<\/p>\n Replisomes require core enzymes: helicase, primase, and DNA polymerases (Pols). Replisomes of both bacteria and eukaryotes require many more proteins not shown here. Textbook illustrations have placed both leading- and lagging-strand DNA Pols behind the helicase. Insights from Gao\u00a0et al.<\/em>reveal the asymmetric juxtaposition of these core enzymes in T7 phage, similar to replisiomes in the eukaryote\u00a0Saccharomyces cerevisiae.<\/em><\/p>\n <\/p>\n <\/p>\n T7 phage is a smart choice for cryo-EM studies because it is the most streamlined replisome known (2<\/em><\/a>). The helicase in T7 phage, gene protein 4 (gp4), forms a hexameric ring, a common feature of all replicative helicases (3<\/em><\/a>,\u00a04<\/em><\/a>). However, unlike other helicases, T7 gp4 also contains a primase located behind the helicase during replication fork progression (2<\/em><\/a>). The T7 DNA Pol, gp5, functions in a 1:1 complex with the 12-kDa bacterial host thioredoxin protein, which increases polymerase processivity (2<\/em><\/a>). Hence, three different proteins comprise the core T7 replisome (gp4, gp5, and thioredoxin). By contrast, the replisome of\u00a0Escherichia coli<\/em>\u2014the bacterial host for T7\u2014contains a dozen different proteins, including a sliding clamp and clamp loader complex for replisome processivity (5<\/em><\/a>).<\/p>\n Despite the different complexity, the proteins of the T7 replisome are homologous to proteins of its\u00a0E. coli<\/em>\u00a0host and thus are representative of the bacterial core replisome. Gao\u00a0et al.<\/em>\u00a0report the 3.2-\u00c5 resolution structure of the T7 replisome in which the gp4 helicase-primase encircles the lagging strand to unwind the parental duplex while also priming the lagging strand. The leading- and lagging-strand DNA Pols connect to and sandwich the helicase between them, an asymmetric arrangement that minimizes single-stranded DNA (ssDNA), which is more susceptible to nucleases and damage than double-stranded DNA (dsDNA).<\/p>\n Multiprotein complexes carry out each step of the \u201ccentral dogma\u201d of genetic information flow: replication, transcription, and translation. Interestingly, proteins of transcription and translation are homologous among Bacteria, Archaea, and Eukarya and thus evolved from a common ancestor. By contrast, the DNA Pol, helicase, and primase of bacterial replisomes share no homology to their eukaryotic counterparts, implying that these replisome enzymes evolved independently, after the evolutionary split of bacteria and eukaryotes (6<\/em><\/a>,\u00a07<\/em><\/a>). The primordial cell possibly used a simpler process of DNA replication, or used an RNA genome.<\/p>\n Surprisingly, the asymmetric arrangement of two DNA Pols that sandwich the helicase was also demonstrated by EM for the eukaryotic replisome of the yeast\u00a0Saccharomyces cerevisiae<\/em>\u00a0(8<\/em><\/a>), albeit at lower resolution than the T7 study by Gao\u00a0et al.<\/em>\u00a0Thus, although \u201cworlds apart\u201d in terms of their independent evolution, the core elements of bacterial and eukaryotic replisomes both contain a helicase between two DNA Pols, although the eukaryotic replisome requires a trimeric scaffolding factor (Ctf4) to help tether the top DNA Pol to the helicase.<\/p>\n Another unexpected feature of the bacterial (T7) and eukaryotic replisomes is that the top DNA Pol functions on the opposite strand: The DNA Pol at the top of T7 helicase replicates the leading strand, whereas the DNA Pol at the top of eukaryotic CMG helicase replicates the lagging strand (see the figure). This is because bacterial helicases encircle the lagging strand, whereas eukaryotic CMG helicase encircles the leading strand (9<\/em><\/a>\u201313<\/em><\/a>). It remains a mystery why this \u201cmirror\u201d arrangement evolved, but both arrangements share a pragmatic logic for replisome function. All replicative helicases split the duplex at their leading edge, with one strand going through the middle of the helicase ring and the other strand deflected off the top of the ring (4<\/em><\/a>). Hence, a DNA Pol at the top of the helicase can immediately duplicate the separated strand.<\/p>\n In bacteria, the primase domain of gp4 is below the helicase for Okazaki fragment extension. In eukaryotes, priming is performed above the CMG helicase by the Pol \u03b1\u2013primase complex, which is secured to the helicase by the Ctf4 scaffold (8<\/em><\/a>,\u00a012<\/em><\/a>\u201314<\/em><\/a>). Pol \u03b1\u2013primase generates an RNA-DNA primer of \u223c25 nucleotides for the lagging strand Pol \u03b4 (13<\/em><\/a>,\u00a014<\/em><\/a>). In eukaryotes, the leading and lagging DNA Pols are encoded by different genes, and the leading strand DNA Pol \u03b5 (14<\/em><\/a>) is located at the bottom of the helicase, which encircles the leading strand and feeds the unwound strand to Pol \u03b5 for continuous leading-strand synthesis (8<\/em><\/a>,\u00a011<\/em><\/a>).<\/p>\n There exist numerous questions for the future. Having a DNA Pol on top of the helicase in T7 suggests that it may be first to encounter DNA damage and DNA-bound proteins before the helicase. Therefore, the DNA Pol arrangement may help replisome progression by detecting lesions or displacing barriers. Single-molecule studies indicate a network of dynamic enzyme exchanges within the replisome of T7 and other replisomes, which may eject or supplement DNA Pols as needed (15<\/em><\/a>). There is much excitement over these new discoveries, and there are many more questions to address in the future than have thus far been answered.<\/p>\n <\/p>\n <\/p>\n (\uc6d0\ubb38: \uc5ec\uae30<\/a>\ub97c \ud074\ub9ad\ud558\uc138\uc694~)<\/p>\n <\/p>\n <\/p>\n","protected":false},"excerpt":{"rendered":"![](\"http:\/\/science.sciencemag.org\/content\/sci\/363\/6429\/814\/F1.medium.gif\")
<\/span><\/a><\/div>\n\n
GRAPHIC: N. DESAI\/SCIENCE<\/em><\/q><\/p>\n
![<\/i>\u00a0Download high-res image<\/span><\/a><\/li>\n<\/i>\u00a0Open in new tab<\/span><\/a><\/li>\n<\/i>\u00a0Download Powerpoint<\/span><\/a><\/li>\n<\/ul>\n<\/div>\n<\/div>Asymmetric organization of core enzymes in replisomes<\/span><\/p>\nReplisomes require core enzymes: helicase, primase, and DNA polymerases (Pols). Replisomes of both bacteria and eukaryotes require many more proteins not shown here. Textbook illustrations have placed both leading- and lagging-strand DNA Pols behind the helicase. Insights from Gao\u00a0et al.<\/em>reveal the asymmetric juxtaposition of these core enzymes in T7 phage, similar to replisiomes in the eukaryote\u00a0Saccharomyces cerevisiae.<\/em><\/p>\nGRAPHIC: N. DESAI\/SCIENCE<\/em><\/q><\/p>\n<\/div>\n<\/figcaption><\/figure>\n <\/p>\n <\/p>\nT7 phage is a smart choice for cryo-EM studies because it is the most streamlined replisome known (2<\/em><\/a>). The helicase in T7 phage, gene protein 4 (gp4), forms a hexameric ring, a common feature of all replicative helicases (3<\/em><\/a>,\u00a04<\/em><\/a>). However, unlike other helicases, T7 gp4 also contains a primase located behind the helicase during replication fork progression (2<\/em><\/a>). The T7 DNA Pol, gp5, functions in a 1:1 complex with the 12-kDa bacterial host thioredoxin protein, which increases polymerase processivity (2<\/em><\/a>). Hence, three different proteins comprise the core T7 replisome (gp4, gp5, and thioredoxin). By contrast, the replisome of\u00a0Escherichia coli<\/em>\u2014the bacterial host for T7\u2014contains a dozen different proteins, including a sliding clamp and clamp loader complex for replisome processivity (5<\/em><\/a>).<\/p>\nDespite the different complexity, the proteins of the T7 replisome are homologous to proteins of its\u00a0E. coli<\/em>\u00a0host and thus are representative of the bacterial core replisome. Gao\u00a0et al.<\/em>\u00a0report the 3.2-\u00c5 resolution structure of the T7 replisome in which the gp4 helicase-primase encircles the lagging strand to unwind the parental duplex while also priming the lagging strand. The leading- and lagging-strand DNA Pols connect to and sandwich the helicase between them, an asymmetric arrangement that minimizes single-stranded DNA (ssDNA), which is more susceptible to nucleases and damage than double-stranded DNA (dsDNA).<\/p>\nMultiprotein complexes carry out each step of the \u201ccentral dogma\u201d of genetic information flow: replication, transcription, and translation. Interestingly, proteins of transcription and translation are homologous among Bacteria, Archaea, and Eukarya and thus evolved from a common ancestor. By contrast, the DNA Pol, helicase, and primase of bacterial replisomes share no homology to their eukaryotic counterparts, implying that these replisome enzymes evolved independently, after the evolutionary split of bacteria and eukaryotes (6<\/em><\/a>,\u00a07<\/em><\/a>). The primordial cell possibly used a simpler process of DNA replication, or used an RNA genome.<\/p>\nSurprisingly, the asymmetric arrangement of two DNA Pols that sandwich the helicase was also demonstrated by EM for the eukaryotic replisome of the yeast\u00a0Saccharomyces cerevisiae<\/em>\u00a0(8<\/em><\/a>), albeit at lower resolution than the T7 study by Gao\u00a0et al.<\/em>\u00a0Thus, although \u201cworlds apart\u201d in terms of their independent evolution, the core elements of bacterial and eukaryotic replisomes both contain a helicase between two DNA Pols, although the eukaryotic replisome requires a trimeric scaffolding factor (Ctf4) to help tether the top DNA Pol to the helicase.<\/p>\nAnother unexpected feature of the bacterial (T7) and eukaryotic replisomes is that the top DNA Pol functions on the opposite strand: The DNA Pol at the top of T7 helicase replicates the leading strand, whereas the DNA Pol at the top of eukaryotic CMG helicase replicates the lagging strand (see the figure). This is because bacterial helicases encircle the lagging strand, whereas eukaryotic CMG helicase encircles the leading strand (9<\/em><\/a>\u201313<\/em><\/a>). It remains a mystery why this \u201cmirror\u201d arrangement evolved, but both arrangements share a pragmatic logic for replisome function. All replicative helicases split the duplex at their leading edge, with one strand going through the middle of the helicase ring and the other strand deflected off the top of the ring (4<\/em><\/a>). Hence, a DNA Pol at the top of the helicase can immediately duplicate the separated strand.<\/p>\nIn bacteria, the primase domain of gp4 is below the helicase for Okazaki fragment extension. In eukaryotes, priming is performed above the CMG helicase by the Pol \u03b1\u2013primase complex, which is secured to the helicase by the Ctf4 scaffold (8<\/em><\/a>,\u00a012<\/em><\/a>\u201314<\/em><\/a>). Pol \u03b1\u2013primase generates an RNA-DNA primer of \u223c25 nucleotides for the lagging strand Pol \u03b4 (13<\/em><\/a>,\u00a014<\/em><\/a>). In eukaryotes, the leading and lagging DNA Pols are encoded by different genes, and the leading strand DNA Pol \u03b5 (14<\/em><\/a>) is located at the bottom of the helicase, which encircles the leading strand and feeds the unwound strand to Pol \u03b5 for continuous leading-strand synthesis (8<\/em><\/a>,\u00a011<\/em><\/a>).<\/p>\nThere exist numerous questions for the future. Having a DNA Pol on top of the helicase in T7 suggests that it may be first to encounter DNA damage and DNA-bound proteins before the helicase. Therefore, the DNA Pol arrangement may help replisome progression by detecting lesions or displacing barriers. Single-molecule studies indicate a network of dynamic enzyme exchanges within the replisome of T7 and other replisomes, which may eject or supplement DNA Pols as needed (15<\/em><\/a>). There is much excitement over these new discoveries, and there are many more questions to address in the future than have thus far been answered.<\/p>\n <\/p>\n <\/p>\n(\uc6d0\ubb38: \uc5ec\uae30<\/a>\ub97c \ud074\ub9ad\ud558\uc138\uc694~)<\/p>\n <\/p>\n <\/p>\n","protected":false},"excerpt":{"rendered":" Replication of the DNA genome is performed by a replisome complex composed of numerous proteins. Cells have duplex DNA genomes, and their replisomes(more…)<\/a><\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_monsterinsights_skip_tracking":false,"_monsterinsights_sitenote_active":false,"_monsterinsights_sitenote_note":"","_monsterinsights_sitenote_category":0,"jetpack_post_was_ever_published":false,"_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":"","jetpack_publicize_message":"","jetpack_publicize_feature_enabled":true,"jetpack_social_post_already_shared":true,"jetpack_social_options":{"image_generator_settings":{"template":"highway","default_image_id":0,"font":"","enabled":false},"version":2}},"categories":[33,34,29,30],"tags":[],"class_list":["post-2712","post","type-post","status-publish","format-standard","hentry","category-do-biology","category-lets-do-chemistry","category-lets-do-science","category-recent-science-news"],"aioseo_notices":[],"jetpack_publicize_connections":[],"jetpack_featured_media_url":"","jetpack-related-posts":[{"id":4191,"url":"https:\/\/biochemistry.khu.ac.kr\/lab\/?p=4191","url_meta":{"origin":2712,"position":0},"title":"Remodeling the genome with DNA twists","author":"biochemistry","date":"October 6, 2019","format":false,"excerpt":"\u00a0 \u00a0 In complex organisms such as humans, a single genetic blueprint can give rise to a multitude of different cell types, from nerve to liver to muscle. Such cellular diversity relies on restricting which portions of genomic DNA are accessible and therefore can be read by cellular machinery. Ultimately,\u2026","rel":"","context":"In "Let's Do Biology!"","block_context":{"text":"Let's Do Biology!","link":"https:\/\/biochemistry.khu.ac.kr\/lab\/?cat=33"},"img":{"alt_text":"","src":"","width":0,"height":0},"classes":[]},{"id":4845,"url":"https:\/\/biochemistry.khu.ac.kr\/lab\/?p=4845","url_meta":{"origin":2712,"position":1},"title":"CRISPR tool modifies genes precisely by copying RNA into the genome & CRISPR: the movie","author":"biochemistry","date":"November 15, 2019","format":false,"excerpt":"\u00a0 \u00a0 The ultimate goal of genome editing is to be able to make any specific change to the blueprint of life. A \u2018search-and-replace\u2019 method for genome editing takes us a giant leap closer to this ambitious goal. \u00a0 \u00a0 Variation in the DNA sequences that constitute the blueprint of\u2026","rel":"","context":"In "Let's Do Biology!"","block_context":{"text":"Let's Do Biology!","link":"https:\/\/biochemistry.khu.ac.kr\/lab\/?cat=33"},"img":{"alt_text":"","src":"","width":0,"height":0},"classes":[]},{"id":3891,"url":"https:\/\/biochemistry.khu.ac.kr\/lab\/?p=3891","url_meta":{"origin":2712,"position":2},"title":"Inserting DNA with CRISPR","author":"biochemistry","date":"July 16, 2019","format":false,"excerpt":"\u00a0 \u00a0 Most prokaryotes rely on the CRISPR-Cas system for adaptive immunity against viruses and mobile elements (1,\u00a02). Small RNAs produced from CRISPR direct Cas effector proteins to seek and destroy nucleic acids from invaders that have complementary target sites (3). There are multiple types of CRISPR, which are defined\u2026","rel":"","context":"In "Let's Do Biology!"","block_context":{"text":"Let's Do Biology!","link":"https:\/\/biochemistry.khu.ac.kr\/lab\/?cat=33"},"img":{"alt_text":"","src":"","width":0,"height":0},"classes":[]},{"id":3361,"url":"https:\/\/biochemistry.khu.ac.kr\/lab\/?p=3361","url_meta":{"origin":2712,"position":3},"title":"When genome editing goes off-target","author":"biochemistry","date":"April 19, 2019","format":false,"excerpt":"\u00a0 \u00a0 Editing DNA in eukaryotic cells with CRISPR-based systems has revolutionized the genome engineering field. Cas (CRISPR-associated) endonucleases are directed to a particular location in the genome by a short guide RNA, providing an easily programmable strategy to target any section of DNA. As of now, two CRISPR-based approaches\u2026","rel":"","context":"In "Let's Do Biology!"","block_context":{"text":"Let's Do Biology!","link":"https:\/\/biochemistry.khu.ac.kr\/lab\/?cat=33"},"img":{"alt_text":"","src":"","width":0,"height":0},"classes":[]},{"id":2672,"url":"https:\/\/biochemistry.khu.ac.kr\/lab\/?p=2672","url_meta":{"origin":2712,"position":4},"title":"CRISPR-Cas9-Based Genome Editing of Human Cells","author":"biochemistry","date":"February 15, 2019","format":false,"excerpt":"\u00a0 \u00a0 CRISPR\/Cas9 systems are engineered versions of the Cas9 protein and guide RNA. \u00a0Typically, they are identical to the\u00a0Streptococcus pyogenes\u00a0type II CRISPR systems, except that a single guide-RNA is used in place of the complementary crRNAs and tracrRNAs of the natural CRISPR system, and the Cas9 protein is codon-optimized\u2026","rel":"","context":"In "Let's Do Biology!"","block_context":{"text":"Let's Do Biology!","link":"https:\/\/biochemistry.khu.ac.kr\/lab\/?cat=33"},"img":{"alt_text":"Genome Editing Overview2","src":"https:\/\/i0.wp.com\/sites.tufts.edu\/crispr\/files\/2014\/11\/Genome-Editing-Overview2-1024x667.png?resize=350%2C200&ssl=1","width":350,"height":200,"srcset":"https:\/\/i0.wp.com\/sites.tufts.edu\/crispr\/files\/2014\/11\/Genome-Editing-Overview2-1024x667.png?resize=350%2C200&ssl=1 1x, https:\/\/i0.wp.com\/sites.tufts.edu\/crispr\/files\/2014\/11\/Genome-Editing-Overview2-1024x667.png?resize=525%2C300&ssl=1 1.5x, https:\/\/i0.wp.com\/sites.tufts.edu\/crispr\/files\/2014\/11\/Genome-Editing-Overview2-1024x667.png?resize=700%2C400&ssl=1 2x"},"classes":[]},{"id":4090,"url":"https:\/\/biochemistry.khu.ac.kr\/lab\/?p=4090","url_meta":{"origin":2712,"position":5},"title":"Emerging uses of DNA mechanical devices","author":"biochemistry","date":"September 18, 2019","format":false,"excerpt":"\u00a0 \u00a0 Modern machines, which are composed of force-generating motors, force sensors, and load-bearing structures, enabled the industrial revolution and are foundational to human civilization. Miniature micromachines are used in countless devices including cell phone microphones, implantable biosensors, and car and airplane accelerometers. Further miniaturization to the nanometer scale would\u2026","rel":"","context":"In "Let's Do Chemistry!"","block_context":{"text":"Let's Do Chemistry!","link":"https:\/\/biochemistry.khu.ac.kr\/lab\/?cat=34"},"img":{"alt_text":"","src":"","width":0,"height":0},"classes":[]}],"jetpack_sharing_enabled":false,"jetpack_shortlink":"https:\/\/wp.me\/p9Xo1j-HK","_links":{"self":[{"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=\/wp\/v2\/posts\/2712","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=2712"}],"version-history":[{"count":3,"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=\/wp\/v2\/posts\/2712\/revisions"}],"predecessor-version":[{"id":2776,"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=\/wp\/v2\/posts\/2712\/revisions\/2776"}],"wp:attachment":[{"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=2712"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=2712"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/biochemistry.khu.ac.kr\/lab\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=2712"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}](\"http:\/\/science.sciencemag.org\/content\/sci\/363\/6429\/814\/F1.large.jpg?download=true\"){kind=link}