Neighboring-Nucleotide Effects on the Rates of Germ-Line Single-Base-Pair Substitution in Human Genes

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1 Am. J. Hum. Genet. 63: , 1998 Neighboring-Nucleotide Effects on the Rtes of Germ-Line Single-Bse-Pir Substitution in Humn Genes Michel Krwczk, Edwrd V. Bll, nd Dvid N. Cooper Institute of Medicl Genetics, University of Wles College of Medicine, Crdiff Summry The spectrum of single-bse-pir substitutions logged in The Humn Gene Muttion Dtbse (HGMD), comprising 7,271 different lesions in the coding regions of 547 different humn genes, ws nlyzed for nerestneighbor effects on reltive muttion rtes. Owing to its retrospective nture, HGMD llows muttion rtes to be estimted only in reltive terms. Therefore, novel methodology ws devised in order to obtin these estimtes in itertive fshion, correcting, t the sme time, for the confounding effects of differentil codon usge nd for the fct tht different types of mino cid replcement come to clinicl ttention with different probbilities. Over nd bove the hypermutbility of CpG dinucleotides, reflected in trnsition rtes five times the bse muttion rte, only subtle nd loclly confined influence of the surrounding DNA sequence on reltive single-bse-pir substitution rtes ws observed, which extended no frther thn 2 bp from the substitution site. A disprity between the two DNA strnds ws evidenced by the fct tht, when substitution rtes were estimted conditionl on the 5 nd 3 flnking nucleotides, significnt rte difference emerged for 10 of 96 possible pirs of complementry substitutionl events. Muttionl bis, fvoring substitutions towrd flnking bses, phenomenon reminiscent of mislignment mutgenesis, ws pprent nd exhibited both directionlity nd reding-frme sensitivity. No specific prepondernce of repet-sequence motifs ws observed in the vicinity of nucleotide substitutions, but moderte correltion between the reltive mutbility nd thermodynmic stbility of DNA triplets emerged, suggesting either inefficient DNA repliction in regions of high stbility or the trnsient stbiliztion of misligned intermedites. Received Februry 9, 1998; ccepted for publiction My 28, 1998; electroniclly published July 10, Address for correspondence nd reprints: Dr. Michel Krwczk, Institute of Medicl Genetics, University of Wles College of Medicine, Heth Prk, Crdiff CF4 4XN, United Kingdom. E-mil: krwczk@crdiff.c.uk 1998 by The Americn Society of Humn Genetics. All rights reserved /98/ $02.00 Introduction The mjority of germ-line muttions in humn genes re thought to result from error-prone endogenous processes involving either chemicl (e.g., methyltion-medited demintion of 5-methylcytosine in CpG dinucleotides), physicl (e.g., DNA slippge), or enzymtic (e.g., postreplictive mismtch repir nd exonucleolytic proofreding) mechnisms (Cooper nd Krwczk 1993). Since the efficiency of these processes is DNA-sequence dependent, it is not surprising tht both the spectrum nd sptil distribution of muttions exhibit bises tht reflect the influence of the locl DNA-sequence environment on germ-line mutbility. A lrge proportion of microdeletions nd microinsertions in humn DNA thus pper to occur s the consequence of repliction slippge medited by the presence of direct or inverted (plindromic) repets in the immedite vicinity (Cooper nd Krwczk 1993). For single-bse-pir substitutions, which constitute the mjority of known lesions cusing humn genetic disese, the influence of the locl DNA-sequence environment on muttion rtes is less cler. Aprt from the well-estblished hypermutbility of CpG dinucleotides tht undergo germ-line trnsition to TG nd CA t frequencies six to seven times the bse muttion rte (Cooper et l. 1995), no cler evidence hs yet been presented for strong nerest-neighbor effect on the nture of inherited nucleotide substitutions in humns in vivo. Previous studies bsed on phylogenetic (e.g., see Golding nd Glickmn 1986) or clinicl dt (e.g., see Todorv nd Dnieli 1997) hve either been purely necdotl or, insted, hve been focused on single genes or gene fmilies. A brod-bsed nlysis of the spectrum of singlebse-pir substitutions hs only recently become possible, through the estblishment of the The Humn Gene Muttion Dtbse (HGMD [Krwczk nd Cooper 1997]), comprehensive literture-bsed collection of muttions either underlying or ssocited with humn inherited disese. Contining 17,200 different nucleotide substitutions from the coding regions of some 550 different genes, HGMD provides n unprlleled source of mteril for the metnlysis of germ-line muttions in humn genes. 474

2 Krwczk et l.: Nucleotide Substitution Rtes 475 Owing to the retrospective nture of HGMD dt, however, nucleotide-substitution rtes cn be ssessed only in reltive terms. Furthermore, for muttion to be represented in HGMD, it must result in phenotype severe enough to hve come to clinicl ttention but, t the sme time, must not be subject to preclinicl selection. In order to tke these considertions into ccount, new nlyticl pproch hs been devised tht models the likelihood tht muttion in HGMD will be observed s function of its consequences t both the DNA nd protein level (Krwczk et l. 1995; Krwczk nd Cooper 1996). In n itertive procedure, estimtes re obtined simultneously for the reltive occurrence rte of given substitution type, with llownce for flnking nucleotides, nd for the reltive clinicl observtion likelihood of its resulting phenotype. The lrge number of different genes nlyzed here ensures tht the bises tht reflect the privte chrcteristics of individul proteins nd tht hmper studies of single genes or gene fmilies re likely to be verged out. Our study revels tht, when neighboring mononucleotides nd dinucleotides re considered s covrites, their effect on single-bse-pir mutgenesis is loclly confined. This finding hs severl importnt prcticl nd theoreticl implictions: considertion of nerestneighbor dependent substitution rtes should help to optimize muttion-serch strtegies, render phylogenetic reconstruction in moleculr-evolutionry studies more relistic (Krwczk et l. 1996), nd llow the privte chrcteristics of gene- or tissue-specific muttionl spectr to be identified, thereby providing the bsis for better understnding of the moleculr mechnisms underlying single-bse-pir substitutions in generl. Mteril nd Methods HGMD HGMD is comprehensive collection of germ-line muttions underlying or ssocited with humn inherited disese, comprising published single-bse-pir substitutions, deletions, duplictions, insertions, nd morecomplex rerrngements in humn nucler genes (Krwczk nd Cooper 1997). Although originlly estblished for reserch purposes (Cooper nd Krwczk 1993), the dtbse hs since cquired much broder utility nd, for this reson, ws mde publicly vilble, through the Internet, in April By November 1997, HGMD contined 7,271 different single-bse-pir substitutions (5,862 missense nd 1,409 nonsense) in the coding regions of 547 different genes. HGMD entries for this ctegory of lesion include the triplet chnge, with n dditionl flnking nucleotide logged when the mutted bse lies in either the first or third position in the triplet. This informtion llows nerest-neighbor effects on substitution rtes to be redily ssessed for the 5 nd 3 nucleotides flnking the site of muttion. For subset of 423 of the genes, reference cdna sequences (strting with the ATG initition codon nd ending with the stop codon) lso were vilble, llowing the nlysis of the broder DNA-sequence context for 6,885 substitutions (94.7% of the totl). It should be noted tht ech nucleotide substitution in HGMD hs been logged only once, in order to void confusion between recurrent nd identicl-by-descent lesions. Although necessitting the systemtic exclusion of multiple independent de novo muttions, this restriction is considered unlikely to bis the estimtes of reltive substitution rtes nd clinicl observtion likelihoods to ny mrked extent, becuse of the lrge smple size. Estimtion of Reltive Single-Bse-Pir Substitution Rtes nd Reltive Clinicl Observtion Likelihoods With the possible exception of biologiclly lethl dominnt conditions, germ-line muttionl spectr ssocited with genetic disese do not llow single-bse-pir substitution rtes to be estimted directly nd in bsolute terms. This shortcoming is due to the fct tht, without extensive hplotyping, discrimintion between recurrent muttion nd identity by descent is impossible. Even if two identicl-by-stte lesions were recognized to be of independent origin, the ctul number of meioses screened for their occurrence would remin unknown. Additionlly, for muttion to be found to be ssocited with disese, it must cuse phenotype sufficiently severe tht it comes to clinicl ttention, but, t the sme time, it must not be selected ginst ntentlly or precliniclly. This implies tht cliniclly observed muttion frequencies my not reflect directly the underlying rtes of occurrence. To disentngle the effects tht muttion nd selection hve on n observed muttionl spectrum such s tht found in HGMD, we hve devised n itertive multivrite procedure (Cooper nd Krwczk 1993; Krwczk et l. 1995; Krwczk nd Cooper 1996) tht tkes into ccount the phenotypic consequences of muttion nd tht estimtes, in reltive terms, single-bse-pir substitution rtes nd clinicl observtion likelihoods of phenotypic consequences. A detiled presenttion of this pproch is given in the Appendix. In brief, the lgorithm ims to mximize the overll likelihood of given muttion smple, ssuming tht the likelihood tht n individul muttion of primry type x nd phenotypic consequence will be observed equls the product of the probbility of occurrence of x t the DNA level, m(x), nd the clinicl observtion likelihood of, L(). Mximum-likelihood estimtes of reltive m nd L vlues, lso corrected for humn genecodon usge nd the redundncy of the genetic code,

3 476 Am. J. Hum. Genet. 63: , 1998 re obtined in itertive fshion. In the present study, muttion type x ws defined either s the nucleotide substitution on its own (e.g., CrT, GrA, or ArT), s the substitution conditionl on flnking mononucleotide (e.g., CGrTG, CNTrANT, or GN 3 ArGN 3 T), or s the substitution conditionl on flnking dinucleotide (e.g., ATCrATA, TN 2 TCrAN 2 TC, or CGN 3 CrCGN 3 A). Phenotypic consequence ws mesured in terms of the chemicl difference between wild-type nd mutnt mino cid residues. This prmeter, originlly devised by Grnthm (1974) to ssess the net effect of mino cid exchnges in evolutionry comprisons, combines the three interdependent properties of composition, polrity, nd moleculr volume in single, continuous quntity. For the ske of simplicity, however, the rnge of chemicl-difference vlues ws divided here into 11 eqully sized intervls, thereby trnsforming into clss vrible; n dditionl clss ws introduced for nonsense muttions. Significnce Assessment (Nerest-Neighbor Effects) The vribility of m(x) nd L() estimtes ws ssessed by bootstrpping, with ech SD determined in 10,000 resmpling simultions (Hjorth 1994). In order to test reltive substitution rtes for potentil strnd difference, muttions were defined in terms of the immeditely 5 nd 3 flnking nucleotides (e.g., x CTGrCAG). Estimtes of m(x) were then compred with m(x c ), the nlogous estimte for the complementry sequence (e.g., x ATCrAGC, nd x c GATrGCT). Differences were deemed to be significnt when m(x) 1 m(x ) c c or m(x)! m(x ) in 9,995 of 10,000 bootstrpping simu- ltions. This threshold ws dopted to llow n overll 95% significnce level for the 96 comprisons involved. The influence of neighboring mononucleotides nd dinucleotides on reltive single-bse-pir substitution rtes ws ssessed s follows. For given substitution x (e.g., x CrA), let x i denote the muttion type defined by mononucleotide or dinucleotide h i flnking substitution x. If it is ssumed tht ll h i re, priori, eqully likely to flnk the wild-type nucleotide in x, then P (h i:x) m(x i)/sm(x j j) cn be interpreted s the posterior probbility of h i, given tht x hs occurred. In the bsence of ny neighboring-nucleotide effects, P(h i :x) 1 1 would equl 4 or 16 for ll mononucleotides or dinucleotides h i, respectively, so tht the Eucliden distnce between P {P} i {P(h i:x)} nd the centroid { i}, with or, d (P, ) [S (P )]2 i 4 i 16 i i i is good mesure of the influence of the flnking mononucleotide or dinucleotide on the reltive rte of x. Whether P ws significntly different from ws gin determined by bootstrpping. When the P vector emerging from the originl dt set ws denoted by P o nd when the vectors from bootstrpping were denoted by P r, P o ws deemed significntly different from when dp,p ( o r)! mx[d(p o, ),d(p r, )], (1) for 19,995 of 10,000 replictes P r. Formul (1) cn be interpreted s P r being closer to P o thn, or t lest on the sme side of s is P o. Since 10 positions surrounding ech of the 12 possible substitutions were considered, this nlysis involved 120 comprisons. The threshold noted bove thus ensured 95% overll significnce level. Significnce Assessment (Codon Usge) In region comprising four mino cid residues both upstrem nd downstrem of missense muttions, the reltive use of mbiguous codons ws tested for significnt fetures, by mens of x 2 sttistic with 1 df. Expected frequencies were clculted from published codon-usge dt for humn genes (Nkmur et l. 1996). Since 55 comprisons were involved, threshold 4 of 0.05/ # 10 ws dopted for the error probbility. Absolute codon usge surrounding nonsense muttions ws relted to the totl 448 HGMD reference cdna sequences. To this end, codon usge ws determined in the reference cdna sequences round codons with the potentil to mutte to termintion codon by single-bse-pir substitution. The frequencies obtined were subsequently weighted, for ech potentilly mutble codon, in terms of its ctul frequency in the HGMD smple. Significnce ws ssessed for ech of the 64 surrounding codons, by mens of x 2 sttistic with 1 df, with doption of threshold of 0.10/ # 10 for the error probbility. A lower overll significnce level of 90% ws chosen, in order to llow for the considerbly smller number of nonsense muttions vilble for nlysis. Significnce Assessment (Muttionl Bis towrd Flnking Nucleotide) The importnce of slippge-medited misincorportion for the occurrence of single-bse-pir substitutions ws ssessed by mens of the reltive proportion of muttions towrd the immeditely 5 or 3 flnking nucleotide. This study necessrily ws limited to sites t which the wild-type nucleotide ws different from the flnking bse. In ddition, CGrTG nd CGrCA trnsitions explicble by the demintion of 5-methylcytosine were discrded (1,675/7,271, or 23% of the totl), since the reltive bundnce of this type of muttion would hve served to obscure ny mechnistic reltionship underlying the remining substitutions. The number of muttions towrd the immeditely 5 or 3 flnking nucleotide tht were expected under the null hypothesis tht

4 Krwczk et l.: Nucleotide Substitution Rtes 477 substitution towrd flnking nucleotide occurs with 1 probbility 3 ws determined s described in the Appendix. Observed frequencies were tested for significnt devition from these expecttions by mens of x 2 sttistic with 1 df. Results Throughout the following sections, shortened form will be used to denote clsses of single-bse-pir substitutions nd their sequence context. For ny oligomers A, B, nd C, (A,B)rC is equivlent to ArC, BrC wheres Ar(B,C) mens ArB, ArC. For exmple, CGr(TG,CA) denotes the clss of CGrTG nd CGrCA trnsitions. Reltive Single-Bse-Pir Substitution Rtes The spectrum of single-bse-pir substitutions within gene-coding regions nd logged in HGMD revels hierrchy of nucleotides tht is cler-cut with respect to their propensity to undergo substitution nmely, G 1 C 1 T 1 A (tble 1). Consistent with previous observtions (Cooper nd Krwczk 1990, 1993), prepondernce of trnsitions (62.5%) over trnsversions (37.5%) ws observed. Most but not ll of this excess cn be ttributed to CGr(TG,CA) muttions, which re redily explicble in terms of methyltion-medited demintion of 5-methylcytosine (5mC) on either the sense or ntisense DNA strnds. This type of lesion ccounts, on its own, for 23.0% of ll substitutions nd for 36.9% of trnsitions. Brekdown of the dt by chromosoml loction reveled, however, tht the proportion of CGr(TG,CA) substitutions ws significntly higher for utosoml genes (1,325/5,296, or 25.0%) thn for X- chromosoml genes (350/1,975, or 17.7%) ( x , 1 df, P! 10 ). In prt, this disprity cn be explined by generlly more pronounced CpG suppression observed in X-linked genes: the verge CpG content ws for the 401 utosoml cdna sequences provided by HGMD nd ws for the 45 X-chromosoml cdnas (Student s t 2.35, 444 df, P!.01). When the CG:GC rtio ws considered, in order to llow for differing G C content in different genes nd/or chromosomes, the respective verge vlues were for the X chromosome nd for utosomes ( t 1.66, 444 df, P!.05). As outlined bove (see the Mteril nd Methods section), muttion frequencies observed in the context of humn inherited disese re unlikely to reflect the true underlying rtes of muttion occurrence. Since different mino cid substitutions hve different effects on protein structure nd function, they necessrily hve come to clinicl ttention (nd thus hve entered HGMD) with Tble 1 Spectrum of Observed Single-Bse-Pir Substitutions in Gene- Coding Regions, Logged in HGMD ORIGINAL NUCLEOTIDE NO. OF SUBSTITUTIONS BY T C A G TOTAL T ) ,237 C 1,632 (940) ) ,343 A ) G ,717 (735) b ) 2,789 Totl 2,452 1,270 2,359 1,190 7,271 Number in prentheses is the proportion of trnsitions tht re CGrTG. b Number in prentheses is the proportion of trnsitions tht re CGrCA. different probbilities. Moreover, codon frequencies differ from one nother, implying tht, in muttionl event, different mino cid residues hve different prior probbilities of being involved. Reltive single-bse-pir substitution rtes corrected for these two confounding fctors re presented in tble 2. Although the rte estimtes re brodly consistent with the frequency dt in tble 1, some differences re nevertheless pprent. Thus, trnsversions (G,A)rT re estimted to occur t only hlf the rtes suggested by their bsolute frequencies. By contrst, the corrected trnsition rte of TrC is some 50% higher thn its frequency-bsed counterprt. A similr, lbeit smller bis lso pplies to the other three trnsitions (nmely, CrT, GrA, nd ArG). Neighboring-Nucleotide Effects It hs been known for some time (Cooper nd Youssoufin 1988) tht the rte of CrT nd GrA trnsitions in humn genes is gretly incresed by the presence of 3 gunine or 5 cytosine residue, respectively, owing to the bundnce of hypermutble methylted CpG dinucleotides (Bird 1986). The question therefore rises s to whether neighboring-nucleotide effect might exist lso for other types of nucleotide substitution. Figure 1 depicts the influence on reltive single-bse-pir substitution rtes tht is exerted by the five nucleotide positions upstrem nd downstrem of potentil muttion site. Ech dt point represents 1 of 12 possible singlebse-pir substitutions. Neighboring-nucleotide effects on reltive muttion rtes re expressed in terms of vector-bsed prmeter, the distnce from centroid, tht mesures, t given position, the devition from independence. The lrger this distnce, the more skewed is the posterior probbility tht prticulr mononucleotides will be observed in the vicinity of given substitution (see the Mteril nd Methods section). The complete dt set is vilble for inspection, on the HGMD Website. Two outliers mrk the strong effects tht the 1 (5 )

5 478 Am. J. Hum. Genet. 63: , 1998 nd 1(3 ) positions hve on GrA nd CrT trnsitions, respectively. A significnt influence lso ws noted, however, for some other substitutions: Tr(C,A), Ar(C,G), nd Gr(T,C) re bised by the nucleotide t position 1, wheres Tr(C,G), Cr(G,A), Ar(T,G), nd GrT re bised by the nucleotide t position 1. Nevertheless, it is evident tht the nerest-neighbor influence decreses mrkedly with distnce from the site of substitution (fig. 1). A significnt, lbeit wek effect ws observed for position 2, but only for five substitutions TrC, CrT, ArG, nd Gr(T,C). Interestingly, the six substitutions significntly influenced by the 1 (5 ) nucleotide cn be mtched with their complementry substitutions being significntly influenced by the 1(3 ) neighbor, nd vice vers. When nerest-neighbor effects were nlyzed with llownce for neighboring dinucleotides rther thn mononucleotides, more substitutions tended to exhibit sttisticlly significnt but weker rte dependency (dt not shown). Nevertheless, the effect gin ws confined to smll region, unlikely to extend beyond positions 2 nd 3. In order to illustrte the impct of the computtionl method described in the Appendix, estimtes of reltive single-bse-pir substitution rtes, simultneously llowing for both flnking nucleotides, lso were clculted, without correction for ny confounding fctors. These estimtes, m (x), simply equted the bsolute number of reports of given muttion type x multiplied by constnt fctor so s to ensure tht the verge m vlue ws unity. When llownce ws mde for multiple testing t n overll 95% level (192 comprisons), differences between the two estimtes were deemed significnt when d m(x) m (x) d j. Here, j denotes the SD of m(x). In totl, m represented significnt overestimte of m for 51 substitutions (fig. 2), none of which involved CGr(TG,CA). On the other hnd, with the exception of CCGrCTG nd GCGrGTG, ll rtes of CGr(TG,CA) trnsitions would hve been significntly underestimted by m. Figure 1 Influence of flnking mononucleotides on bis-corrected reltive single-bse-pir substitution rtes. Ech circle represents one of 12 possible substitutions. The verticl xis mesures the importnce of flnking position for the respective substitution rte in terms of vector-bsed prmeter ( Distnce from Centroid ; see the Mteril nd Methods section). Substitutions with significnt neighboring-nucleotide effect re denoted by the blckened circles. A Strnd Difference in Single-Bse-Pir Substitution Rtes Inspection of the reltive rtes of CGr(TG,CA) trnsitions s estimted conditionl on either the upstrem or downstrem nucleotide, respectively (tble 3), suggests tht methyltion-medited demintion of CpG dinucleotides is significntly bised by the 5 flnking nucleotide on the noncoding DNA strnd (tble 3, rows 1 4) but not on the coding strnd (tble 3, rows 5 8). By contrst, the nucleotide immeditely downstrem of CpG ppers to be significnt for CGrTG trnsitions, irrespective of the DNA strnd involved. Here, 3 denine residues re ssocited with the highest reltive substitution rtes (tble 3, rows 9 12 nd 13 16). Also included in tble 3 (rows nd 20 22) re reltive CNGr(TNG,CNA) trnsition rtes, llowing us to ddress the question s to whether cytosine methyltion nd consequent high-frequency demintion might lso Tble 2 Reltive Single-Bse-Pir Substitution Rtes in Humn Nucler Genes Cusing Inherited Disese ORIGINAL NUCLEOTIDE RELATIVE SUBSTITUTION RATE SD T C A G T ) C ) A ) G ) Bsed on HGMD dt nd corrected for confounding effects s described in the Appendix. The estimtes re unitless nd hve been scled so tht their verge, tken over ll 12 substitution types, is unity.

6 Krwczk et l.: Nucleotide Substitution Rtes 479 c Only for N AN / T ( P.105) could strnd dif- ference in reltive substitution rte possibly be ruled out. At first sight, the higher rte for CGrCA thn for CGrTG might pper to be inconsistent with the dt presented in tble 1 (rtio of bsolute frequencies 735: 940, or.78). This discrepncy is, however, explicble in terms of CGrTG trnsitions being more likely to disrupt protein function thn re CGrCA trnsitions (CGArTGA trnsitions crete termintion codons, wheres no CGrCA trnsition yields nonsense muttion). Figure 2 Bis-corrected versus uncorrected estimtes of reltive single-bse-pir substitution rtes, conditionl on 5 nd 3 flnking mononucleotides. x muttion type; m(x) corrected estimte of reltive substitution rte, derived s described in the Appendix; nd m (x) uncorrected estimte, bsed solely on the observed frequency of x. Pirs for which Fm(x) m (x)f is 13.5 SD of m(x) re denoted by crosses (#). occur t these motifs. Although the reltive rtes of CNGrTNG nd CNGrCNA trnsition re not substntilly higher thn the verge (i.e., unity) when N G is excluded, flnking-nucleotide effect nevertheless is noted: the dt re consistent with CNG undergoing trnsition to TNG, on both strnds, t 50% higher rte when N A thn when N T or N C. Depicted in figure 3 re reltive single-bse-pir substitution rtes tking into ccount the single nucleotides immeditely flnking, on its 5 nd 3 sides, the mutted bse. Ech dt point represents pir of muttions, with one substitution being the complementry homologue of the other (e.g., GCArGTA vs. TGCrTAC). Were substitution rtes identicl on both DNA strnds, then the dt points should pproximte the 45 line. However, 10 significnt outliers were identified (fig. 3), nd these re listed in tble 4. Purines nd pyrimidines were involved in five cses ech, with G nd T consistently showing rtes of substitution higher thn those for C nd A. Interestingly, one substitution pir involves CGr(TG,CA) trnsitions, nd the GrA rte ws found to exceed tht of CrT. The sme reltionship holds for the other three CGNrCAN/N c CGrN c TG pirs (where N c denotes the bse complementry to N), lthough the corresponding rte differences filed to ttin sttisticl significnce once multiple testing hd been llowed for. Nevertheless, individul error probbilities were.001 for c c N TN / A nd.002 for N GN / C, indictive of similrly strong bis for these substitution pirs. Reltive Clinicl Observtion Likelihoods In the process of computing reltive single-bse-pir substitution rtes conditionl on both the 5 nd 3 flnking nucleotides, we lso estimted the reltive clinicl observtion likelihoods of different mino cid replcements s clssified by the chemicl difference between the respective wild-type nd mutnt residues. A stedy increse in clinicl observtion likelihood with incresing chemicl difference ws pprent (fig. 4). Moreover, nonsense muttions were found to be more thn twice Tble 3 Neighboring-Nucleotide Effects on Reltive CGr(TG,CA) nd CNGr(TNG,CNA) Trnsition Rtes Substitution Reltive Substitution Rte SD CGTrCAT CGCrCAC CGArCAA CGGrCAG ACGrATG GCGrGTG TCGrTTG CCGrCTG TCGrTCA CCGrCCA ACGrACA GCGrGCA CGArTGA CGGrTGG CGTrTGT CGCrTGC CTGrCTA CAGrCAA CGGrCGA CAGrTAG CTGrTTG CCGrTCG Bsed on HGMD dt nd corrected for confounding effects s described in the Appendix. The estimtes re unitless nd hve been scled so tht they yield n verge of unity for the respective dinucleotide position; reltive rtes therefore cn be relted to one nother only within subcolumn.

7 480 Am. J. Hum. Genet. 63: , 1998 codons but one (CTA) tht were ssocited with nonsense muttions lso were found to be (reltively) bundnt round mino cid replcements. However, only one codon (GGG) ws underrepresented in both dt sets. Thus, no evidence ws found for the existence of muttion-protective sequence motifs. The observed pucity of glycine residues round nonsense muttions my be suggestive of structurl/functionl constrints rther thn of muttion repression. Figure 3 Bis-corrected reltive single-bse-pir substitution rtes, conditionl on 5 nd 3 flnking mononucleotides. Ech circle represents pir of substitution rtes, m(x) nd m(x c ), with one substitution (x c ) being the complementry homologue of the other x. Pirs with significntly different nerest-neighbor dependent rte estimtes re denoted by crosses (#). s likely to come to clinicl ttention s the most extreme missense muttions (chemicl difference ) nd were three times more likely to come to clinicl ttention thn the verge mino cid chnge. Codon Usge round Substitution Sites An lterntive mens to study the importnce of the immedite DNA-sequence environment for single-bsepir mutgenesis would be to try to identify muttionssocited sequence motifs directly. At lest for missense muttions, however, this type of nlysis would hve been seriously hmpered by the difficulty in controlling for the selective effects of the mino cid sequence context. It therefore ws decided to confine the investigtion of missense muttions to surrounding synonymous codon usge tht is, by comprison of the reltive frequencies only for neighboring triplets encoding one nd the sme mino cid (tble 5). Nonsense muttions, on the other hnd, re, on verge, three times more likely to come to clinicl ttention thn re missense muttions (see bove), suggesting tht selection cting on the immedite mino cid sequence context of such lesions is less likely to confound their observed spectrum. Generl codon usge therefore ws nlyzed for significnt fetures round sites of nonsense muttion (tble 6). A totl of 15 codons were found to be significntly over- or underrepresented in the vicinity (i.e., four mino cid residues upstrem nd downstrem) of missense muttions. Intriguingly, considerble overlp with the results obtined for nonsense muttions ws noted: ll A Role for Slippge-Medited Misincorportion? Kunkel (1985) proposed model tht sought to explin through trnsient mislignment of the primer templte, cused by looping out of single templte bse ( mislignment mutgenesis ) nucleotide misincorportion during DNA repliction. If this mechnism of slippge-medited misincorportion were to ply n importnt role in the genertion of single-bse-pir substitutions in humn genes, then substntil proportion of muttions in the HGMD dt set should exhibit identity between the newly introduced bse nd one of the bses immeditely flnking the site of muttion. The observed nd expected frequencies presented in tble 7 show tht this is indeed the cse, but only t certin codon positions. Muttion towrd the 5 flnking nucleotide hs occurred significntly more often thn expected t the second position of the codon but not t the first or lst position; muttion towrd the 3 flnking bse is fvored t the first position of codon but is disfvored t the second position. These findings suggest muttion mechnism, t position 1 nd position 2 in the codon (both of which re criticl in the specifiction of the encoded mino cid residue), tht is bised towrd Tble 4 Strnd Difference in Reltive Single-Bse-Pir Substitution Rtes Originl Substitution (Reltive Substitution Rte SD ) Wtson Crick Homologue (Reltive Substitution Rte SD ) GGTrGTT ( ) ACCrAAC ( ) TGGrTAG ( ) CCArCTA ( ) CGGrCAG ( ) CCGrCTG ( ) CTTrCCT ( ) AAGrAGG ( ) CTCrCCC ( ) GAGrGGG ( ) TGCrTCC ( ) GCArGGA ( ) CTGrCCG ( ) CAGrCGG ( ) GGTrGAT ( ) ACCrATC ( ) CTGrCAG ( ) CAGrCTG ( ) CTTrCGT ( ) AAGrACG ( ) Bsed on HGMD dt nd corrected for confounding effects, s described in the Appendix. The estimtes re unitless nd hve been scled so tht their verge, tken over ll 192 substitution types, is unity. Only substitutions for which one reltive-rte estimte ws consistently lrger thn its counterprt in 19,995/10,000 bootstrp simultions re included.

8 Krwczk et l.: Nucleotide Substitution Rtes 481 such q ( n 2 or 3). Both thermodynmic prmeters of triplet show significnt positive correltion with m v (fig. 5) when they re tested by mens of Spermn rnk correltion coefficient (DH 0.23, P!.05; DG 0.35, P!.005). This implies tht ny increse in stbility of the immedite DNA-sequence environment increses the likelihood tht given nucleotide will undergo substitution. The fct tht this correltion is stronger for DG thn for DH( DGt0 K) is indictive of temperturedependent reltionship. Discussion Figure 4 Reltive clinicl observtion likelihood of mino cid substitutions, s function of chemicl difference between substituting nd wild-type residues. SDs of estimtes determined by bootstrpping (10,000 simultions) re demrcted by verticl brs. the nucleotide t the other position. Inspection of the genetic code revels tht such bis invribly serves to void the de novo introduction of termintion codons. Mutbility nd Thermodynmic Stbility The stbility nd melting behvior of DNA duplex depends criticlly on its primry nucleotide sequence. More specificlly, Bresluer et l. (1986) demonstrted tht, thermodynmiclly, DNA duplex structures cn be considered s the sum of their nerest-neighbor pirwise interctions. From the clorimetric study of 19 DNA oligomers nd 9 DNA polymers, Bresluer et l. (1986) compiled tbles of the trnsition enthlpy chnges (DH) nd the trnsition free-energy chnges (DG) ssocited with disruption, t 1 M NCl, 37 C, nd ph 7.0, of ny of the 10 possible Wtson-Crick nerest-neighbor interctions. The sum of these pirwise vlues, tken over the primry DNA sequence, ws found to correlte strongly with the experimentlly observed DH nd DG of 12 DNA oligomers. In order to ssess whether the nerest-neighbor influence on single-bse-pir mutgenesis is ssocited with thermodynmic DNA stbility, we ttempted to relte DH nd DG of given triplet, q q 1q0q 1, to the verge reltive muttion rte t the centrl nucleotide 1 of the triplet, q 0,m v(q) n 7 q m(q 1q0q 1 r q 1qq 1). Here, summtion is over ll q ( q0, excluding CGr(TG,CA) trnsitions, nd n is the number of Single-bse-pir substitutions cusing humn genetic disese my rise vi number of different endogenous mechnisms. Perhps the most importnt nd best understood is the demintion of 5-methylcytosine, the most bundnt chemiclly modified bse in vertebrte genomes nd one tht is confined lmost exclusively to CpG dinucleotides (Cooper nd Krwczk 1993). The high rte of 5mC demintion nd consequent replcement by thymine (Shen et l. 1994) renders the CpG dinucleotide hot spot for germ-line muttion in vertebrte genomes. Another mutgenesis model often invoked to ccount mechnisticlly for germ-line nucleotide substitutions in humns involves the misincorportion of noncomplementry nucleotides s consequence of trnsient templte-primer mislignment ( mislignment mutgenesis ; Kunkel 1990, 1992). Mislignment is thought to be medited by short direct or inverted sequence repets in the immedite vicinity of the lesions. In somtic tissues, 5mC demintion lso ppers to be n importnt mechnism of single-bsepir substitution (Hollstein et l. 1991; Tornletti nd Pfeifer 1995). Indeed, the reltive rte of mitotic cncerssocited CGr(TG,CA) trnsitions observed in the TP53 gene, the most widely mutted gene in humn tumorigenesis, is very similr to the overll germ-line rte observed in other humn genes (Krwczk et l. 1995). Some somtic muttions my occur, teleologiclly speking, by design for exmple, the vrious preprogrmmed hot spots for single-bse-pir mutgenesis in the vrible (V) regions of the mmmlin immunoglobulin genes. Their hypermutbility, potentiting the genertion of diversity in the immune response, ppers to be trgeted minly towrd short RGYW ( R A/G; Y T/C; nd W A/T) nd TAA motifs (Rogozin nd Kolchnov 1992). In vitro dt further suggest the concomitnt existence, in V regions, of muttionl cold-spot motifs (e.g., TAGA; Lin et l. 1997). Wht ll the bove mechnisms hve in common is tht the muttionl bis involved is DNA-sequence dependent. However, since the vilble evidence for sequence-dependent muttionl bis either hs been bsed on in vitro dt or hs relted only to smll number

9 482 Am. J. Hum. Genet. 63: , 1998 Tble 5 Codons Significntly Over-/Underrepresented mong 4 Amino Acid Residues Surrounding Missense Muttions CODON CGr(TG,CA) EXCLUDED ( N 4,318) CGr(TG,CA) INCLUDED ( N 5,556) No. of Codons Observed Over-/Underrepresenttion No. of Codons (%) x 2 Observed Over-/Underrepresenttion (%) x 2 CGA GGT CCT GGT CTT b GAA , b ACT b AAT b AAC b CTG 1, , b GAG 1, , b GTC GCG b GGG CCG Compred with mbiguous codon usge in humn genes (Nkmur et l. 1996). b Vlue is not significnt. of motifs or genes (see bove), it is uncler whether there could be specific DNA sequence motifs tht re frequently or even invribly ssocited with single-bsepir substitutions in humn genes, cusing inherited disese. With the estblishment of HGMD, it becme possible to nswer this question by utiliztion of lrge number of known nucleotide substitutions in wide vriety of different genes. In terms of their reltive frequency of occurrence, the most importnt ctegory of single-bse-pir substitution in HGMD is represented by CrT nd GrA trnsitions within CpG dinucleotides; some 23% of ll single-bsepir substitutions found within the coding regions of humn genes re of this type. When, through considertion of reltive mutbilities, llownce is mde for the confounding effects of codon usge nd differentil clinicl observtion likelihoods, this proportion trnsltes into men trnsition rte, for either CGrTG or CGrCA, tht is five times higher thn the bse muttion rte. This represents considerble downwrd djustment of our erlier estimte of 7.4, which ws derived from ninefold-smller smple (Cooper nd Krwczk 1993). It reflects the disppernce of n initil reporting bis in the humn moleculr-genetics literture, bis tht most likely ws due to the limited number of muttion-detection techniques vilble during the 1980s nd erly 1990s. Before 1993, 31% of ll published single-bse-pir substitutions in humn genes were CGr(TG,CA). This vlue dropped to some 24% in , before reching its current level of 21%. Intriguingly, the proportion of CGr(TG,CA) trnsitions is significntly higher for utosoml genes (25.0%) thn for X-linked genes (17.7%), finding tht directly reflects the significntly lower frequency of CpG in the coding sequences of X-linked genes (2.9%), compred with tht in utosoml genes (3.7%). The lower CpG frequency in X-chromosoml genes my itself be consequence of generlly incresed level of DNA methyltion, n increse tht results from the recruitment of this postsynthetic modifiction to ply role in X inctivtion (Hornstr nd Yng 1994; Jmieson et l. 1996). For CpG dinucleotides to be hypermutble in the context of genetic disese, they must be methylted in the germ line. Since it cnnot be excluded tht the efficiency of both DNA methyltrnsferse ction (Bolden et l. 1985; Smith 1994; Smith nd Bker 1997) nd G:T mismtch repir (Sibght-Ullh nd Dy 1993) re influenced by sequence motifs flnking the CpG dinucleotide, the question rises s to whether, by virtue of their DNA sequence context, some CpG sites my be intrinsiclly more mutble thn others. Significnt differences in the reltive muttion rte of CpG dinucleotides, depending on flnking nucleotides, indeed were noted in the present study (tble 3). These results re consistent with those of Ollil et l. (1996), who noted preference for 5 pyrimidines nd 3 purines flnking mutted CpG dinucleotides, lbeit in much smller dt set derived from publicly vilble locus-specific muttion dtbses. Our findings might, however, pper to conflict with those of Cly et l. (1995), who reported tendency towrd 5 Cnd3 G in CpG-poor, noncoding regions

10 Krwczk et l.: Nucleotide Substitution Rtes 483 Tble 6 Codons Significntly Over-/Underrepresented mong 4 Amino Acid Residues Surrounding Nonsense Muttions CODON CGr(TG,CA) EXCLUDED ( N 998) CGr(TG,CA) INCLUDED ( N 1,328) No. of Codons Observed Over-/Underrepresenttion No. of Codons (%) x 2 Observed Over-/Underrepresenttion (%) x 2 CGA b CTA b ACT CTT b GTT b GAA GGA b GGG b GGT b b Compred with 448 reference (humn) cdna sequences. Vlue is not significnt. of vertebrte DNA, indictive of lower muttion rte of CCGG. Such reltionship is not immeditely pprent from the HGMD dt. However, even if protective effect such s tht suggested by Cly et l. (1995) were exclusively confined to prticulr CpG-contining oligomer, nothing would be known regrding its overll efficiency in coding regions. Were this efficiency low, other nerest-neighbor effects might esily hve obscured its influence on the observed muttionl spectrum in humn genes. An lterntive possibility is tht subset of methylted CpG dinucleotides might be more prone to demintion, perhps by virtue of their occurrence in low-melting-temperture domin (the rte of 5mC demintion in single-strnded DNA is 28-fold higher thn tht in double-strnded DNA [Ehrlich et l. 1986]). To test this postulte, we ssessed the frequency of A nd T residues flnking CGArTGA muttions. Although A/ T richness ws found to be significntly incresed in the vicinity of such lesions, compred with tht in control DNA sequences (50.4% vs. 47.6%), this elevtion ws not deemed to be strong enough to suggest ny prticulr propensity of the mutted regions to become trnsiently single strnded. 5mC is known to occur t low frequency in non- CpG dinucleotides within triplets of the form CpNpG (Woodcock et l. 1988; Clrk et l. 1995; Ky et l. 1997), nd muttions t these sites hve been reported in the NF1 gene nd the BRCA1 gene (Rodenhiser et l. 1996, 1997). This could imply tht methyltionmedited CrT nd GrA trnsitions occur t CpNpG triplets in humn genes. However, since the reltive rtes of CpNpGrTpNpG ( N ( G) trnsition nd CpNpGrCpNpA ( N ( C) trnsition tht hve been derived from the present study re not substntilly higher thn the verge substitution rte conditionl on the next-but-one nucleotide, we my conclude tht methyltion-medited demintion t such triplets hs not contributed significntly to the muttionl spectrum observed in HGMD. Some studies hve proposed strnd bis in either CpG demintion frequency or T:G mismtch repir, with CrT trnsitions outnumbering GrA trnsitions (Skndlis et l. 1994; Leder et l. 1995). In terms of their observed frequencies, such reltionship lso holds for the collection of single-bse-pir substitutions tht hve been nlyzed in the present study (tble 1). However, none of the previous studies llowed either for codon usge or for the mgnitude of mino cid exchnge, so tht the skewed trnsition frequencies observed probbly reflected observtionl bis rther thn intrinsic differences in muttion rte. When reltive substitution rtes re considered, CGrCA is estimted to occur t probbility tht is 1.4-fold higher thn tht for CGrTG (tble 3). Although the sme substitution types pper to be subject to next neighbor-effects on the coding nd noncoding DNA strnds, the quntittive differences in non- CpG single-bse-pir substitution rtes observed here confirm tht the two DNA strnds re not fully equivlent in terms of their rtes nd ptterns of muttion (Wu nd Med 1987). There re severl possible (nd non mutully exclusive) resons for this strnd symmetry. First, the four nucler DNA polymerses, ech ssocited with its own distinctive muttionl spectrum, my be differentilly involved in the synthesis of the leding nd lgging strnds during DNA repliction (Kunkel 1992; Bmbr et l. 1997). Second, since the trnscriptionl elongtion complex is symmetricl (Kinz nd Roberts 1992), muttion rtes my differ between trnscribed nd nontrnscribed strnds, on ccount of either unequl exposure to DNA dmge or differentil repir. Not only my the trnsiently single-

11 484 Am. J. Hum. Genet. 63: , 1998 strnded nontrnscribed DNA strnd be prticulrly vulnerble to muttion (e.g., by methyltion-medited demintion; Beletskii nd Bhgwt 1996), but trnscription-coupled repir (Hnwlt 1994; Drpkin et l. 1994; Bhti et l. 1996), process tht corrects lesions specificlly on the trnscribed DNA strnd, lso could ccount for muttion-rte differences between trnscribed nd nontrnscribed strnds. Both these mechnisms would predict higher muttion rte for the nontrnscribed s opposed to the trnscribed DNA strnd. This hypothesis is, however, impossible to test on the bsis of muttion dt logged in HGMD. Serches for sequence motifs potentilly ssocited with muttionl hot spots were lrgely unsuccessful. This includes polypyrimidine runs (x5 bp) nd the deletion hot spot consensus sequence (TGRRKR), two motifs previously found to be strongly ssocited with the occurrence of microdeletions (X20 bp) in humn genes (Cooper nd Krwczk 1993). This notwithstnding, two codons (GAA nd ACT) were found to be overrepresented in the vicinity of both missense nd nonsense muttions other thn CGr(TG,CA) wheres GGG ws underrepresented. The biologicl mening of these ssocitions, however, which point towrd muttionl effect rther thn towrd observtionl bis, remins unknown. No prepondernce of direct or inverted repets ws noted in the vicinity of muttions t non-cpg dinucleotides. There is thus no evidence tht mislignment, medited by repetitive sequences surrounding substitution site, contributes substntilly to the observed muttionl spectrum in humn genes. However, subtle neighboring-nucleotide effect reminiscent of mislignment-mutgenesis models nevertheless ws noted; substntil proportion of observed single-bse-pir substitutions exhibited identity between the newly introduced bse nd one of the bses immeditely flnking the site of muttion. Since this effect occurred only t distnce Figure 5 Thermodynmic stbility nd reltive mutbility of dsdna triplets. m v (q) bis-corrected verge reltive substitution rte t the centrl nucleotide of triplet q; DH(q) chnge in trnsition enthlpy (blckened squres) ssocited with the disruption of q; nd DG(q) chnge in trnsition-free energy t 37 C(unblckened squres). of 1 bp nd in the bsence of surrounding repet sequences, it is potentilly explicble in terms of mislignment mutgenesis involving highly loclized DNA slippge, misincorportion, nd relignment events, t the repliction fork, between templte nd primer (Kunkel 1990, 1992). Intriguingly, the muttionl bis towrd next neighbors occurred only t specific codon positions nd exhibited directionlity. Since such phenomenon is unlikely to be explicble in terms of the primry muttionl event, it is probbly ssocited with the DNA-repir process. Implicit in this ssumption, however, is tht the DNA-repir mchinery is ble to recognize the reding Tble 7 Possible Role for Slippge-Medited Misincorportion SUBSTITUTIONS b 5 Flnking 3 Flnking POSITION N F E(F) x 2 N F E(F) x 2 1 1, , c 2 1, c 2, c Overll 4,114 1,374 1, d 4,155 1,266 1, Of muttion in codon. b N number of substitutions, other thn CGr(TG,CA), for which the mutted nucleotide differs from the flnking nucleotide; F number of substitutions towrd flnking nucleotide; nd E(F) expecttion of F, under model of rndom muttion (for detils, see text). c Vlue is significnt (, or 3 P!.05/ # 10 ). d Vlue is significnt ( P!.05/2, or.025).

12 Krwczk et l.: Nucleotide Substitution Rtes 485 frme nd to utilize this informtion s cue in effecting the repir of DNA. Consistent with such reltionship, the observed correction bis would operte in such wy s to remove newly introduced termintion codons. The bility of the DNA-repir mechnism to tke the reding frme into ccount, thereby minimizing the effects of muttion, would hve hd positive selective vlue becuse of the reltively deleterious nture of infrme termintion codons. Evidence for reding-frme sensitivity in the DNA-repir process hs come from our previous observtion tht reltive single-bse-pir substitution rtes re bised towrd the voidnce of those replcements tht (i) chnge the chemicl chrcteristics of the encoded mino cid residue substntilly nd (ii) hve high likelihood of coming to clinicl ttention (Krwczk nd Cooper 1996). We concluded from this finding tht, by considertion of the genetic code, selection hd optimized the DNA repir mechnism in such wy s to void the most hzrdous of mino cid replcements, ctegory tht certinly would include nonsense muttions. The thermodynmic stbility of DNA triplets ws found to be positively correlted with the verge reltive rte t which the centrl nucleotide of triplet undergoes substitution, finding tht implies tht higher rther thn lower DNA duplex stbility renders gene region more prone to single-bse-pir substitution. Consistently, the bsence of flnking repet elements noted for the muttions nlyzed in the present study suggests tht extensive strnd slippge (which would require the DNA to be single strnded) is unlikely to ply n importnt role in the genertion of single-bse-pir substitutions. A high degree of thermodynmic stbility could, in principle, impir DNA repliction, in vrious wys. First, the likelihood tht DNA helicses would be incpble of unwinding the two DNA strnds correctly or efficiently my be expected to be higher in regions tht re more stble (Chen et l. 1992). Second, temporry renneling of the two ntive DNA strnds during repliction might be fvored nd could be more enduring in such regions. In both cses, DNA polymerse ctivity would be seriously impeded by loclized doublestrnded DNA structures, which could result in either the cesstion of polymeriztion or the skipping of one or more nucleotides, leving gp in the nscent DNA strnd. Miscorrection during the postreplictive repir of such nicks would then introduce single-bse-pir substitution. Alterntively, the observed correltion could reflect the incresed stbility of t lest some slippge-medited mislignments during repliction of the ntive nd nscent DNA strnds, llowing enough time for misincorportion of noncomplementry nucleotide. In this cse, however, the thermodynmic stbilities of the misligned structures must be comprble to those of the wild-type triplets, n ssumption for which there is currently no evidence. Finlly, since mutgenesis depends not only on polymerse misincorportion but lso on 3 exonucleolytic proofreding, the correltion observed between triplet stbility nd mutgenicity cn be interpreted lso s n effect of surrounding bse-pir stbility inhibiting proofreding (Petrusk nd Goodmn 1985). The clinicl observtion likelihood of n mino cid substitution is positively correlted with the chemicl difference between the respective wild-type nd mutnt mino cid residue, the highest chnce of coming to clinicl ttention being observed for nonsense muttions. A prmeter closely relted to chemicl difference hs been employed recently, by Rodin et l. (1998), to mesure the selective effects of mino cid replcements in the somtic, germ-line, nd evolutionry spectr of p53 muttions. On the bsis of the implicit ssumption tht their prmeter ws positively correlted with clinicl severity, Rodin et l. concluded tht, owing to the structure of the genetic code, the likelihood of clinicl observtion should be much higher for CGrTG trnsitions thn for CGrCA trnsitions. Indeed, the rtio of the two trnsition types turned out to be good indictor of the selective pressure ginst loss-of-function muttions in criticl regions of the p53 protein. Thus, the findings of Rodin et l. (1998) not only justify the use of chemicl difference in the present context but lso emphsize tht cliniclly observed spectr of single-bsepir substitutions re likely to be bised by substitutiondependent clinicl observtion likelihoods. In summry, our nlysis of the lrgest vilble collection of germ-line single-bse-pir substitutions in humn genes hs, for the first time, llowed us to disentngle the reltive effects tht selection nd muttion hve on the muttionl spectrum observed in humn inherited diseses. It hs been shown tht the reltive rtes t which such lesions occur t the DNA level is significntly influenced by the surrounding sequence context. However, with regrd to the existence of sequence motifs tht might serve to promote mutgenesis, only subtle nd very loclized effects were noted over nd bove the well-estblished hypermutbility of CpG dinucleotides. It will be most interesting to determine whether these effects, mnifesting here in the form of nerest-neighbor dependent substitution rtes nd codon usge round muttionl sites, lso occur in noncoding DNA nd in DNA from other species or cn be reproduced in vitro. Similrly, our hypothesis of both reding-frme sensitive DNA-repir bis nd reltionship between DNA duplex stbility nd mutbility will require further independent verifiction by, for exmple, comprtive evolutionry or in vitro studies. It is nevertheless hoped tht our tenttive conclusions my serve to guide the design of future experiments iming to iden-