title: Stability of the human polymerase δ holoenzyme and its implications in lagging strand DNA synthesis.
abstract: In eukaryotes, DNA polymerase δ (pol δ) is responsible for replicating the lagging strand template and anchors to the proliferating cell nuclear antigen (PCNA) sliding clamp to form a holoenzyme. The stability of this complex is integral to every aspect of lagging strand replication. Most of our understanding comes from Saccharomyces cerevisae where the extreme stability of the pol δ holoenzyme ensures that every nucleobase within an Okazaki fragment is faithfully duplicated before dissociation but also necessitates an active displacement mechanism for polymerase recycling and exchange. However, the stability of the human pol δ holoenzyme is unknown. We designed unique kinetic assays to analyze the processivity and stability of the pol δ holoenzyme. Surprisingly, the results indicate that human pol δ maintains a loose association with PCNA while replicating DNA. Such behavior has profound implications on Okazaki fragment synthesis in humans as it limits the processivity of pol δ on undamaged DNA and promotes the rapid dissociation of pol δ from PCNA on stalling at a DNA lesion.
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, PA 16802.
authors.forename: Mark
authors.initials: M
authors.lastname: Hedglin
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, PA 16802.
authors.forename: Binod
authors.initials: B
authors.lastname: Pandey
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, PA 16802 sjb1@psu.edu.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D004247:DNA / Q000096:biosynthesis*; D004258:DNA Polymerase III / Q000737:chemistry* / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004795:Enzyme Stability; D031541:Fluorescence Resonance Energy Transfer; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism; D051818:Replication Protein C / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D017931:DNA Primers; D004277:DNA, Single-Stranded; D020035:Holoenzymes; D018809:Proliferating Cell Nuclear Antigen; D051716:Replication Protein A; D004247:DNA; D004258:DNA Polymerase III; D051818:Replication Protein C
keywords: DNA polymerase delta; PCNA; lagging strand; stability; translesion DNA synthesis