Ad­her­ing To The 'Repli­con Model' The Sloppy Way

by Christoph

Sixty years ago Ja­cob, Bren­ner and Cuzin de­vised their 'Rep­licon Model', in­spir­ing and use­ful guide­line for repli­cation re­search ever since. Ac­cord­ing to the model, a 'Repli­con' is a ge­netic el­e­ment repli­cated from a sin­gle 'Repli­ca­tor' – repli­ca­tion ori­gin, in mod­ern terms – and repli­ca­tion is trig­gered by a pos­i­tive trans-act­ing fac­tor, the 'Ini­tia­tor' (see Fig­ure 1). One hall­mark of the 'Rep­licon Model' was the pos­tu­la­tion of a pos­i­tive reg­u­la­tor: at the time of its pub­li­ca­tion gene reg­u­la­tion was mostly thought about in terms of neg­a­tive reg­u­la­tion or repres­sion, in­spired by the sem­i­nal lac operon par­a­digm.

Fig­ure 1. 'Cir­cles in the Sand': A draw­ing in the sand of a repli­con as pre­sented in Ja­cob et al. (1963). The main cir­cle rep­re­sents the repli­con, the square box rep­re­sents the repli­ca­tor, and the ar­row in­di­cates where the ini­tia­tor pro­tein is en­coded, which, when syn­the­sized, binds to the repli­ca­tor (Fig­ure pro­vided by M. Méchali). Source

A Mat­ter of Lan­guage

Many bac­te­ria have a sin­gle chro­mo­so­mal repli­ca­tion ori­gin, oriC, which has been iden­ti­fied and stud­ied in E. coli (Gammapro­teobac­te­ria), Bacil­lus sub­tilis (Fir­mi­cutes), Caulobact­der cres­cen­tus (Alphaproteobac­teria), He­li­cobac­ter py­lori (Ep­silon­pro­teo­ba­te­ria), Myco­bacterium tu­ber­cu­lo­sis and Strep­to­myces coeli­color (Acti­nobac­te­ria), to name just some fa­vored model or­gan­isms. The 'Ini­tia­tor' in bac­te­ria is the DnaA pro­tein. (Al­most) all se­quenced bac­te­r­ial genomes have dnaA genes and all DnaA pro­teins are ho­mologs that be­long to a dis­tinct sub­class of the AAA+ AT­Pases. (Al­most) all bac­te­ria em­ploy a set of con­served repli­ca­tion fac­tors for initia­tion, strand sep­a­ra­tion, prim­ing, clamp­ing, and dis­con­tin­u­ous DNA syn­the­sis. De­spite this rel­a­tive sim­plic­ity, the pre- and post-ini­ti­a­tion mech­a­nisms that en­sure the 'once and only once' chromo­some repli­ca­tion per cell cy­cle turned out to be not only in­tri­cate but as­ton­ish­ingly vari­able among the cases stud­ied. Us­ing a metaphor one might say that with re­spect to repli­ca­tion, all bac­te­ria speak Eng­lish, us­ing the same gram­mar and syn­tax but each branch with a rather unique lo­cal di­alect in their vo­cab­u­lary. Just like some­one from In­ver­ness, Florida would face prob­lems get­ting along in In­ver­ness, Scot­land.

Fig­ure 2. H. vol­canii repli­ca­tion ori­gins. Source

This is bi­ol­ogy, so there are ex­cep­tions. Pseudomonas species (Gammapro­teobac­te­ria) have two func­tional oriCs, how­ever they are lo­cated at a close dis­tance to each other. Many of the in­sect en­dosym­biont bac­te­ria with highly re­duced genomes lack one or more genes of the repli­ca­tion ma­chin­ery – in­clud­ing dnaA genes – and it re­mains to be fig­ured out how they repli­cate their chro­mosomes. And there are (at least) two species of Fuso­bacteria that lack dnaA genes but have RepA-type plas­mid ini­tia­tor genes, in­trigu­ingly at the chro­mo­so­mal po­sition that is usu­ally "oc­cu­pied" by dnaA. So far, no gross con­tra­dic­tions to the 'Repli­con Model'.

Stick­ing to the metaphor, ar­chaea and eu­karya speak French. The gram­mar and syn­tax of repli­cation – ini­ti­a­tion, strand sep­a­ra­tion, prim­ing, clamp­ing, dis­con­tin­u­ous DNA syn­the­sis – are fairly sim­i­lar to those of the bac­te­ria. On the other hand, they use a com­pletely dif­fer­ent vo­cab­u­lary: the fac­tors are func­tional ho­mologs of their bac­te­r­ial coun­ter­parts but be­long to dif­fer­ent pro­tein fa­milies. The chro­mo­some of Py­ro­coc­cus abyssi (Eu­r­yarchaeota) is repli­cated from a sin­gle oriC, whereas three dif­fer­ent oriCs are used to repli­cate the sin­gle chro­mo­some of Sul­folobus species (Cre­nar­chaeota). Eu­karia usu­ally have a plethora of ori­gins along their chro­mo­somes. But here again no gross con­tra­dic­tions to the 'Repli­con Model'.

If One Ori­gin Is Good, Are Sev­eral Bet­ter?

Haloferax vol­canii (Eu­r­yarchaeota, Halobac­te­ria) sports sev­eral repli­ca­tion ori­gins on its main chro­mo­some, all of which have been mapped by Thorsten Allers' lab. For the afi­ciona­dos: of other ge­netic and phys­i­cal tech­niques, they used the tra­di­tional minichro­mo­some ap­proach, that is, de­ter­min­ing whether a pre­sump­tive ori­gin can drive the repli­ca­tion of a se­lec­table ori­gin-less plas­mid. In their present study, these re­searchers asked to what ex­tent each of these mul­ti­ple ori­gins con­tributes to chro­mo­some repli­ca­tion. To this end, they turned DNA se­quenc­ing into an an­a­lyt­i­cal tool.

This in­ge­nious twist needs prob­a­bly a brief ex­pla­na­tion. In 'shot­gun' DNA se­quenc­ing of genomes, chro­mo­so­mal DNA – usu­ally ob­tained from sta­tion­ary cul­tures – is chopped down to bits and pie­ces that are then se­quenced. The short reads are as­sem­bled to the full genome se­quence by ap­propriate soft­ware. Plot­ting the num­ber of reads for a given base vs. its genome po­si­tion gives a rough mea­sure for the 'se­quenc­ing depth', i.e. the re­li­a­bil­ity of the ob­tained data. If, how­ever, chro­mo­so­mal DNA is pre­pared and se­quenced from an asyn­chro­nously grow­ing cul­ture, plot­ting the num­ber of reads for a given base vs. its genome po­si­tion in­di­cates repli­ca­tion start points seen as peaks on a graph. The rea­son be­ing that DNA frag­ments close to an ac­tive repli­ca­tion ori­gin oc­cur with higher fre­quency.

Fig­ure 3. RadA re­com­bi­nase is es­sen­tial in an ΔoriC1,2,3,pHV4 mu­tant. radA was placed un­der con­trol of the tryp­to­phan-in­ducible Pt­naA pro­moter, in oriC1 and ΔoriC1,2,3,pHV4 strains (H1637 and H1642). The for­mer grows slowly in the ab­sence of tryp­to­phan whereas the lat­ter is in­vi­able. Ab­sence of tryp­to­phan does not af­fect the growth of oriC1 and ΔoriC1,2,3,pHV4 con­trol strains (H26 and H1546); the ΔtrpA con­trol strain (H53) is aux­otrophic for tryp­to­phan. Source

De­tour: The prin­ci­ple here used of "count­ing gene co­pies" was de­vised early on for work con­firm­ing that bac­terial chro­mo­some repli­ca­tion is se­quen­tial, that is, it starts at a site we call ori­gin and pro­ceeds bidi­rec­tion­ally to a ter­mi­nus.  In 1963, Yoshikawa and Sueoka mea­sured the num­ber of copies of var­i­ous genes in Bacil­lus sub­tilis us­ing ge­netic trans­for­ma­tion. They in­ci­den­tally discov­ered that, when grow­ing rapidly, bac­te­ria un­dergo multi­fork repli­ca­tion to cope with the prob­lem that cell divi­sion takes less time than repli­cat­ing the chro­mo­some.

All H. vol­canii ori­gins could thus be shown to con­tribute to chro­mo­some repli­ca­tion al­beit with dif­fer­ing ef­fi­ca­cies (see Fig­ure 2, up­per part). In sub­se­quent pair­wise growth com­pe­ti­tion ex­per­i­ments, sin­gle ori­gin dele­tion strains grew slower than the wild type par­ent but to some sur­prise, the dele­tion of two ore three ori­gins re­sulted in strains that grew slightly bet­ter than the wild type! In­deed, a strain hav­ing all ori­gins deleted grew ~7.5% bet­ter than the wild type (see Fig­ure 3). Stranger things are hap­pen­ing here.

Fig­ure 4. Pair­wise growth com­pe­ti­tion as­says com­par­ing wild type (WT) and ori­gin dele­tion strains. Source

In the strain whose ori­gins had all been deleted, Allers and cowork­ers did not de­tect novel peaks, which would have sug­gested that silent ori­gins had been ac­ti­vated. But they no­ticed a wider zone of copy num­ber en­rich­ment in the 2,250 kb re­gion near the rrnB ri­bo­so­mal RNA operon (see Fig­ure 2, lower part). Since it is known that highly tran­scribed DNA is as­so­ci­ated with el­e­vated re­com­bination, they as­sumed that D‑loops and R‑loops in the rrnB re­gion could fa­cil­i­tate the ini­ti­a­tion of repli­ca­tion much the same way as en­ter­tained by phage T4 (see p. 335 in Weigel and Seitz (2006)). To con­firm this, they placed the re­com­bi­na­tion gene radA un­der con­trol of a tryp­to­phan-in­ducible pro­moter. In the ab­sence of tryp­to­phan, when this pro­moter is tightly re­pressed, ori­gin-less cells failed to grow, an ef­fect res­cued by the ad­di­tion of tryp­to­phan (see Fig­ure 4).

Sim­ple It Isn't

The ob­ser­va­tion that the rate of growth cor­re­lates in­versely with the ac­tiv­ity of re­main­ing ori­gins sug­gested to these re­searchers that re­com­bi­na­tion-de­pen­dent repli­ca­tion is more ef­fi­cient than ori­gin-de­pen­­dent repli­ca­tion, but the for­mer has a lower affin­ity for the replica­tive he­li­case MCM, which is rate-lim­it­ing due to its low abun­dance. Why then is there ori­gin-de­pen­dent repli­ca­tion at all? These work­ers hy­poth­e­size that ori­gin-de­pen­dent repli­ca­tion al­lows for a tighter reg­u­la­tion of the co­or­di­na­tion be­tween repli­ca­tion and chro­mo­some seg­re­ga­tion. This would be cru­cial in bac­te­ria like E. coli, which un­der fast growth con­di­tions need to main­tain a fixed ploidy in over­lap­ping cell cy­cles. H. vol­canii, how­ever, is highly poly­ploid with ~20 chro­mo­somes per cell, which al­lows for a sto­chas­tic dis­tri­b­u­tion of chromo­somes to daugh­ter cells. The au­thors en­vi­sion the pos­sibility that an an­ces­tral, ori­gin-lack­ing H. vol­canii chro­mo­some was vir­tu­ally 'hi­jacked' by re­lated self­ish repli­cons con­tain­ing a repli­ca­tion ori­gin and an ini­tia­tor gene in close vicin­ity. With time, the cou­pling of repli­ca­tion to seg­re­ga­tion evolved to en­sure their prop­a­ga­tion. This at the cost of a slight­ly re­duced growth fit­ness as ob­served for the "wild type" in com­par­i­son to the ori­gin-dele­tion mu­tants.

To come full cir­cle: al­ready be­fore the on­set of the new mil­len­nium, Tokio Ko­goma showed that in E. coli chro­mo­some repli­ca­tion is pos­si­ble in sdrA mu­tants lack­ing dnaA or oriC or both. The lack of RNase H in the sdrA mu­tant leads to a pro­longed half life of mRNA·DNA hy­brids and the recom­binational re­pair of such struc­tures is suf­fi­cient to sus­tain chro­mo­some repli­ca­tion. Ko­goma called this process con­sti­tu­tive sta­ble DNA repli­ca­tion (cSDR). As was found by Hawkins et al. for H. vol­canii radA mu­tants lack­ing the ori­gins, cSDR in E. coli is cru­cially de­pen­dent on the RecA recombi­nation pro­tein. It ap­pears that the "three Rs" (repli­ca­tion, re­pair, re­com­bi­na­tion) are nicely inter­twined mech­a­nis­ti­cally in the ar­chaea too and ­– most likely – also in the eu­karya. A fi­nal re­mark: the 'Repli­con Model' does not pre­clude the ex­is­tence of such in­tri­cate "backup sys­tems".

 

Ref­er­ence

Hawkins M, Malla S, Blythe MJ, Nieduszyn­ski CA, Allers T. (2013). Ac­cel­er­ated growth in the ab­sence of DNA repli­ca­tion ori­gins. Na­ture, 503 (7477), 544–547 PMID 24185008