Resolving Individual Steps of Okazaki-fragment Maturation at a Millisecond Timescale

Overview

Journal Nat Struct Mol Biol

Specialties Cell Biology
Molecular Biology

Date 2016 Apr 12

PMID 27065195

Citations 68

Authors

Joseph L Stodola

Peter M Burgers

Affiliations

Soon will be listed here.

Abstract

DNA polymerase delta (Pol δ) is responsible for elongation and maturation of Okazaki fragments. Pol δ and the flap endonuclease FEN1, coordinated by the PCNA clamp, remove RNA primers and produce ligatable nicks. We studied this process in the Saccharomyces cerevisiae machinery at millisecond resolution. During elongation, PCNA increased the Pol δ catalytic rate by >30-fold. When Pol δ invaded double-stranded RNA-DNA representing unmatured Okazaki fragments, the incorporation rate of each nucleotide decreased successively to 10-20% that of the preceding nucleotide. Thus, the nascent flap acts as a progressive molecular brake on the polymerase, and consequently FEN1 cuts predominantly single-nucleotide flaps. Kinetic and enzyme-trapping experiments support a model in which a stable PCNA-DNA-Pol δ-FEN1 complex moves processively through iterative steps of nick translation, ultimately completely removing primer RNA. Finally, whereas elongation rates are under dynamic dNTP control, maturation rates are buffered against changes in dNTP concentrations.

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References

Gomes X, Gary S, Burgers P . Overproduction in Escherichia coli and characterization of yeast replication factor C lacking the ligase homology domain. J Biol Chem. 2000; 275(19):14541-9. DOI: 10.1074/jbc.275.19.14541. View

Gomes X, Burgers P . Two modes of FEN1 binding to PCNA regulated by DNA. EMBO J. 2000; 19(14):3811-21. PMC: 313985. DOI: 10.1093/emboj/19.14.3811. View

Jin Y, Obert R, Burgers P, Kunkel T, Resnick M, Gordenin D . The 3'-->5' exonuclease of DNA polymerase delta can substitute for the 5' flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability. Proc Natl Acad Sci U S A. 2001; 98(9):5122-7. PMC: 33174. DOI: 10.1073/pnas.091095198. View

Bhagwat M, Nossal N . Bacteriophage T4 RNase H removes both RNA primers and adjacent DNA from the 5' end of lagging strand fragments. J Biol Chem. 2001; 276(30):28516-24. DOI: 10.1074/jbc.M103914200. View

Raghuraman M, Winzeler E, Collingwood D, Hunt S, Wodicka L, Conway A . Replication dynamics of the yeast genome. Science. 2001; 294(5540):115-21. DOI: 10.1126/science.294.5540.115. View

Kao H, Henricksen L, Liu Y, Bambara R . Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate. J Biol Chem. 2002; 277(17):14379-89. DOI: 10.1074/jbc.M110662200. View

Lu X, Tan C, Zhou J, You M, Carastro L, Downey K . Direct interaction of proliferating cell nuclear antigen with the small subunit of DNA polymerase delta. J Biol Chem. 2002; 277(27):24340-5. DOI: 10.1074/jbc.M200065200. View

Bermudez V, MacNeill S, Tappin I, Hurwitz J . The influence of the Cdc27 subunit on the properties of the Schizosaccharomyces pombe DNA polymerase delta. J Biol Chem. 2002; 277(39):36853-62. DOI: 10.1074/jbc.M202897200. View

Ayyagari R, Gomes X, Gordenin D, Burgers P . Okazaki fragment maturation in yeast. I. Distribution of functions between FEN1 AND DNA2. J Biol Chem. 2002; 278(3):1618-25. DOI: 10.1074/jbc.M209801200. View

10.

Chabes A, Georgieva B, Domkin V, Zhao X, Rothstein R, Thelander L . Survival of DNA damage in yeast directly depends on increased dNTP levels allowed by relaxed feedback inhibition of ribonucleotide reductase. Cell. 2003; 112(3):391-401. DOI: 10.1016/s0092-8674(03)00075-8. View

11.

Chapados B, Hosfield D, Han S, Qiu J, Yelent B, Shen B . Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair. Cell. 2004; 116(1):39-50. DOI: 10.1016/s0092-8674(03)01036-5. View

12.

Kunkel T, Sabatino R, Bambara R . Exonucleolytic proofreading by calf thymus DNA polymerase delta. Proc Natl Acad Sci U S A. 1987; 84(14):4865-9. PMC: 305206. DOI: 10.1073/pnas.84.14.4865. View

13.

Tsurimoto T, Stillman B . Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4. Proc Natl Acad Sci U S A. 1990; 87(3):1023-7. PMC: 53402. DOI: 10.1073/pnas.87.3.1023. View

14.

Burgers P . Saccharomyces cerevisiae replication factor C. II. Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases delta and epsilon. J Biol Chem. 1991; 266(33):22698-706. View

15.

Capson T, Peliska J, Kaboord B, Frey M, LIVELY C, Dahlberg M . Kinetic characterization of the polymerase and exonuclease activities of the gene 43 protein of bacteriophage T4. Biochemistry. 1992; 31(45):10984-94. DOI: 10.1021/bi00160a007. View

16.

Henricksen L, Umbricht C, Wold M . Recombinant replication protein A: expression, complex formation, and functional characterization. J Biol Chem. 1994; 269(15):11121-32. View

17.

Podust V, Podust L, Muller F, Hubscher U . DNA polymerase delta holoenzyme: action on single-stranded DNA and on double-stranded DNA in the presence of replicative DNA helicases. Biochemistry. 1995; 34(15):5003-10. DOI: 10.1021/bi00015a011. View

18.

Sugimoto N, Nakano S, Yoneyama M, Honda K . Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes. Nucleic Acids Res. 1996; 24(22):4501-5. PMC: 146261. DOI: 10.1093/nar/24.22.4501. View

19.

Eissenberg J, Ayyagari R, Gomes X, Burgers P . Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase delta and DNA polymerase epsilon. Mol Cell Biol. 1997; 17(11):6367-78. PMC: 232488. DOI: 10.1128/MCB.17.11.6367. View

20.

Montecucco A, Rossi R, Levin D, Gary R, Park M, Motycka T . DNA ligase I is recruited to sites of DNA replication by an interaction with proliferating cell nuclear antigen: identification of a common targeting mechanism for the assembly of replication factories. EMBO J. 1998; 17(13):3786-95. PMC: 1170714. DOI: 10.1093/emboj/17.13.3786. View