THE JOURNAL OF I NVESTIGATI VE DERMATOLOGY Vol. 60, No. G Copy rig h t@ 1973 by The Will iams & Wilkins Co. Printed in U.S.A.

MOLECULAR ASPECTS OF GENETIC RECOMBINATION AND THEIR RELEVANCE TO CUTANEOUS BIOLOGY* D. MARTIN CARTER, M.D., PH.D. t

INTRODUCTION spaced. In " fin e-structure" mapping, recom bina­ Through ge netic recombination, a fundam ental tiO n fr equency is considered a fu nction of t he biologic process t hat reassorts linked ue netic distance between recombining markers (Benze r, markers, offspring can ex press ge netic traits t hat 1961 ). Genetic maps actuall y re present t he linear were not present together in either parent. Ind i­ structure of t he DNA molec ule. T he agreement vidual progeny may be mult ice llul ar orua nisms between ge netic maps a nd t he physical distance single cell s, bacteria, viruses, or molebc ul es oi· along t he DNA mo lecul e is ve ry high in bacteria DNA. To medical biologists, recombination is and bacteriophage (Hogness et al. , 1966). At t he usually identifi ed with the process of crossin rr-ove r present t ime, recombination data from hi rr her in w hic h genetic material i s exchanged b etween organisms are much less helpful in definin ub t he homologous chromosomes durinu meiosis. Genetic process in molecul ar terms. T his may r e fl ect~ less recombination is now f elt to bhave even wid er precise knowledge of eukaryotic chromosome significa n ce, having been implicated in such di­ structure regarding t he li near red upl ication of verse biologic phenomena as the circul ari zation of genes and whether all DNA is fun ctional (Call an, the b acteri al chromosome before repli cation t he 1967; T homas, 1970). It wo uld be naiv e to assume insertion of t he DNA of temperate viruses' in to t hat all organisms perform recombination in an host D A , DNA-mediated b acteri al transforma­ identical m a nner, a nd distinctions between organ­ tion , and as t he ge neral m eans by whi ch ue netic Isms have a lready been delineated. However it var iability is generated (see reviews by Bodmer is ge nerally assumed that t he mechanism f or 'all and Darlington, 1969; Davern , 1971). recombination events, in all species, preserves many co mmon features. Whatever m odel is de­ GENE FUNCTION IN RECOMBINATION: A REFLECTION vised f or r eco mbination, it must accoun t for at OF DNA STRUCTURE least t':"o characteristic features of t he process: synapsts, the a li gnment or pai ring of homologous U nderstanding t he organi zation of t he DNA chromosome segments; and exchange, t he recom­ molecule, we can co rre late rather precisely t h e bining o f information h eld in DNA of t he homo­ chemical structure of the materi al storinu ue netic logs . information with some of t he ge netic e~e~1ts af­ fecting t he ex pression of t his information . BREAKING AN D HEJOINI NG VERSUS COPY CHOICE Chromosomes, which co ntain ge nes in a linear For many years, two array, are composed primarily o f D NA. The well ­ classic t heori es for recom­ b~nati o n vied for acceptance . Breaking known dou ble heli x can be depicted in a number and joining of homologous molec of ways (Fig. 1). Repeating spirals can s imulate ul es. a t heo ry origin all y pro­ posed b y J anssens ( the t hree-dimensiona l structure o f t he ri gid DNA 1909), was proposed to explain cytologic observations of m molec ule (la), or the molecule can be opened-out eiotic chi asmata. J ans­ sens suggested t hat chr diagrammaticall y, to reveal t he covalently linkecl omoso mal coiling produced sufficient tension to suga r-phos phate " backbone" of each strand and break chromatids and that homologs rejoined to the h ydrogen-bonded pairs of nucleotides holdinu produce reciprocally recom­ binan t proge the t wo strands together ( 1c). Further abbrev i a~ ny. J anssens' model could not ade­ quately account for t tion can reduce a diagram of t he DNA molecul e to he pa iring of DNA molecules; but even more t roubleso me, since two lines with cross-li nks (1b). Each of t hese chromosome s ize is constant, was diagrams s hows t hat t h"e intact molec ul e co ntains its require ment t hat a me­ chani cal process should two complementary polynucleotide c hains of op­ produce breaking o f ho­ mologous chromatids at prec isely t h posite pol a rity held together b y hydrogen b onds. e same in ter­ nucleot id e sites. _Ge net ic. ma ps _have been constructed f or many Another model, known as copy microrganisms from studies of t he freq uency choice was ori gin all y s uggested b y Belling (1931). He !'inked with which r ecombinant progeny occur in crosses recombination wi t h chromosome repli catio between parents with all eli c markers at adjacent n so that only the in fo rmation of both parents, but not or far-spaced sites on t he c hromosome. There is t he actual parental D NA , would be linked in the less crossing over between mutations located close DNA of recombinant pr togeth er t han b etween t hose more distantly ogeny. T he model suggests that an enzyme, DNA polymerase, cha nges pa­ This work was s upported in part by USP HS Grant. rental template when synthesizin g re co mbina nt ·a. ROl C A 12496-01. molecul es. T he major difference between these . • From t h e D epartment or Derm atology, Ya le Uni ve r­ stty School of Med1c1ne, New Have n, Co nn ecticut . two models is t he importance attached to DNA . t Howard Hughes Medi cal Investigator, Yale Uni ve r­ repli cation: t he co py c hoice t heo ry is completely Si ty. dependent upon DNA replication wh ereas t he 369 370 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

(a) 3' 5' (b) 5' 3' cha ins; (2) pairing homologous segmen ts; (3) repairing t he recombina nt molecule by trimming excess DNA a nd fill ing in missing sequen ces; a nd (4) closing t he polynucleotide chain (Table I ). The a bove should be viewed as t he sine qua non of recombination but not necessarily a ll t he steps fo r whi ch every conceivabl e molecular m odel must m ake provision . 3' 5' ® 5' Enzymes for Breahing and J oining le i ® ,--(" (d) p~ :: ~® The growin g number of enzymes from variou biologic systems t hat are capable of acting upon ®Y)----@ : : c ,-{"" r .. ~® different kinds of DNA s ubstrates (Richardson . 1969) provides t he tools with which molecula r ®~ :: tB-C(® model building can proceed. Acting under their specified condit ions upon the backbone of t he ,---{" ®~c ::~-· ® DNA molecul e, the various nucleases, polymer­ 5' ® ases, a nd li gases can break or rejoin the suga r­ 3' 5' phosphate linkages between adjacent nucleotides FIG. l. Diagramatic representations o f the double (Fig. 2) . Table I shows how t hese enzymic acti\'i­ helic al structure of DNA . A = adenine, T = thymine, G ties might contribute to recombination b y brea k­ = guanine, C = cytosin e; 5' and 3' refer to the polarity of ing a nd rejoining. Figure 3 represents the kind of the suga r-ph os phate backbone. theorizing engaged in by a number of con tempo­ rary biologists (e.g., Thomas, 1966; Meselson. breaking a nd joining theory is not. M eselson and 1967; a nd Whitehouse, 1968). others using de nsity gradient techniques demon­ The sorts of enzymi c properties required for strated t h at recombinant progeny of phage recombination b y breaking a nd rejoining mig h t be la mbda can be composed almost ent irely of pa­ expected to participate in other biologic processes ren tal materia l (Meselson and Weigle, 1961; Kel­ affecting D NA. Best known of these are DNA lenberger et aL, 1961) and clearl y establi shed that re plication and t he repair of injuries to DNA recombina nt progeny DN A m olecules can be pro­ induced by ult raviolet light . Among t he fi rst duced by t he breaking and rejoining of parental recom bination-defective mutants iden tified for E. D N A. S ubsequent studies of phage lambda co li were those fr om a g roup of organisms excep­ showed that extens ive new DNA synthesis can tionally sensit ive to irradia tion by ul traviolet light also accompa ny breaking a nd rejoining in recom­ (Howa rd -Fla nders a nd Theriot, 1966). Among t he bination (Stahl a nd Stahl, 197 1). few enzymes known to be required for recombina­ t ion are those listed in Table II. These have b een THE H YBRID OVERLAP AND SYNAPSIS id ent ifi ed from careful genetic a n d biochemical Breaking a nd joining of DNA can t hus explain evaluation of recombination defective mutant m t he exchange step in recombination, but what several microorganis ms . m echanism has sufficient precision to keep the m olecular size of progeny molecules constant? The Reco mbination Enzym es of Phage Lambda What permits the prec ise pairing of DNA in The clearest data establis hing a certain enzymic syn a psis before excha nge? Double-stranded DNA activity with recombination h ave been gath ered molecules would not be expected to ali gn chemi­ for phage la mbda . The structural genes specifying cally, but t he pairing o f comple men tary, s ingle la mbda exonuclease a nd beta protein m ap di­ strands of portions of homologous molecul es might rectly in the recombination (rec) loci on t he phage offer t he precision required for promoting recom­ chromosome (Shulman et al. , 1970). The biologic bination events t hat produce progeny o f uniform properties o!' beta protein re ma in unknown. T he size. S upport ing t his expectation is t he c he mical a nd genetic id entification of a heterozygous, bi­ TABLE I parental area called the hybrid overlap region in Possible s teps in genetic reco mbination the progeny of several species of microbial recom­ bination events (see Bodmer a nd Darlington, Step Enzyme 1969). a. breakage end onuclease STEPS IN RECOMBINATION b. pai rin g exonuclease c. repairing Reduced to its minimal elements, recombina­ 1) trimming exo nu clease or endo- tion by breakage and rejoining requires at least 4 nuclease steps for the incorporation of n ew information and 2) filling polymerase the restoration of the recombina nt molecul e o f d. closing li gase DNA: ( 1) breaking t he parental polynucleotide GENETIC RECOMBINATION 371

(a) Endonuclease one at a t ime (Carter and Radding, 197 1). The enzyme binds to but cannot hydrolyze DN A at \_ in tern al in terrupt ions (ni cks o r gaps) in D A • (Radding a nd Carter, 197 1). These propert ies led I I I I I I I - ll II II to t he proposal of a model for t he role o f la mbda exonuclease in ge netic recombination that seems (b) Exonuclease to have been co nfirmed (Cassuto and Radding, -...... 1971) . The importance of t he work is t hat it approximates reco mbination in vitro a nd shows • I I II I I I - IIIII t he a bili ty of lambda exonuclease to perform two tasks in recombination (Fig. 4). Since t he e nzy me (c) ON A Polymerase T ABLE II I. Enzymes clearly associated with genetic recombination I I - II IIIII Organism Enzy me (d) Polynucleotide ligase E. coli AT P -dependent nuclease Buttin and Wright ( 1968) Barbour and Clark ( 1970) n• Ill I I 1111111 Oishi (1969 ) - Phage T -4 Gene 32 binding protein F IG. 2. E nzymes that act upon D NA substrates to Alberts (1968) break a nd rejoin the s ugar-phosphate linkages of the Phage la mbda Exonu Backbo n e. e = enzyme. clease and beta protein S hulman et al. (1970) (a) 3' (b) ' , T"lllrrl ,.,1 I'TTII,..,II"Tll l-rriiTTI I-rrll 3 -rlll\\lrrrrr-llrTI TTII.-IIn-1 5 5' ..L.LLL..LO Exo nuc lease prepares fragme nts of DNA for 3 - 3· ~~ rejoininQ by base pairinQ : •• '11 1 Ill!I l [!!Till 5 5' ~ exo DNA . fraQment Ql!t!! l !il l l! i ltillil/ll! i li i lll i ll!l! l ll l l!l!l 3 ' - 3 ' (c) ;. ~ 11 111111111 Ill lllllllllllll ll lli l 1ii!illlill0 3' DNA fraQmen t exo s' ~a Qtlllllllllllllllllllil (e) 3' (f) 3' , rrrlllJrrn[[ [-rrr[]] 1([0J i lllli!l!!!!l!!lll!!ll!l!ii!!Q s' lJIIlllLJirn + 5

(g) 3' l (h) 3' • ,...II"'l llrnllrrlllrrrlI]J]JJ ! be s' J I I1 !II 1111111111 5 F IG. 3 . A hy pothetical general model for reco mbina­ II II II l tion by breaking a nd joining wi th some new DNA lllllllllllljj;j;j'j;i!lc)nrll •I I(~II II II II synth esis . ( a) Two homologo us DNA m olec ul es ; (b) 1 icks a re introduced at non -id entical n earby sites by endonuclease (0); (c) sin gle strands " unwind "' from l d each molecule and homologs pair as DNA polymerase (e ) acts at nicks; (d ) endonuclease (0) acts again at one a nd n i cks are re c oQnized as a siQna l for site on eac h p a rent producing one reco mbinant (e) and the enzyme to stop. two fragm e n ts t hat pair to produce an additional recom­ bi nan t (f). R epa ir with poly merase a nd li gase gives two intact r ecom binants (g) and (h). i l l ! I I! Ill! I II! illliiilli !l I I Qurrrrt Iiiii I tQttllll!il I

FIG. 4. A model for the role of la mbda e:transduction. defi cient in other functions t hat require t he e n­ S uch specul ation h as been corroborated b y the zymic propert ies used in recombinat ion . AL each recent resul ts of M erril and colleagues (1971). step in t he chain, there o ught to be patients wit h P atients wi t h galactosemia lack the tra nsferase genetic defects alt hough total l ack of some func­ enzyme required f or the production of UD P galac­ t ions might be in co mpatible wit h life. Cutaneous tose (K alckar et a l. , 1956) and in infancy d evelop biologists need to identify t hose circumstances m ajor nutri t ional p roblems sometimes resu lt ing in t hat require breaking and rejoining o f D NA and, death. M erri l and colleagues grew in culture when breaking o r j oining seem to be defective, to fibrobl asts from a patie nt wi t h galactosemia. see whether r ecombinational defects are a lso pres­ They also pre pa red suspensions of bacteriopha ae e nt. M aking use of the methodology developed for la mbda ca rrying E. coli ge nes for t he b acte r i~ microbial systems, we can a nt icipate s igni!'icant transferase enzyme. The huma n galactosemic fi­ progress in t his fi eld. brobl asts, deficient in tra nsferase, were exposed to a g reat excess of phage-carrying bacteria l t rans­ Repair of UV Injuries to DNA ferase genes, and evidence was found for stable A defect in t he repair of ul t raviolet-induced cha nges in t he properties of t he galactosemic cell . injury to D NA has been elegant ly defined f or the An a ppreciable a mount (0.2%) of t he R NA m ade he ri table d isease of photosensit ivity, xeroderma by t he cell s seemed to have been specified b y the p ig men tosu m (Cleaver, 1968) . Patie nts wi t h bacteri al virus genome. Even more tart ling was xeroderma pigmentosum appa rent ly lack t he nor­ t he presence of a near-norma l a mount of t ra nsfer­ m al degree of endonucleo lytic activity required for ase activity in the galactosemic fibroblasts after the excision of UV-induced p yrimidine dimers in exposure to the bacteri al virus. One can conclude DN A (C leaver, 1969). What is the relationship t hat bacteri al genetic information w as expressed between such a defect in t he repair of UV -induced by m ammali a n cell s. T wo questions a rise: (1) Had injury a nd a propens ity for m aligna nt disease? the bacteri al D NA been in corporated into m am­ Cells o f p atients wi t h xeroderma pigmentosum malia n D N A b y recombination, a nd if so was it cannot modify certain DNA viruses to which t hey in corporated into nuclear D N A or into mitochon­ have been ex posed (Aaronson a nd Lytle, 1970). drial or other episomal D NA? (2) In what kinds of Wit hout having a s pecifi c defect in t heir recombi­ clinical condit ions can we hope for a "gene ther­ national m achinery, perhaps t hese cells a re less apy" that would ut ilize recombinationa l event ? a ble to defend against t he a d verse cellula r effects CONCLUSIONS of viral or other foreign genomes int roduced into t heir DNA b y recom bin ation a nd a re t hus more Genetic recombination i s a general mean s of prone to express pot ent ially " maligna nt" informa­ cha ngin g t he order of linked genetic m arkers by tion. breaking a nd rejoining D NA. It is accomplis h ed t hrough t he action of enzymes t hat act upon and DNA Viruses Incorporating into and Excising re model the physical structure o f D N A. Because From Ce lls of its general b iologic importance, we a re n ot Likewise, viral t ransformation and t he induc­ startled to encounter r ecombination in cuta neou t ion of latent viruses require breaking and rejoin ­ events that require breaking and rejoining of in g. T he wa rt virus, a D N A virus, must become D NA. Alterted to the mecha nis ms involved, we stably in corporated into t he genome o f t he cells it hope to identify more s kin diseases result ing from infects if t he c lonal n ature o f the cutaneous war t, recombinational d efects. M ost speculatively , we a ri s in g from one t ransformed cell , is to be ex­ mi ght furt her h ope to devise procedures that would pl ained (Murray et al. , 1971) . T he release of t he attempt, by recombination, to correct defects in he rpes s implex virus from i ts latent state of the genetic information of cutaneous cells. residence in cutaneous cells likewi se requires a n exc is iona l event a nd uses nucleases of s imila r REFERENCES nature to t hose required in recom,b inatio n. T o Aaronso n, S. A., and Lytl e, C. D. (1970). Decreased host what other circumstan ces a re persons suscept ible ce ll reactivation of irradi ated SV 40 virus in GENETIC RECOMBINATION 373

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