12/6/17 1 2 Single-molecule studies contrast ordered DNA replication with 3 stochastic translesion synthesis 4 5 6 7 Gengjing Zhao1, Emma S. Gleave1 & Meindert H. Lamers1,2* 8 9 10 11 1MRC laboratory of Molecular Biology, Francis Crick Avenue, Cambridge 12 Biomedical Campus, Cambridge, CB2 0QH, United Kingdom. 13 2Current address: Leiden University Medical Center, Einthovenweg 20, 2300 RC 14 Leiden, The Netherlands 15 *For Correspondence: [email protected] 16 17 1 12/6/17 18 ABSTRACT 19 High fidelity replicative DNA polymerases are unable to synthesize past DNA 20 adducts that result from diverse chemicals, reactive oxygen species or UV light. To 21 bypass these replication blocks, cells utilize specialized translesion DNA polymerases 22 that are intrinsically error prone and associated with mutagenesis, drug resistance, and 23 cancer. How untimely access of translesion polymerases to DNA is prevented is 24 poorly understood. Here we use co-localization single-molecule spectroscopy 25 (CoSMoS) to follow the exchange of the E. coli replicative DNA polymerase Pol 26 IIIcore with the translesion polymerases Pol II and Pol IV. We find that in contrast to 27 the toolbelt model, the replicative and translesion polymerases do not form a stable 28 complex on one clamp but alternate their binding. Furthermore, while the loading of 29 clamp and Pol IIIcore is highly organized, the exchange with the translesion 30 polymerases is stochastic and is not determined by lesion-recognition but instead a 31 concentration-dependent competition between the polymerases. 32 2 12/6/17 33 INTRODUCTION 34 To ensure faithful replication of the genomic DNA, replicative DNA polymerases 35 have a narrow active site that limits the incorporation of incorrect nucleotides. In 36 addition, rare nucleotide mis-incorporations into the primer strand prevent further 37 DNA synthesis and a 3'-5' exonuclease is required to remove the misincorporated 38 nucleotides [1]. In contrast, when the polymerase encounters a lesion on the template 39 strand in the form of a modified base caused by diverse chemicals, reactive oxygen 40 species, or UV light [2,3], the high-fidelity replicative DNA polymerases are stalled. 41 To bypass these replication blocks, all cells harbor multiple specialized translesion 42 DNA polymerases [4] that have more open active sites and are therefore able to 43 accommodate bulky DNA adducts and continue DNA synthesis. As a result of their 44 more open active sites, the translesion polymerases are error prone and consequently 45 associated with increased mutagenesis, drug resistance, and cancer [5,6]. Therefore, 46 the access of the translesion polymerases to DNA needs to be tightly controlled, but 47 how this is achieved has been the subject of debate. 48 The 'toolbelt' model [7] predicts that in E. coli the replicative DNA 49 polymerase Pol III and the translesion DNA polymerase Pol IV bind simultaneously 50 to the DNA sliding clamp , a dimeric, ring-shaped protein that encircles the DNA 51 and provides processivity to the replicative DNA polymerases [8]. This way, the 52 translesion polymerase functions in a manner analogous to the proofreading 53 exonuclease: when the replicative polymerase inserts an incorrect nucleotide into the 54 nascent strand, it will be removed by the proofreading exonuclease, whereas when the 55 polymerase encounters a lesion on the template strand, the DNA is transferred to the 56 translesion polymerase that can bypass the lesion. Thus both the exonuclease and 57 translesion polymerase act as 'tools' that enable the replicative polymerase to 58 overcome potential roadblocks to DNA replication. The toolbelt model, which was 3 12/6/17 59 originally based on steady-state Förster Energy Resonance Transfer (FRET) 60 experiments that showed the simultaneous binding of the replicative and translesion 61 polymerases to the -clamp, has found support in several subsequent studies [9-11]. 62 However, all these studies used bulk studies that due to the asynchronous nature 63 cannot separate out the sequential steps during a reaction [12]. More recently, single 64 molecule approaches have also been used [13,14], but in these experiments the 65 exchange of polymerases was inferred indirectly through the change in speed of DNA 66 synthesis and therefore it cannot be determined whether the polymerases bind 67 simultaneously. In addition, recent studies reveal that during DNA replication in E. 68 coli the two binding pockets of the dimeric -clamp are occupied by the replicative 69 DNA polymerase Pol III and the associated proofreading exonuclease [15,16]. The 70 cryo-EM structure of the trimeric Pol III-exonuclease-clamp (, , ) complex [17] 71 also shows that most of the clamp is covered, leaving no space for a second 72 polymerase. Therefore, it remains controversial whether the replicative and 73 translesion polymerases can co-localize on a single clamp. 74 Consequently, an alternative view to the toolbelt model is that the translesion 75 DNA polymerases compete for binding to clamp-DNA through 'mass action', as 76 evidenced by the fact that the bypass of a N2-acetylaminofluorene guanine adduct by 77 Pol V or Pol II depends on the relative concentrations of the two polymerases [18,19], 78 and that the concentrations of Pol IV and Pol V are dramatically increased during the 79 bacterial SOS DNA damage response [20]. 80 Regardless of the model, the DNA sliding clamp plays a pivotal role in 81 controlling access of the translesion polymerases to the DNA. However, the control 82 for access to the clamp-DNA is complicated by the fact that on the lagging strand, 83 DNA synthesis is discontinuous and every ~1000 base pairs a new clamp is loaded, 4 12/6/17 84 followed by the binding of the replicative DNA polymerase. Due to its closed circular 85 shape, the clamp must be loaded and unloaded onto the DNA by the dedicated clamp 86 loader complex (/-complex in bacteria, RFC in archaea and eukaryotes) [21,22]. 87 Once the clamp is loaded onto primed DNA, the replicative polymerase associates 88 and initiates DNA synthesis. How the repeated loading and unloading of the clamp 89 and replicative polymerase Pol III on the lagging strand is coordinated, while 90 simultaneously preventing the untimely association of the translesion polymerases has 91 not been studied so far. 92 Here, we use co-localization single molecule spectroscopy (CoSMoS) [23] to 93 directly visualize the loading of the E. coli clamp loader (/-complex), the DNA 94 sliding clamp , the replicative DNA polymerase Pol III, the proofreading 95 exonuclease , as well as the exchange with the translesion polymerases Pol II and Pol 96 IV. The multi-color CoSMoS experiments enable us to follow the binding and 97 dissociation of multiple proteins in real-time on a single DNA molecule, which makes 98 it the most suitable method to discriminate between simultaneous or sequential 99 binding of different molecules on a DNA substrate. Our work shows that the 100 translesion polymerases Pol II and Pol IV do not form a stable complex with the 101 replicative polymerase Pol III on the clamp-DNA and therefore the clamp does not 102 function as a molecular toolbelt. Furthermore, we find that the sequential activities of 103 the replication proteins clamp loader, clamp, and Pol III are highly organized while 104 the exchange with the translesion polymerases Pol II and Pol IV is disordered and 105 determined by mass action through concentration-dependent competition for the 106 hydrophobic groove on the surface of the -clamp. Hence, our results provide a 107 unique insight into the temporal organization of the events in DNA replication and 108 translesion synthesis, and contrast the highly organized replication events with 109 stochastic polymerase exchange during translesion synthesis. 5 12/6/17 110 111 RESULTS 112 Preparation of DNA substrates and fluorescently labeled proteins 113 The ring-shaped E. coli -clamp is capable of threading and unthreading itself 114 on free DNA ends and therefore we attached a primer-template DNA substrate to a 115 glass surface and blocked its free end with monovalent streptavidin (Figure 1A). 116 Subsequently, the binding of fluorescently labeled proteins to DNA was followed by 117 two- or three-color total internal reflection fluorescence microscopy (see Materials 118 and Methods) with a frame rate of 0.44 and 0.66 seconds (s), respectively, on ~800 119 well separated DNA molecules per field of view (Figure 1B-C). Proteins were 120 fluorescently labeled via maleimide-cysteine crosslinking or enzymatically via an N- 121 terminal Ybbr tag [24], and the fluorescently labeled proteins retained wild-type 122 activity as indicated by polymerase processivity assay (Figure 1-figure supplement 123 1A-C). For detection of Pol IIIcore (, , ), we fluorescently labeled the subunit. 124 For the clamp loader complexes (3' and 3'), the fluorescent label was placed on 125 the ' subunit. The lifetime of the each of the fluorophores was measured individually 126 on DNA-bound clamps (Atto 488 274.4 ± 16.5 s, Atto 565: 145.7 ± 7.5 s, Atto 647N: 127 93.0 ± 7.4 s) (Figure 1-figure supplement 1D-F). In most experiments, after initial 128 detection of the DNA molecules, the fluorophore (Atto 488) on the DNA was 129 bleached so that the same color could be re-used on one of the proteins. The bleaching 130 of the DNA fluorophore has no effect on the lifetime of Pol IIIcore on clamp-DNA 131 (on without bleaching 18.1 ± 1.6, on with bleaching 16.8 ± 1.8) (Figure 1-figure 132 supplement 1G-H). 133 134 6 12/6/17 135 Clamp loading and unloading are distinct processes 136 The isolated -clamp shows no interaction with the end-blocked DNA (Figure 137 2-figure supplement 1A-B) When combined with the clamp loader complex 138 (31'1), frequent clamp loading events are observed where the loader and clamp 139 arrive at the DNA simultaneously or in two adjacent frames (Figure 2A-B), due to the 140 sequential data acquisition of the three laser channels (further explained in Figure 2- 141 figure supplement 1C-E).