ~

L

/s (b)

Y

0.05 0.0

0.025 0.025

I.(3 q3

20

40

o0mn

I 0.01

Time

6:)

l,

I 0.4

X i

Fro. 2. - - Replication of the homopolymer Poly (dC) : incorporation and excision o[ A, a non-complementary nueleotide. Fig. 2a shows the i n c o r p o r a t i o n of A into p o l y m e r

dy/dx

i 0.2

I 0.02

I 0.6 x 10-4M

(curve y) a n d the a c c u m u l a t i o n of dAMP (curve x) while the c o m p l e m e n t a r y s u b s t r a t e (dGTP) is b e i n g consumed. T h e probabil'ity for a n A r e s i d u e to escape from exoisio~ a f t e r a n i n c o r p o r a t i o n decreases as t h e coneenCration of dGTP is deere,asing. The resulCs can be i n t e r p r e t e d in t e r m s of the simplest model e q u a t i o n (Eq. 8). This gives : k ~ / k ~ = 5.4 X 104. Unless otherwise indicated, t h e conditions are the same as for Fig. 1 Poly(dC) : 0.66 o.d.u./ml ; e n z y m e : 4 u n i t s / m l dGTP : 8 × 10-sM ; dATP : 4 × 10-TM.

TABLE III.

Competition b e t w e e n correct and incorrect nucleotide on AMV DNA p o l y m e r a s e [[rom Battula and Loeb, ref. 77]. Ratio ol correct IT) to incorrect (C) snbstrate concentrations

Incorporation ol T

Incorporation of C

3.33 2.0 1.25 0.833 0.602 0.407

65.5 59.0 63.0 63.0 65.0 64.0

0.020 0.036 0.040 0.092 0.107 0.154

n e g l i g i b l e [76]. If t w o s u b s t r a t e s A a n d B a t c o n c e n t r a t i o n s [A] a n d [B] c o m p e t e o n a w e l l : b e h a ving polymerase one should have, under steadystate conditions : A incorporated [A] D = -- c o n s t a n t (9) B incorporated [B]

BIOCHIMIE, 19'78, 60, n ° 10.

Discrimination Tint [dTTP] .Cine [dCTP] 983 820 1096 834 1007 1023

p o l y m e r a s e [ 7 7 ; see t a b l e I I I ] . S i m i l a r l y , E n g e l and yon Hippel studied the competition between A and N 6 -methyladenine for incorporation into P o l y ( d A - d T ) w i t h E. coli D N A p o l y m e r a s e 1, at r a t h e r h i g h c o n c e n t r a t i o n s of s u b s t r a t e s (4 × 10 -4 M), m a k i n g t h e e x c i s i o n a c t i v i t y i n e f f i c i e n t .

1092

F . B e r n a r d i a n d J. N i n i o .

T h e y also o b s e r v e d a r o u g h c o n s t a n c y f o r t h e e x p r e s s i o n D [78]. D v a r i e s l i k e t h e r e c i p r o c a l of t h e r a t i o i n E q . (7) u n d e r c o n d i t i o n s w h e r e e x c i s i o n r e m a i n s m o d e r a t e _ i.e., t h e p r o b a b i l i t y of going back twice in succession may be neglected. F o r a t r e a t m e n t of t h e , i n c o r p o r a t i o n r a t i o s i n a situation where multiple excisions occur (the ¢ p e e l b a c k >> p r o b l e m ) , see [60] o r [61].

PYROPHOSPHOROLYSIS.

A t e q u i l i b r i u m , t h e p r o p o r t i o n s of t h e v a r i o u s products of c h e m i c a l reactions are related through the equilibrium constants. The presence of p y r o p h o s p h a t e in a polymerization reaction s h o u l d b r i n g t h e r e a c t i o n c l o s e r to e q u i l i b r i u m .

PRO CF,S SIVITY.

T h e a d v a n t a g e of a d i s t r i b u t i v e over a processive one for fighting

w a s p o i n t e d o u t [716]. C o n s i d e r t h a t t h e r e a r e some sequences along the template ~vhich bind t h e D N A p o l y m e r a s e p o o r l y . T h e m e c h a n i s m of polymerization will be distributive along these sequences and error-rates should be most sensit i v e t h e r e to e n v i r o n m e n t a l m a n i p u l a t i o n s .

mechanism tautomeries

1.o' Cb)

Y

0.8

0.0 .50 0.4

.25 0.2

o~

6t0 Time

~

~

I 0.1

l~o~nn

X

I 0.2

1-

(c)

dy/dx

/

(d)

o.s~

X)

[dCTP] I

0.2 xlO-SM

I

0.4

I 0.6

I 0.8

I 1.o

I

0

1/[dCTP] I

106

I

2.106

I

3.1060

Fro. 3. - - Evidence for some kinetic complexity in the polymerization~excision interplay.

The h o m o p o l y m e r Poly(dC) is replicated in the presence of dGTP a n d dCTP. W e follow here the i n c o r p o r a t i o n of C (curve y) a n d the p r o d u c t i o n of dCMP (curve x). The i n t e r p r e t a t i o n , in t e r m s of Eq. (12) w h i c h corresponds to a k i n e t i c amplification or a kinetic p r o o f r e a d i n g m e c h a n i s m gives a ~ 0.9 az~d B ---- 4.6 X 106. E x p e r i m e n t a l conditions : MnC12, t{131 a n d Tris as in figure 1 ; Poly (d13), 0.3'3 o.d.u./ml ; dGTP, 4 X 10-5M ; dCTP, 1O-SM • enzyme, 2~ u n i t s / m l . BIOCHIMIE, 1978, 60, ~° 10.

A c c u r a c y of DNA replication. Note h o w e v e r that e q u i l i b r i u m c a n n o t be reached s i m u l t a n e o u s l y for A.T a n d G,C base-pairs. This makes it difficult to p r e d i c t the effect of i n c r e a sing p y r o p h o s p h a t e c o n c e n t r a t i o n s on accuracy. E r r o r s m a y increase at certain positions a n d decrease at others. V i e w i n g a p o r t i o n of sequence as a succession of stretches in ~ h i c h i n c o r p o r a tions occur a l t e r n a t i v e l y close and far from equil i b r i u m m a y suggest hitherto u n s u s p e c t e d effects.

KINETIC AMPLIFICATION. There are in p r i n c i p l e clever enzyme mechanisms nvhich p e r m i t to achieve a high specificity w i t h relatively modest means. These k i n e t i c p r o o f r e a d i n g or k i n e t i c a m p l i f i c a t i o n mechanisms [31, 69, 79, 80] have been a b u n d a n t l y commented, most often in a m i s l e a d i n g m a n n e r , a n d one finds i n the litterature several m i s c o n c e p t i o n s about the distinctive p r o p e r t i e s of such schemes. F o r this reason, the r e a d e r who wishes to avoid m i s i n t e r p r e t a t i o n s should read carefully the original p u b l i c a t i o n s . The parable of the f i s h e r m a n [81] and the c o i n - m a c h i n e analogy [82] give intuitive equivalents to k i n e t i c amplification. The key concept a r o u n d w h i c h the schemes revolve is that of the m a x i m u m specificity that m a y be extracted from a given step of a reaction, given the k i n e t i c constants for this step. I n the preceding section, we c o m p a r e d the p r o b a b i l i t i e s of escape from excision for two c o m p e t i n g nucleotides and saw i) that the ratio of the p r o b a b i l i t i e s could be altered b y c h a n g i n g n o n specifically the c o n s t a n t h 1 it) that there was a limit b e y o n d w h i c h the ratio could not increase, given by k~AP/k~. The kinetic p r o o f r e a d i n g of kinetic amplification schemes are designed in such a w a y as to allow d i s c r i m i n a t i o n to go b e y o n d the <> (here, the ratio o,f the k2's) p r o v i d e d that the non-specific constants (the equivalent of h 1) fulfill c e r t a i n conditions. Some other recent proposals on h o w d i s c r i m i n a t i o n is achieved [83-851 do not deal w i t h the above specific issue, a n d should not be c o n s i d e r e d as i n c o m p a t i b l e w i t h the kinetic amplification concept. W h e n applied to DNA polymerases, Hopfield's scheme [791 m a y be w r i t t e n as :

~d.

et•

,/~

tfl, n , 8 ~"

(1)

{'~,~,:l.y m"

while the delayed-escape model BIOCH1MIE, 1978, 60, n ° 10.

~,n.~ (11) n -t.

n

rr÷~.

We w i l l show elsewhere that the two schemes, although very different physically, give rise to the same k i n d of predictions. W h e n a homopolym e r is replicated w i t h a substrate S, one should have, in the absence of p y r o p h o s p b o r o l y s i s :

dx

1 +

~

=

ct

~ IS]

(12)

w h e r e a and ~ are two constants, x denotes as before the a m o u n t of nucleoside m o n o p h o s p h a t e p r o d u c e d in the course of the p o l y m e r i s a t i o n reaction, w h e t h e r r e s u l t i n g from the excision of a p r e v i o u s l y i n c o r p o r a t e d residue, or from the direct cleavage of dN~TP into dNMP. However the two schemes give different p r e d i c t i o n s w h e n a p p l i e d to the fate of a second competing substrate. It is conseivable that some DNA-dependent ATPases are actually s u b u n i t s of DNA polymerases. By w o r k i n g together, some k i n d of k i n e t i c a m p l i f i c a t i o n might be achieved, as proposed by Alberts et al. [86]. We have found a system in w h i c h the observed kinetics seem to c o n f o r m to Eq. [12]. We replicate Poly (dC) with dGTP as substrate, in the presence of dCTP. W h e n enough Poly (riG) is formed, w e begin to see the i n c o r p o r a t i o n of C residues into polymer, a n d the p r o d u c t i o n of dCMP. The e x p e r i m e n t was done in the absence of a pyrophosphatase, a n d the deviation in the results from the p r e d i c t i o n of Eq. [12] is possibly due to the i n c i d e n c e of p y r o p h o s p h o r o l y s i s . We have n o w started w i t h M. Dorizzi a n d M. Saghi the study of the p o l y m e r i z a t i o n / e x c i s i o n kinetics w i t h E. colt DNA polymerase 1, in the presence or absence of pyrophosphatase, a n d hope to be able in the near future to b r i n g definite i n f o r m a tions on the m e c h a n i s m s of i n c o r p o r a t i o n and excision. REFERENCES.

•~'t'4

xx

I

1093

[60] becomes

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