TY - JOUR
T1 - Dynamic Assembly and Disassembly of the Human DNA Polymerase δ Holoenzyme on the Genome In Vivo
AU - Drosopoulos, William C.
AU - Vierra, David A.
AU - Kenworthy, Charles A.
AU - Coleman, Robert A.
AU - Schildkraut, Carl L.
N1 - Funding Information:
We thank Kirstin Eckert and Stephen Benkovic for helpful advice. This work was supported by National Institutes of Health/National Institute of General Medical Sciences grant 5R01-GM045751 (C.L.S.) and National Institutes of Health/National Institute on Drug Abuse grant 5U01-DA047729 as part of the 4D Nucleome project (R.A.C.). D.A.V. was supported by Training Grant T-32 NIH 5T32AG023475 , and C.A.K. was supported by a NIH Medical Scientist Training Program grant T32GM007288 .
Funding Information:
We thank Kirstin Eckert and Stephen Benkovic for helpful advice. This work was supported by National Institutes of Health/National Institute of General Medical Sciences grant 5R01-GM045751 (C.L.S.) and National Institutes of Health/National Institute on Drug Abuse grant 5U01-DA047729 as part of the 4D Nucleome project (R.A.C.). D.A.V. was supported by Training Grant T-32 NIH 5T32AG023475, and C.A.K. was supported by a NIH Medical Scientist Training Program grant T32GM007288. W.C.D. R.A.C. and C.L.S. designed and supervised the study. W.C.D. generated constructs and cell lines. W.C.D. D.A.V. and C.A.K. performed experiments. R.A.C. and D.A.V. performed computational analyses of data. W.C.D. and R.A.C. analyzed the results. W.C.D. wrote the manuscript. D.A.V. contributed to the initial draft. W.C.D. R.A.C. and C.L.S. edited the manuscript. The authors declare no competing interests.
Publisher Copyright:
© 2020 The Authors
PY - 2020/2/4
Y1 - 2020/2/4
N2 - Human 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.
AB - Human 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.
KW - DNA polymerase delta holoenzyme
KW - PCNA
KW - live cell single molecule tracking
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U2 - 10.1016/j.celrep.2019.12.101
DO - 10.1016/j.celrep.2019.12.101
M3 - Article
C2 - 32023453
AN - SCOPUS:85078739217
SN - 2211-1247
VL - 30
SP - 1329-1341.e5
JO - Cell Reports
JF - Cell Reports
IS - 5
ER -