Dynamic Assembly and Disassembly of the Human DNA Polymerase δ Holoenzyme on the Genome In Vivo

William C Drosopoulos,David A Vierra,Charles A Kenworthy,Robert A Coleman,Carl L Schildkraut

doi:10.1016/j.celrep.2019.12.101

William C Drosopoulos, David A Vierra + Show 3 more

Open Access

https://doi.org/10.1016/j.celrep.2019.12.101

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Journal: Cell Reports	Publication Date: Feb 1, 2020
Citations: 21	License type: cc-by

Affiliation: Albert Einstein College of Medicine

Abstract

SUMMARYHuman DNA polymerase delta (Pol δ) forms a holoenzyme complex with the DNA sliding clamp proliferating cell nuclear antigen (PCNA) to perform its essential roles in genome replication. Here, we utilize live-cell single-molecule tracking to monitor Pol δ holoenzyme interaction with the genome in real time. We find holoenzyme assembly and disassembly in vivo are highly dynamic and ordered. PCNA generally loads onto the genome before Pol δ. Once assembled, the holoenzyme has a relatively short lifetime on the genome, implying multiple Pol δ binding events may be needed to synthesize an Okazaki fragment. During disassembly, Pol δ dissociation generally precedes PCNA unloading. We also find that Pol δ p125, the catalytic subunit of the holoenzyme, is maintained at a constant cellular level, indicating an active mechanism for control of Pol δ levels in vivo. Collectively, our studies reveal that Pol δ holoenzyme assembly and disassembly follow a predominant pathway in vivo; however, alternate pathways are observed.

Highlights

The eukaryotic genome is replicated primarily by an ensemble of three B-family DNA polymerases, namely, Pol a, polymerase delta (Pol d), and Pol ε (Burgers and Kunkel, 2017)
Leading strand synthesis is mainly performed by Pol ε, which extends the nascent strand in a continuous uninterrupted manner, whereas the lagging strand is copied in a discontinuous fashion as short (100–250 nt) Okazaki fragments by Pol d (Burgers and Kunkel, 2017)
Establishment of a System for Real-Time Imaging of Pol d Holoenzyme Assembly and Disassembly in Living Human Cells We have developed an approach to track the interactions of Pol d with nuclear chromatin and proliferating cell nuclear antigen (PCNA) based on the detection of fluorescently labeled Pol d and PCNA

Summary

Introduction

The eukaryotic genome is replicated primarily by an ensemble of three B-family DNA polymerases, namely, Pol a, Pol d, and Pol ε (Burgers and Kunkel, 2017). Active Pol d complexes can be found in cells as heterotetramers, Pol d4 (p125/p68/p50/p12), or heterotrimers, Pol d3 (p125/p68/ p50) (Figure 1A; Lee et al, 2012). Pol d4 has been shown to have higher polymerase activity than Pol d3 in vitro (Meng et al, 2010; Podust et al, 2002), implying that the d4 complex could be the major replicative assembly of Pol d. Pol d3 is generated in vivo by targeted degradation of the p12 subunit in response to UV, alkylating agents, and replication stress (Lee et al, 2012; Zhang et al, 2007), implying it is involved in DNA damage response. Pol d3 is formed during S phase in unperturbed cells, suggesting that it plays an important role in DNA replication (Chea et al, 2012; Zhang et al, 2013)

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