In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. We present the high-resolution cryo-EM structure of the human processive Pol δ-DNA-PCNA complex in the absence and presence of FEN1. Pol δ is anchored to one of the three PCNA monomers through the C-terminal domain of the catalytic subunit. The catalytic core sits on top of PCNA in an open configuration while the regulatory subunits project laterally. This arrangement allows PCNA to thread and stabilize the DNA exiting the catalytic cleft and recruit FEN1 to one unoccupied monomer in a toolbelt fashion. Alternative holoenzyme conformations reveal important functional interactions that maintain PCNA orientation during synthesis. This work sheds light on the structural basis of Pol δ's activity in replicating the human genome.

Protein-induced fluorescence enhancement (PIFE) is a popular tool for characterizing protein-DNA interactions. PIFE has been explained by an increase in local viscosity due to the presence of the protein residues. This explanation, however, denies the opposite effect of fluorescence quenching. This work offers a perspective for understanding PIFE mechanism and reports the observation of a phenomenon that we name protein-induced fluorescence quenching (PIFQ), which exhibits an opposite effect to PIFE. A detailed characterization of these two fluorescence modulations reveals that the initial fluorescence state of the labeled mediator (DNA) determines whether this mediator-conjugated dye undergoes PIFE or PIFQ upon protein binding. This key role of the mediator DNA provides a protocol for the experimental design to obtain either PIFQ or PIFE, on-demand. This makes the arbitrary nature of the current experimental design obsolete, allowing for proper integration of both PIFE and PIFQ with existing bulk and single-molecule fluorescence techniques.

In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. We present the high-resolution cryo-EM structure of the human processive Pol δ-DNA-PCNA complex in the absence and presence of FEN1. Pol δ is anchored to one of the three PCNA monomers through the C-terminal domain of the catalytic subunit. The catalytic core sits on top of PCNA in an open configuration while the regulatory subunits project laterally. This arrangement allows PCNA to thread and stabilize the DNA exiting the catalytic cleft and recruit FEN1 to one unoccupied monomer in a toolbelt fashion. Alternative holoenzyme conformations reveal important functional interactions that maintain PCNA orientation during synthesis. This work sheds light on the structural basis of Pol δ's activity in replicating the human genome.Three DNA polymerases (Pols), a, d and e replicate the genomic DNA in eukaryotes, with the latter two possessing the proofreading exonuclease activity required for high fidelity DNA synthesis 1,2 . Pol d replicates the lagging strand and may share a role with Pol e in replicating the leading strand [3][4][5][6][7][8][9][10] . In contrast to the continuous leading strand synthesis, the lagging strand is synthesized discontinuously in ~200 nucleotide (nt)-long Okazaki fragments, which are then ligated to form the contiguous lagging strand 2 .Synthesis of each Okazaki fragment starts with the low fidelity Pol a synthesizing a ~30 nt RNA/DNA initiator primer. Replication factor C (RFC) then loads the homotrimeric clamp PCNA at the primer/template (P/T) junction 11 . PCNA encircles the duplex DNA 3 and tethers Pol d to the DNA enhancing its processivity from few nucleotides to hundreds of nucleotides per DNA binding event [12][13][14] . PCNA has also been shown to increase the nucleotide incorporation rate of Pol d 15 . Additionally, the interaction of PCNA with Pol d is critical for coordinating its transient replacement by other PCNA partner proteins. In the maturation of Okazaki fragments, Pol d invades the previously synthesized Okazaki fragments to gradually displace the RNA-DNA primers for their removal by the PCNA-bound FEN1 16 . In translesion DNA synthesis, Pol d is transiently replaced by a PCNA-bound translesion DNA polymerase to ensure the continuation of DNA replication 17 .Mammalian Pol d consists of a catalytic subunit and three regulatory subunits ( Figure 1a). The catalytic subunit (p125) harbours the polymerase and exonuclease activities, and a metal-binding C-terminal domain (CTD). The regulatory subunits (p50, also referred to as the B-subunit, p66 and p12) are required for optimal activity of the holoenzyme 18 and there is evidence of different context-specific subassemblies of Pol d in vivo [19][20][21] . In particular, DNA damage or replication stress triggers the degradation of the p12 subunit, resulting in the formation of a three-subunit enzyme with an...

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Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage.