Separable, Ctf4-mediated recruitment of DNA Polymerase α for initiation of DNA synthesis at replication origins and lagging-strand priming during replication elongation

Autoři: Sarina Y. Porcella aff001;  Natasha C. Koussa aff001;  Colin P. Tang aff001;  Daphne N. Kramer aff001;  Priyanka Srivastava aff001;  Duncan J. Smith aff001
Působiště autorů: Department of Biology, New York University, New York, NY, United States of America aff001;  Department of Biology, New York University, New York, NY,United States of America aff001
Vyšlo v časopise: Separable, Ctf4-mediated recruitment of DNA Polymerase α for initiation of DNA synthesis at replication origins and lagging-strand priming during replication elongation. PLoS Genet 16(5): e32767. doi:10.1371/journal.pgen.1008755
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008755


During eukaryotic DNA replication, DNA polymerase alpha/primase (Pol α) initiates synthesis on both the leading and lagging strands. It is unknown whether leading- and lagging-strand priming are mechanistically identical, and whether Pol α associates processively or distributively with the replisome. Here, we titrate cellular levels of Pol α in S. cerevisiae and analyze Okazaki fragments to study both replication initiation and ongoing lagging-strand synthesis in vivo. We observe that both Okazaki fragment initiation and the productive firing of replication origins are sensitive to Pol α abundance, and that both processes are disrupted at similar Pol α concentrations. When the replisome adaptor protein Ctf4 is absent or cannot interact with Pol α, lagging-strand initiation is impaired at Pol α concentrations that still support normal origin firing. Additionally, we observe that activation of the checkpoint becomes essential for viability upon severe depletion of Pol α. Using strains in which the Pol α-Ctf4 interaction is disrupted, we demonstrate that this checkpoint requirement is not solely caused by reduced lagging-strand priming. Our results suggest that Pol α recruitment for replication initiation and ongoing lagging-strand priming are distinctly sensitive to the presence of Ctf4. We propose that the global changes we observe in Okazaki fragment length and origin firing efficiency are consistent with distributive association of Pol α at the replication fork, at least when Pol α is limiting.

Klíčová slova:

DNA replication – Galactose – Gel electrophoresis – Chromatin – Nucleosomes – Saccharomyces cerevisiae – Synthesis phase – Southern blot


1. Kunkel TA, Burgers PM (2008) Dividing the workload at a eukaryotic replication fork. Trends Cell Biol 18: 521–527. doi: 10.1016/j.tcb.2008.08.005 18824354

2. Clausen AR, Lujan SA, Burkholder AB, Orebaugh CD, Williams JS, Clausen MFet al. (2015) Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation. Nat Struct Mol Biol 22: 185–191. doi: 10.1038/nsmb.2957 25622295

3. Daigaku Y, Keszthelyi A, Muller CA, Miyabe I, Brooks T, Retkute Ret al. (2015) A global profile of replicative polymerase usage. Nat Struct Mol Biol 22: 192–198. doi: 10.1038/nsmb.2962 25664722

4. Pursell ZF, Isoz I, Lundstrom EB, Johansson E, Kunkel TA (2007) Yeast DNA polymerase epsilon participates in leading-strand DNA replication. Science 317: 127–130. doi: 10.1126/science.1144067 17615360

5. Reijns MA, Kemp H, Ding J, de Procé SM, Jackson AP, Taylor MS (2015) Lagging-strand replication shapes the mutational landscape of the genome. Nature 518: 502–506. doi: 10.1038/nature14183 25624100

6. Garbacz MA, Lujan SA, Burkholder AB, Cox PB, Wu Q, Zhou ZXet al. (2018) Evidence that DNA polymerase δ contributes to initiating leading strand DNA replication in Saccharomyces cerevisiae. Nat Commun 9: 858. doi: 10.1038/s41467-018-03270-4 29487291

7. Yeeles JT, Janska A, Early A, Diffley JF (2017) How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication. Mol Cell 65: 105–116. doi: 10.1016/j.molcel.2016.11.017 27989442

8. Miyabe I, Mizuno K, Keszthelyi A, Daigaku Y, Skouteri M, Mohebi S et al. (2015) Polymerase δ replicates both strands after homologous recombination-dependent fork restart. Nat Struct Mol Biol 22: 932–938. doi: 10.1038/nsmb.3100 26436826

9. Aria V, Yeeles JTP (2018) Mechanism of Bidirectional Leading-Strand Synthesis Establishment at Eukaryotic DNA Replication Origins. Mol Cell

10. Taylor MRG, Yeeles JTP (2018) The Initial Response of a Eukaryotic Replisome to DNA Damage. Molecular Cell 70: 1067–1080. doi: 10.1016/j.molcel.2018.04.022 29944888

11. Balakrishnan L, Bambara RA (2013) Okazaki fragment metabolism. Cold Spring Harb Perspect Biol 5:; doi: 10.1101/cshperspect.a010173 23378587

12. Ogawa T, Okazaki T (1980) Discontinuous DNA replication. Annu Rev Biochem 49: 421–457. doi: 10.1146/ 6250445

13. Smith DJ, Whitehouse I (2012) Intrinsic coupling of lagging-strand synthesis to chromatin assembly. Nature 483: 434–438. doi: 10.1038/nature10895 22419157

14. Pourkarimi E, Bellush JM, Whitehouse I (2016) Spatiotemporal coupling and decoupling of gene transcription with DNA replication origins during embryogenesis in C. elegans. Elife 5:

15. Devbhandari S, Jiang J, Kumar C, Whitehouse I, Remus D (2017) Chromatin Constrains the Initiation and Elongation of DNA Replication. Mol Cell 65: 131–141. doi: 10.1016/j.molcel.2016.10.035 27989437

16. Kurat CF, Yeeles JT, Patel H, Early A, Diffley JF (2017) Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates. Mol Cell 65: 117–130. doi: 10.1016/j.molcel.2016.11.016 27989438

17. Wu CA, Zechner EL, Reems JA, McHenry CS, Marians KJ (1992) Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. V. Primase action regulates the cycle of Okazaki fragment synthesis. J Biol Chem 267: 4074–4083. 1740453

18. Yadav T, Whitehouse I (2016) Replication-Coupled Nucleosome Assembly and Positioning by ATP-Dependent Chromatin-Remodeling Enzymes. Cell Rep

19. Kunkel TA (2011) Balancing eukaryotic replication asymmetry with replication fidelity. Curr Opin Chem Biol 15: 620–626. doi: 10.1016/j.cbpa.2011.07.025 21862387

20. Glover TW, Berger C, Coyle J, Echo B (1984) DNA polymerase alpha inhibition by aphidicolin induces gaps and breaks at common fragile sites in human chromosomes. Hum Genet 67: 136–142. doi: 10.1007/BF00272988 6430783

21. Lemoine FJ, Degtyareva NP, Lobachev K, Petes TD (2005) Chromosomal translocations in yeast induced by low levels of DNA polymerase a model for chromosome fragile sites. Cell 120: 587–598. doi: 10.1016/j.cell.2004.12.039 15766523

22. Song W, Dominska M, Greenwell PW, Petes TD (2014) Genome-wide high-resolution mapping of chromosome fragile sites in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 111: E2210–8. doi: 10.1073/pnas.1406847111 24799712

23. Kouprina N, Kroll E, Bannikov V, Bliskovsky V, Gizatullin R, Kirillov Aet al. (1992) CTF4 (CHL15) mutants exhibit defective DNA metabolism in the yeast Saccharomyces cerevisiae. Mol Cell Biol 12: 5736–5747. doi: 10.1128/mcb.12.12.5736 1341195

24. Borges V, Smith DJ, Whitehouse I, Uhlmann F (2013) An Eco1-independent sister chromatid cohesion establishment pathway in S. cerevisiae. Chromosoma 122: 121–134. doi: 10.1007/s00412-013-0396-y 23334284

25. Hanna JS, Kroll ES, Lundblad V, Spencer FA (2001) Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion. Mol Cell Biol 21: 3144–3158. doi: 10.1128/MCB.21.9.3144-3158.2001 11287619

26. Gambus A, van Deursen F, Polychronopoulos D, Foltman M, Jones RC, Edmondson RD et al. (2009) A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase alpha within the eukaryotic replisome. EMBO J 28: 2992–3004. doi: 10.1038/emboj.2009.226 19661920

27. Tanaka H, Katou Y, Yagura M, Saitoh K, Itoh T, Araki H et al. (2009) Ctf4 coordinates the progression of helicase and DNA polymerase alpha. Genes Cells 14: 807–820. doi: 10.1111/j.1365-2443.2009.01310.x 19496828

28. Fumasoni M, Zwicky K, Vanoli F, Lopes M, Branzei D (2015) Error-Free DNA Damage Tolerance and Sister Chromatid Proximity during DNA Replication Rely on the Polalpha/Primase/Ctf4 Complex. Mol Cell 57: 812–823. doi: 10.1016/j.molcel.2014.12.038 25661486

29. Villa F, Simon AC, Ortiz Bazan MA, Kilkenny ML, Wirthensohn D, Wightman M et al. (2016) Ctf4 Is a Hub in the Eukaryotic Replisome that Links Multiple CIP-Box Proteins to the CMG Helicase. Mol Cell 63: 385–396. doi: 10.1016/j.molcel.2016.06.009 27397685

30. Zhu W, Ukomadu C, Jha S, Senga T, Dhar SK, Wohlschlegel JA et al. (2007) Mcm10 and And-1/CTF4 recruit DNA polymerase alpha to chromatin for initiation of DNA replication. Genes Dev 21: 2288–2299. doi: 10.1101/gad.1585607 17761813

31. Muzi Falconi M, Piseri A, Ferrari M, Lucchini G, Plevani P, Foiani M (1993) De novo synthesis of budding yeast DNA polymerase alpha and POL1 transcription at the G1/S boundary are not required for entrance into S phase. Proc Natl Acad Sci U S A 90: 10519–10523. doi: 10.1073/pnas.90.22.10519 8248139

32. Haruki H, Nishikawa J, Laemmli UK (2008) The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes. Mol Cell 31: 925–932. doi: 10.1016/j.molcel.2008.07.020 18922474

33. Foltman M, Evrin C, De Piccoli G, Jones RC, Edmondson RD, Katou Yet al. (2013) Eukaryotic replisome components cooperate to process histones during chromosome replication. Cell Rep 3: 892–904. doi: 10.1016/j.celrep.2013.02.028 23499444

34. Evrin C, Maman JD, Diamante A, Pellegrini L, Labib K (2018) Histone H2A-H2B binding by Pol α in the eukaryotic replisome contributes to the maintenance of repressive chromatin. EMBO J 37:

35. McGuffee SR, Smith DJ, Whitehouse I (2013) Quantitative, Genome-Wide Analysis of Eukaryotic Replication Initiation and Termination. Mol Cell 50: 123–135. doi: 10.1016/j.molcel.2013.03.004 23562327

36. Petryk N, Kahli M, d’Aubenton-Carafa Y, Jaszczyszyn Y, Shen Y, Silvain M et al. (2016) Replication landscape of the human genome. Nat Commun 7: 10208. doi: 10.1038/ncomms10208 26751768

37. Ivessa AS, Lenzmeier BA, Bessler JB, Goudsouzian LK, Schnakenberg SL, Zakian VA (2003) The Saccharomyces cerevisiae helicase Rrm3p facilitates replication past nonhistone protein-DNA complexes. Mol Cell 12: 1525–1536. doi: 10.1016/s1097-2765(03)00456-8 14690605

38. Osmundson JS, Kumar J, Yeung R, Smith DJ (2017) Pif1-family helicases cooperatively suppress widespread replication-fork arrest at tRNA genes. Nat Struct Mol Biol 24: 162–170. doi: 10.1038/nsmb.3342 27991904

39. Samora C, Saksouk J, Goswami P, Wade B, Singleton M, Bates P et al. (2016) Ctf4 Links DNA Replication with Sister Chromatid Cohesion Establishment by Recruiting the Chl1 Helicase to the Replisome. Molecular Cell 63: 371–384. doi: 10.1016/j.molcel.2016.05.036 27397686

40. Simon AC, Zhou JC, Perera RL, van D, Frederick, Evrin C, Ivanova MEet al. (2014) A Ctf4 trimer couples the CMG helicase to DNA polymerase α in the eukaryotic replisome. Nature 510: 293–297. doi: 10.1038/nature13234 24805245

41. Sasaki M, Kobayashi T (2017) Ctf4 Prevents Genome Rearrangements by Suppressing DNA Double-Strand Break Formation and Its End Resection at Arrested Replication Forks. Molecular Cell 66: 533–545.e5. doi: 10.1016/j.molcel.2017.04.020 28525744

42. Kwan EX, Wang XS, Amemiya HM, Brewer BJ, Raghuraman MK (2016) rDNA Copy Number Variants Are Frequent Passenger Mutations in Saccharomyces cerevisiae Deletion Collections and de Novo Transformants. G3 (Bethesda) 6: 2829–2838.

43. Shyian M, Mattarocci S, Albert B, Hafner L, Lezaja A, Costanzo Met al. (2016) Budding Yeast Rif1 Controls Genome Integrity by Inhibiting rDNA Replication. PLoS Genet 12: e1006414. doi: 10.1371/journal.pgen.1006414 27820830

44. Yoshida K, Bacal J, Desmarais D, Padioleau I, Tsaponina O, Chabes Aet al. (2014) The histone deacetylases sir2 and rpd3 act on ribosomal DNA to control the replication program in budding yeast. Mol Cell 54: 691–697. doi: 10.1016/j.molcel.2014.04.032 24856221

45. Ciccia A, Elledge SJ (2010) The DNA damage response: making it safe to play with knives. Mol Cell 40: 179–204. doi: 10.1016/j.molcel.2010.09.019 20965415

46. Osborn AJ, Elledge SJ (2003) Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53. Genes Dev 17: 1755–1767. doi: 10.1101/gad.1098303 12865299

47. Szyjka SJ, Viggiani CJ, Aparicio OM (2005) Mrc1 is required for normal progression of replication forks throughout chromatin in S. cerevisiae. Mol Cell 19: 691–697. doi: 10.1016/j.molcel.2005.06.037 16137624

48. Dubarry M, Lawless C, Banks AP, Cockell S, Lydall D (2015) Genetic Networks Required to Coordinate Chromosome Replication by DNA Polymerases α, δ, and ε in Saccharomyces cerevisiae. G3 (Bethesda) 5: 2187–2197.

49. Shah KA, Shishkin AA, Voineagu I, Pavlov YI, Shcherbakova PV, Mirkin SM (2012) Role of DNA polymerases in repeat-mediated genome instability. Cell Rep 2: 1088–1095. doi: 10.1016/j.celrep.2012.10.006 23142667

50. Van Esch H, Colnaghi R, Freson K, Starokadomskyy P, Zankl A, Backx L et al. (2019) Defective DNA Polymerase α-Primase Leads to X-Linked Intellectual Disability Associated with Severe Growth Retardation, Microcephaly, and Hypogonadism. Am J Hum Genet 104: 957–967. doi: 10.1016/j.ajhg.2019.03.006 31006512

51. Bellelli R, Borel V, Logan C, Svendsen J, Cox DE, Nye E et al. (2018) Polε Instability Drives Replication Stress, Abnormal Development, and Tumorigenesis. Mol Cell

52. Chen YH, Keegan S, Kahli M, Tonzi P, Fenyö D, Huang TTet al. (2019) Transcription shapes DNA replication initiation and termination in human cells. Nat Struct Mol Biol 26: 67–77. doi: 10.1038/s41594-018-0171-0 30598550

53. Hamperl S, Bocek MJ, Saldivar JC, Swigut T, Cimprich KA (2017) Transcription-Replication Conflict Orientation Modulates R-Loop Levels and Activates Distinct DNA Damage Responses. Cell 170: 774–786.e19. doi: 10.1016/j.cell.2017.07.043 28802045

54. Tran PLT, Pohl TJ, Chen CF, Chan A, Pott S, Zakian VA (2017) PIF1 family DNA helicases suppress R-loop mediated genome instability at tRNA genes. Nat Commun 8: 15025. doi: 10.1038/ncomms15025 28429714

55. Dicarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res

56. Jiang C, Pugh BF (2009) A compiled and systematic reference map of nucleosome positions across the Saccharomyces cerevisiae genome. Genome Biol 10: R109. doi: 10.1186/gb-2009-10-10-r109 19814794

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