Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA

Lancey, Claudia; Tehseen, Muhammad; Bakshi, S. K.; Percival, Matthew; Takahashi, Masateru; Sobhy, Mohamed A.; Raducanu, Vlad-Stefan; Blair, Kerry; Muskett, Frederick W.; Ragan, Timothy J.; Crehuet, Ramón; Hamdan, Samir M.; Biasio, Alfredo De

doi:10.1038/s41467-021-26251-6

Cited by 28 publications

(37 citation statements)

References 94 publications

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“…For all reconstituted reactions, the median length of the reaction products at a particular timepoint was estimated using a modified version of the median analysis presented in refs. 15 , 88 . At each experimental timepoint, product intensities were integrated between the 1-nt SD (or NT) product and the top or bottom of the corresponding lane.…”

Section: Methodsmentioning

confidence: 99%

Mechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics

et al. 2022

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The final steps of lagging strand synthesis induce maturation of Okazaki fragments via removal of the RNA primers and ligation. Iterative cycles between Polymerase δ (Polδ) and Flap endonuclease-1 (FEN1) remove the primer, with an intermediary nick structure generated for each cycle. Here, we show that human Polδ is inefficient in releasing the nick product from FEN1, resulting in non-processive and remarkably slow RNA removal. Ligase 1 (Lig1) can release the nick from FEN1 and actively drive the reaction toward ligation. These mechanisms are coordinated by PCNA, which encircles DNA, and dynamically recruits Polδ, FEN1, and Lig1 to compete for their substrates. Our findings call for investigating additional pathways that may accelerate RNA removal in human cells, such as RNA pre-removal by RNase Hs, which, as demonstrated herein, enhances the maturation rate ~10-fold. They also suggest that FEN1 may attenuate the various activities of Polδ during DNA repair and recombination.

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Section: Methodsmentioning

confidence: 99%

Mechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics

et al. 2022

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“…In the eukaryotic replisome, when DNA is damaged, high-fidelity polymerases stall and the PCNA clamp, which is responsible for providing binding sites for the polymerases, is mono-ubiquitylated. This facilitates exchange of the replicative polymerase for a TLS polymerase and retention of TLS polymerase to the damaged sites ( Boehm et al, 2016 ; Lancey et al, 2021 ). These dynamics must be tightly regulated due to the low-fidelity of TLS polymerases.…”

Section: Protein Dynamics Provide Pathways For Lesion Bypassmentioning

confidence: 99%

“…The structural basis of the interaction of TLS polymerases with both DNA and unmodified or mono-ubitquitylated PCNA, and therefore the mechanism of TLS polymerase recruitment to sites of DNA damage, remain poorly understood. In 2021, Lancey et al (2021) identified this gap in knowledge and used cryo-EM to investigate.…”

Section: Cryo-electron Microscopymentioning

confidence: 99%

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Observing protein dynamics during DNA-lesion bypass by the replisome

Wilkinson

Spenkelink

Oijen

2022

Front. Mol. Biosci.

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Faithful DNA replication is essential for all life. A multi-protein complex called the replisome contains all the enzymatic activities required to facilitate DNA replication, including unwinding parental DNA and synthesizing two identical daughter molecules. Faithful DNA replication can be challenged by both intrinsic and extrinsic factors, which can result in roadblocks to replication, causing incomplete replication, genomic instability, and an increased mutational load. This increased mutational load can ultimately lead to a number of diseases, a notable example being cancer. A key example of a roadblock to replication is chemical modifications in the DNA caused by exposure to ultraviolet light. Protein dynamics are thought to play a crucial role to the molecular pathways that occur in the presence of such DNA lesions, including potential damage bypass. Therefore, many assays have been developed to study these dynamics. In this review, we discuss three methods that can be used to study protein dynamics during replisome–lesion encounters in replication reactions reconstituted from purified proteins. Specifically, we focus on ensemble biochemical assays, single-molecule fluorescence, and cryo-electron microscopy. We discuss two key model DNA replication systems, derived from Escherichia coli and Saccharomyces cerevisiae. The main methods of choice to study replication over the last decades have involved biochemical assays that rely on ensemble averaging. While these assays do not provide a direct readout of protein dynamics, they can often be inferred. More recently, single-molecule techniques including single-molecule fluorescence microscopy have been used to visualize replisomes encountering lesions in real time. In these experiments, individual proteins can be fluorescently labeled in order to observe the dynamics of specific proteins during DNA replication. Finally, cryo-electron microscopy can provide detailed structures of individual replisome components, which allows functional data to be interpreted in a structural context. While classic cryo-electron microscopy approaches provide static information, recent developments such as time-resolved cryo-electron microscopy help to bridge the gap between static structures and dynamic single-molecule techniques by visualizing sequential steps in biochemical pathways. In combination, these techniques will be capable of visualizing DNA replication and lesion encounter dynamics in real time, whilst observing the structural changes that facilitate these dynamics.

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“…N-HelQ proteins did not bind to heparin, collecting in flow-through fractions. Purification of human DNA polymerase d complex (PolD1-4) was as described in [28], and DNA polymerase k (PolK) as in [34].…”

Section: Protein Purificationsmentioning

confidence: 99%

Human HelQ and DNA polymerase δ interact to halt DNA synthesis and stimulate DNA single-strand annealing

Liu

Lever

Cubbon

et al. 2022

Preprint

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DNA strand breaks can be repaired by base pairing with unbroken homologous DNA, forming a template for new DNA synthesis that patches over the break site. In eukaryotes multiple DNA break repair pathways utilize DNA polymerase δ to synthesise new DNA from the end of the broken DNA strand. Here we show that DNA synthesis by human Pol δ is halted by the HelQ DNA repair protein directly targeting isolated Pol δ or Pol δ in complex with PCNA and RPA. The mechanism of inhibition by HelQ is independent of DNA binding and maps to a region of HelQ that also modulates DNA binding by RPA. Interaction of HelQ with the POLD3 subunit of Pol δ stimulated DNA single-strand annealing activity of HelQ. The data suggest a mechanism for HelQ restraining the extent of DNA synthesis across multiple DNA break repair pathways.

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Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA

Cited by 28 publications

References 94 publications

Mechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics

Mechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics

Observing protein dynamics during DNA-lesion bypass by the replisome

Human HelQ and DNA polymerase δ interact to halt DNA synthesis and stimulate DNA single-strand annealing

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