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DNA Replication Through Strand Displacement During Lagging Strand DNA Synthesis in Saccharomyces Cerevisiae

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DNA Replication Through Strand Displacement During Lagging Strand DNA Synthesis in Saccharomyces Cerevisiae G C A T T A C G G C A T genes Review DNA Replication Through Strand Displacement During Lagging Strand DNA Synthesis in Saccharomyces cerevisiae Michele Giannattasio 1,2,* and Dana Branzei 1,3,* 1 IFOM (Fondazione Istituto FIRC di Oncologia Molecolare), 20139 Milan, Italy 2 Dipartimento di Oncologia ed Emato-Oncologia, Universita’ degli Studi di Milano, 20122 Milan, Italy 3 Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche (IGM-CNR), Via Abbiategrasso 207, 27100 Pavia, Italy * Correspondence: [email protected] (M.G.); [email protected] (D.B.) Received: 11 January 2019; Accepted: 18 February 2019; Published: 21 February 2019 Abstract: This review discusses a set of experimental results that support the existence of extended strand displacement events during budding yeast lagging strand DNA synthesis. Starting from introducing the mechanisms and factors involved in leading and lagging strand DNA synthesis and some aspects of the architecture of the eukaryotic replisome, we discuss studies on bacterial, bacteriophage and viral DNA polymerases with potent strand displacement activities. We describe proposed pathways of Okazaki fragment processing via short and long flaps, with a focus on experimental results obtained in Saccharomyces cerevisiae that suggest the existence of frequent and extended strand displacement events during eukaryotic lagging strand DNA synthesis, and comment on their implications for genome integrity. Keywords: DNA replication; lagging strand DNA synthesis; Okazaki fragment processing; strand displacement DNA synthesis; DNA helicases; flap endonucleases 1. The Replication Fork and the DNA Synthesis Apparatus With the scope of introducing the proteins involved in lagging strand DNA synthesis in the context of the replication fork and replisome, we begin with a brief introduction of DNA replication initiation. However, due to the limited extent of this introduction, we re-direct readers interested in the mechanisms and regulations of DNA replication origin activation, replisome assembly and structure to recent reviews on this topic [1–4]. DNA replication initiates from specific regions on the chromosomes, known as origins of replication, which are well defined in Saccharomyces cerevisiae by the presence of an autonomously replicating sequence (ARS), but are less defined in vertebrates [3,5–8]. Briefly, origin licensing in S. cerevisiae starts in M/G1 with the loading of the MCM2-7 (Minichromosome Maintenance Complex) complex on an ARS sequence bound by ORC (Origin Recognition Complex). For DNA replication to initiate, an inactive double hexamer of Mcm2-7 needs to be activated and separated into single hexamers, each of them in complex with Cdc45 (Cell division cycle 45) and GINS (GO, Ichi, Ni and San complex), to form the replicative CMG (Cdc45-Mcm2-7-GINS) helicase [1]. Formation of an active CMG complex is promoted by S-CDK (Cyclin Dependent Kinase)-dependent phosphorylation of Sld2 (Synthetic Lethal with Dpb11-1 number 2) and Sld3 (Synthetic Lethal with Dpb11-1 number 3) [9], and by DDK (Dbf4-dependent kinase)-dependent phosphorylation of MCM [3,10,11]. MCM appears to be the only essential DDK target for replisome assembly and origin activation in an in vitro system reproducing DNA replication origin firing using purified S. cerevisiae proteins [12]. DDK phosphorylation of Mcm4/6 generates binding sites for Sld3 [13], which recruits Genes 2019, 10, 167; doi:10.3390/genes10020167 www.mdpi.com/journal/genes Genes 2019, 10, 167 2 of 25 Cdc45. Phosphorylated MCM acts together with CDK-phosphorylated Sld2 and Sld3 to trigger the recruitment of firing factors, which remodel the MCM double hexamer to form the CMG helicase [5]. How this remodeling occurs is not fully understood [14]. Sld3 to trigger the recruitment of firing factors, which remodel the MCM double hexamer to form the DNA polymerase " is recruited to MCM through Dpb11 (DNA polymerase B possible subunit CMG helicase [5]. How this remodeling occurs is not fully understood [14]. 11)-mediatedDNA interactions polymerase withε is recruited CDK-phosphorylated to MCM through Sld3 Dpb11 and (DNA Sld2, whilepolymerase Cdc45 B recruitmentpossible subunit to MCM requires11)-mediated DDK-dependent interactions phosphorylation with CDK-phosphorylat of MCM [ed3, 5Sld3]. Ctf4 and (Chromosome Sld2, while Cdc45 Transmission recruitment Fidelity to 4) and MCM10MCM requires participate DDK-dependent in the recruitment phosphorylation of DNA polymerase of MCM [3,5].α to Ctf4 chromatin (Chromosome and to theTransmission CMG complex for initiationFidelity 4) of and DNA MCM10 replication participate [15, 16in ].the MCM10 recruitment also of has DNA a crucial polymerase role inα to the chromatin activation and of to the the CMG helicaseCMG for complex replication for initiation initiation of [12 DNA,14]. replication DNA polymerase [15,16]. MCM10α also interactsalso has directlya crucial withrole MCMin the [17], but theactivation detailed of mechanism the CMG helicase of Pol αforrecruitment replication initiation to the replisome [12,14]. DNA is not polymerase fully elucidated. α also Furthermore,interacts α evidencedirectly from with both MCMin [17], vitro but[12 the,18 detailed] and in mechanism vivo [19] systemsof Pol recruitment indicate that to the Ctf4 replisome is dispensable is not fully for Pol α-Primaseelucidated. -mediated Furthermore, priming. evidence Rather, from it seems both in that vitro Ctf4 [12,18] is important and in vivo for [19] parental systems histones indicate transfer that to Ctf4 is dispensable for Pol α-Primase -mediated priming. Rather, it seems that Ctf4 is important for the newly replicated lagging strand filament [20]. DNA polymerase δ is recruited to the replisome parental histones transfer to the newly replicated lagging strand filament [20]. DNA polymerase δ is throughrecruited interaction to the replisome with PCNA through (proliferating interaction cell with nuclear PCNA antigen),(proliferating which cell isnuclear loaded antigen), on the which growing 3’ ends ofis loaded the newly on the synthesized growing 3’ filaments ends of the by newly the replication synthesized factor filaments C (RFC) by the [7,21 replication]. In Figure factor1, a schematizedC (RFC) view[7,21]. of a replication In Figure fork1, a andschematized the replisome view isof shown.a replication The antiparallel fork and the nature replisome of the is DNA shown. double The helix and 5’–3’antiparallel direction nature of the of DNAthe DNA synthesis double at helix the replicationand 5’–3’ direction fork impose of the that DNA one synthesis newly synthesized at the strandreplication of the replication fork impose fork that isone polymerized newly synthesized in a continuous strand of the way replication (leading fork strand is polymerized filament), in a while the othercontinuous is subjected way (leading to discontinuous strand filament), DNA while synthesis the other (lagging is subjected strand filament)to discontinuous [22]. Upon DNA DDK and CDK-mediatedsynthesis (lagging phosphorylation strand filament) events,[22]. Upon MCM DDK double and CDK-mediated hexamers get phosphorylation activated, the DNAevents, at the originMCM is unwound double hexamers and Okazaki get activated, fragments the composed DNA at the of origin RNA is and unwound DNA initiator and Okazaki primers fragments (DNAi) are composed of RNA and DNA initiator primers (DNAi) are synthesized both on the leading and synthesized both on the leading and lagging strands [7,23] by DNA polymerase α-Primase (Figure1). lagging strands [7,23] by DNA polymerase α-Primase (Figure 1). FigureFigure 1. Schematic 1. Schematic structure structure of of the the replisome replisome andand the DNA DNA replication replication fork fork of S. of cerevisiae.S. cerevisiae. DNADNA polymerasepolymerase" synthesizes ε synthesizes the leadingthe leading strand strand continuously continuously while while DNA DNA polymerase polymeraseα -Primaseα-Primase and and DNA δ polymeraseDNA polymeraseδ carry out laggingcarry out strand lagging synthesis strand synthesis in a discontinuous in a discontinuous way creating way creating Okazaki Okazaki fragments α (OFs).fragments DNA polymerase (OFs). DNAα-Primase polymerase makes -Primase the RNA makes primer the and RNA synthesizes primer and the synthesizes DNA initiator the DNA fragment. initiator fragment. After this event, Pol α falls off from the 3’ end of the growing OF at the replication After this event, Pol α falls off from the 3’ end of the growing OF at the replication fork and PCNA fork and PCNA (Proliferating Cell Nuclear Antigen) is loaded by the RFC (Replication Factor C) (Proliferating Cell Nuclear Antigen) is loaded by the RFC (Replication Factor C) complex leading to complex leading to a DNA polymerase switch between Pol α and Pol δ on the lagging strand. The a DNA polymerase switch between Pol α and Pol δ on the lagging strand. The growing 3’ end of growing 3’ end of the previous OF, which is being synthesized by Pol δ will encounter the 5’ end of the previousthe last OF, OF, which which contains is being the synthesized RNA-DNA initiators by Pol δsegmentswill encounter previously the synthesized 5’ end of the by Pol last α OF,. This which containsencounter the RNA-DNA will lead to initiators the processing segments and maturation previously of synthesized the OF (see Figure by Pol 2).α . This encounter will lead to the processing and maturation of the OF (see Figure2). Genes 2019, 10, 167 3 of 25 It was recently demonstrated that during the first
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