Mechanism of Lagging-Strand DNA Replication in Eukaryotes

  • Joseph L. Stodola
  • Peter M. BurgersEmail author
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1042)


This chapter focuses on the enzymes and mechanisms involved in lagging-strand DNA replication in eukaryotic cells. Recent structural and biochemical progress with DNA polymerase α-primase (Pol α) provides insights how each of the millions of Okazaki fragments in a mammalian cell is primed by the primase subunit and further extended by its polymerase subunit. Rapid kinetic studies of Okazaki fragment elongation by Pol δ illuminate events when the polymerase encounters the double-stranded RNA-DNA block of the preceding Okazaki fragment. This block acts as a progressive molecular break that provides both time and opportunity for the flap endonuclease 1 (FEN1) to access the nascent flap and cut it. The iterative action of Pol δ and FEN1 is coordinated by the replication clamp PCNA and produces a regulated degradation of the RNA primer, thereby preventing the formation of long-strand displacement flaps. Occasional long flaps are further processed by backup nucleases including Dna2.


DNA replication Lagging strand Okazaki fragment maturation DNA polymerase α-primase DNA polymerase δ Flap endonuclease 1 Dna2 



The research in the authors’ laboratory is supported by grants from the US National Institutes of Health (GM032431, GM083970, and GM118129 to P.B). J.S. was supported in part by a grant from the USA-Israel Binational Science Foundation (2013358).


  1. Agarkar VB, Babayeva ND, Pavlov YI, Tahirov TH (2011) Crystal structure of the C-terminal domain of human DNA primase large subunit: implications for the mechanism of the primase-polymerase alpha switch. Cell Cycle 10(6):926–931. PubMedPubMedCentralCrossRefGoogle Scholar
  2. Ayyagari R, Gomes XV, Gordenin DA, Burgers PM (2003) Okazaki fragment maturation in yeast. I. Distribution of functions between FEN1 AND DNA2. J Biol Chem 278(3):1618–1625PubMedCrossRefGoogle Scholar
  3. Bae SH, Choi E, Lee KH, Park JS, Lee SH, Seo YS (1998) Dna2 of Saccharomyces cerevisiae possesses a single-stranded DNA-specific endonuclease activity that is able to act on double-stranded DNA in the presence of ATP. J Biol Chem 273(41):26880–26890PubMedCrossRefGoogle Scholar
  4. Bae SH, Bae KH, Kim JA, Seo YS (2001a) RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes. Nature 412(6845):456–461PubMedCrossRefGoogle Scholar
  5. Bae SH, Kim JA, Choi E, Lee KH, Kang HY, Kim HD, Kim JH, Bae KH, Cho Y, Park C, Seo YS (2001b) Tripartite structure of Saccharomyces cerevisiae Dna2 helicase/endonuclease. Nucleic Acids Res 29(14):3069–3079PubMedPubMedCentralCrossRefGoogle Scholar
  6. Balakrishnan L, Bambara RA (2010) Eukaryotic lagging strand DNA replication employs a multi-pathway mechanism that protects genome integrity. J Biol Chem 286(9):6865–6870. R110.209502 [pii] 10.1074/jbc.R110.209502 PubMedPubMedCentralCrossRefGoogle Scholar
  7. Balakrishnan L, Bambara RA (2013) Flap endonuclease 1. Annu Rev Biochem 82:119–138. PubMedPubMedCentralCrossRefGoogle Scholar
  8. Baranovskiy AG, Zhang Y, Suwa Y, Babayeva ND, Gu J, Pavlov YI, Tahirov TH (2015) Crystal structure of the human primase. J Biol Chem 290(9):5635–5646. PubMedCrossRefGoogle Scholar
  9. Baranovskiy AG, Babayeva ND, Zhang Y, Gu J, Suwa Y, Pavlov YI, Tahirov TH (2016a) Mechanism of concerted Rna-DNA primer synthesis by the human primosome. J Biol Chem.
  10. Baranovskiy AG, Zhang Y, Suwa Y, Gu J, Babayeva ND, Pavlov YI, Tahirov TH (2016b) Insight into the human DNA primase interaction with template-primer. J Biol Chem 291(9):4793–4802. PubMedCrossRefGoogle Scholar
  11. Budd ME, Campbell JL (1997) A yeast replicative helicase, Dna2 helicase, interacts with yeast FEN-1 nuclease in carrying out its essential function. Mol Cell Biol 17(4):2136–2142PubMedPubMedCentralCrossRefGoogle Scholar
  12. Budd ME, Choe W-C, Campbell J (1995) DNA2 encodes a DNA helicase essential for replication of eukaryotic chromosomes. J Biol Chem 270(45):26766–26769PubMedCrossRefGoogle Scholar
  13. Budd ME, Choe W, Campbell JL (2000) The nuclease activity of the yeast DNA2 protein, which is related to the RecB-like nucleases, is essential in vivo. J Biol Chem 275(22):16518–16529PubMedCrossRefGoogle Scholar
  14. Budd ME, Reis CC, Smith S, Myung K, Campbell JL (2006) Evidence suggesting that Pif1 helicase functions in DNA replication with the Dna2 helicase/nuclease and DNA polymerase delta. Mol Cell Biol 26(7):2490–2500PubMedPubMedCentralCrossRefGoogle Scholar
  15. Bullock PA, Seo YS, Hurwitz J (1991) Initiation of simian virus 40 DNA synthesis in vitro. Mol Cell Biol 11(5):2350–2361PubMedPubMedCentralCrossRefGoogle Scholar
  16. Burgers PM (2009) Polymerase dynamics at the eukaryotic DNA replication fork. J Biol Chem 284(7):4041–4045PubMedPubMedCentralCrossRefGoogle Scholar
  17. Burgers PM, Kunkel TA (2017) Eukaryotic DNA replication fork. Annu Rev Biochem.
  18. Burgers PM, Gordenin D, Kunkel TA (2016) Who is leading the replication fork, Pol epsilon or Pol delta? Mol Cell 61(4):492–493. PubMedPubMedCentralCrossRefGoogle Scholar
  19. Clausen AR, Lujan SA, Burkholder AB, Orebaugh CD, Williams JS, Clausen MF, Malc EP, Mieczkowski PA, Fargo DC, Smith DJ, Kunkel TA (2015) Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation. Nat Struct Mol Biol 22(3):185–191. nsmb.2957 [pii] 10.1038/nsmb.2957 PubMedPubMedCentralCrossRefGoogle Scholar
  20. Daigaku Y, Keszthelyi A, Muller CA, Miyabe I, Brooks T, Retkute R, Hubank M, Nieduszynski CA, Carr AM (2015) A global profile of replicative polymerase usage. Nat Struct Mol Biol 22(3):192–198. PubMedPubMedCentralCrossRefGoogle Scholar
  21. Devbhandari S, Jiang J, Kumar C, Whitehouse I, Remus D (2017) Chromatin constrains the initiation and elongation of DNA replication. Mol Cell 65(1):131–141. PubMedCrossRefGoogle Scholar
  22. Duxin JP, Dao B, Martinsson P, Rajala N, Guittat L, Campbell JL, Spelbrink JN, Stewart SA (2009) Human Dna2 is a nuclear and mitochondrial DNA maintenance protein. Mol Cell Biol 29(15):4274–4282. MCB.01834-08 [pii] 10.1128/MCB.01834-08 PubMedPubMedCentralCrossRefGoogle Scholar
  23. Duxin JP, Moore HR, Sidorova J, Karanja K, Honaker Y, Dao B, Piwnica-Worms H, Campbell JL, Monnat RJ Jr, Stewart SA (2012) Okazaki fragment processing-independent role for human Dna2 enzyme during DNA replication. J Biol Chem 287(26):21980–21991. PubMedPubMedCentralCrossRefGoogle Scholar
  24. Eki T, Matsumoto T, Murakami Y, Hurwitz J (1992) The replication of DNA containing the simian virus 40 origin by the monopolymerase and dipolymerase systems. J Biol Chem 267(11):7284–7294PubMedGoogle Scholar
  25. Ganai RA, Zhang XP, Heyer WD, Johansson E (2016) Strand displacement synthesis by yeast DNA polymerase epsilon. Nucleic Acids Res.
  26. Garg P, Stith CM, Sabouri N, Johansson E, Burgers PM (2004) Idling by DNA polymerase delta maintains a ligatable nick during lagging-strand DNA replication. Genes Dev 18(22):2764–2773PubMedPubMedCentralCrossRefGoogle Scholar
  27. Georgescu RE, Langston L, Yao NY, Yurieva O, Zhang D, Finkelstein J, Agarwal T, O’Donnell ME (2014a) Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork. Nat Struct Mol Biol 21(8):664–670. PubMedPubMedCentralCrossRefGoogle Scholar
  28. Georgescu RE, Yao N, Indiani C, Yurieva O, O’Donnell ME (2014b) Replisome mechanics: lagging strand events that influence speed and processivity. Nucleic Acids Res 42(10):6497–6510. PubMedPubMedCentralCrossRefGoogle Scholar
  29. Georgescu RE, Schauer GD, Yao NY, Langston LD, Yurieva O, Zhang D, Finkelstein J, O’Donnell ME (2015) Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation. Elife 4:e04988. PubMedPubMedCentralCrossRefGoogle Scholar
  30. Gomes XV, Burgers PM (2001) ATP utilization by yeast replication factor C. I. ATP-mediated interaction with DNA and with proliferating cell nuclear antigen. J Biol Chem 276(37):34768–34775PubMedCrossRefGoogle Scholar
  31. Grasby JA, Finger LD, Tsutakawa SE, Atack JM, Tainer JA (2012) Unpairing and gating: sequence-independent substrate recognition by FEN superfamily nucleases. Trends Biochem Sci 37(2):74–84. PubMedCrossRefGoogle Scholar
  32. Howes TR, Tomkinson AE (2012) DNA ligase I, the replicative DNA ligase. Subcell Biochem 62:327–341. PubMedCrossRefGoogle Scholar
  33. Jin YH, Obert R, Burgers PM, Kunkel TA, Resnick MA, Gordenin DA (2001) 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 98(9):5122–5127PubMedPubMedCentralCrossRefGoogle Scholar
  34. Jin YH, Ayyagari R, Resnick MA, Gordenin DA, Burgers PM (2003) Okazaki fragment maturation in yeast. II. Cooperation between the polymerase and 3′-5′-exonuclease activities of Pol delta in the creation of a ligatable nick. J Biol Chem 278(3):1626–1633PubMedCrossRefGoogle Scholar
  35. Jin YH, Garg P, Stith CM, Al Refai H, Sterling J, Weston L, Kunkel T, Resnick MA, Burgers PM, Gordenin DA (2005) The multiple biological roles for the 3′-5′-exonuclease of DNA polymerase d require switching between the polymerase and exonuclease domains. Mol Cell Biol 25:461–471PubMedPubMedCentralCrossRefGoogle Scholar
  36. Johansson E, Dixon N (2013) Replicative DNA polymerases. Cold Spring Harb Perspect Biol 5(6):1–14. 5/6/a012799 [pii] 10.1101/cshperspect.a012799 CrossRefGoogle Scholar
  37. Johnson RE, Klassen R, Prakash L, Prakash S (2015) A major role of DNA polymerase delta in replication of both the leading and lagging DNA strands. Mol Cell 59(2):163–175. S1097-2765(15)00439-6 [pii] 10.1016/j.molcel.2015.05.038 PubMedPubMedCentralCrossRefGoogle Scholar
  38. Johnson RE, Klassen R, Prakash L, Prakash S (2016) Response to Burgers et al. Mol Cell 61(4):494–495. PubMedCrossRefGoogle Scholar
  39. Kadyrov FA, Genschel J, Fang Y, Penland E, Edelmann W, Modrich P (2009) A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair. Proc Natl Acad Sci U S A 106(21):8495–8500. PubMedPubMedCentralCrossRefGoogle Scholar
  40. Kaiser MW, Lyamicheva N, Ma W, Miller C, Neri B, Fors L, Lyamichev VI (1999) A comparison of eubacterial and archaeal structure-specific 5′-exonucleases. J Biol Chem 274(30):21387–21394PubMedCrossRefGoogle Scholar
  41. Kang HY, Choi E, Bae SH, Lee KH, Gim BS, Kim HD, Park C, MacNeill SA, Seo YS (2000) Genetic analyses of Schizosaccharomyces pombe dna2(+) reveal that dna2 plays an essential role in Okazaki fragment metabolism. Genetics 155(3):1055–1067PubMedPubMedCentralGoogle Scholar
  42. Kang YH, Lee CH, Seo YS (2010) Dna2 on the road to Okazaki fragment processing and genome stability in eukaryotes. Crit Rev Biochem Mol Biol 45(2):71–96. PubMedCrossRefGoogle Scholar
  43. Kao HI, Henricksen LA, Liu Y, Bambara RA (2002) Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate. J Biol Chem 277(17):14379–14389PubMedCrossRefGoogle Scholar
  44. Kao HI, Campbell JL, Bambara RA (2004) Dna2p helicase/nuclease is a tracking protein, like FEN1, for flap cleavage during Okazaki fragment maturation. J Biol Chem 279(49):50840–50849. PubMedCrossRefGoogle Scholar
  45. Kilkenny ML, De Piccoli G, Perera RL, Labib K, Pellegrini L (2012) A conserved motif in the C-terminal tail of DNA polymerase alpha tethers primase to the eukaryotic replisome. J Biol Chem 287(28):23740–23747. M112.368951 [pii] 10.1074/jbc.M112.368951 PubMedPubMedCentralCrossRefGoogle Scholar
  46. Kilkenny ML, Longo MA, Perera RL, Pellegrini L (2013) Structures of human primase reveal design of nucleotide elongation site and mode of Pol alpha tethering. Proc Natl Acad Sci U S A 110(40):15961–15966. PubMedPubMedCentralCrossRefGoogle Scholar
  47. Klinge S, Nunez-Ramirez R, Llorca O, Pellegrini L (2009) 3D architecture of DNA Pol alpha reveals the functional core of multi-subunit replicative polymerases. EMBO J 28(13):1978–1987. emboj2009150 [pii] 10.1038/emboj.2009.150 PubMedPubMedCentralCrossRefGoogle Scholar
  48. Koc KN, Stodola JL, Burgers PM, Galletto R (2015) Regulation of yeast DNA polymerase delta-mediated strand displacement synthesis by 5′-flaps. Nucleic Acids Res 43(8):4179–4190. gkv260 [pii] 10.1093/nar/gkv260 PubMedPubMedCentralCrossRefGoogle Scholar
  49. Koc KN, Singh SP, Stodola JL, Burgers PM, Galletto R (2016) Pif1 removes a Rap1-dependent barrier to the strand displacement activity of DNA polymerase delta. Nucleic Acids Res 44(8):3811–3819. PubMedPubMedCentralCrossRefGoogle Scholar
  50. Koh KD, Balachander S, Hesselberth JR, Storici F (2015) Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA. Nat Methods 12(3):251–257. nmeth.3259 [pii] 10.1038/nmeth.3259 PubMedPubMedCentralCrossRefGoogle Scholar
  51. Kuchta RD, Stengel G (2010) Mechanism and evolution of DNA primases. Biochim Biophys Acta 1804(5):1180–1189. PubMedCrossRefGoogle Scholar
  52. Kuchta RD, Reid B, Chang LM (1990) DNA primase. Processivity and the primase to polymerase alpha activity switch. J Biol Chem 265(27):16158–16165PubMedGoogle Scholar
  53. Kumar S, Burgers PM (2013) Lagging strand maturation factor Dna2 is a component of the replication checkpoint initiation machinery. Genes Dev 27(3):313–321. gad.204750.112 [pii] 10.1101/gad.204750.112 PubMedPubMedCentralCrossRefGoogle Scholar
  54. Kumaran S, Kozlov AG, Lohman TM (2006) Saccharomyces cerevisiae replication protein A binds to single-stranded DNA in multiple salt-dependent modes. Biochemistry 45(39):11958–11973. PubMedPubMedCentralCrossRefGoogle Scholar
  55. 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(1):117–130. PubMedPubMedCentralCrossRefGoogle Scholar
  56. Larrea AA, Lujan SA, Nick McElhinny SA, Mieczkowski PA, Resnick MA, Gordenin DA, Kunkel TA (2010) Genome-wide model for the normal eukaryotic DNA replication fork. Proc Natl Acad Sci U S A 107(41):17674–17679. 1010178107 [pii] 10.1073/pnas.1010178107 PubMedPubMedCentralCrossRefGoogle Scholar
  57. Lee KH, Kim DW, Bae SH, Kim JA, Ryu GH, Kwon YN, Kim KA, Koo HS, Seo YS (2000) The endonuclease activity of the yeast Dna2 enzyme is essential in vivo. Nucleic Acids Res 28(15):2873–2881PubMedPubMedCentralCrossRefGoogle Scholar
  58. Levikova M, Cejka P (2015) The Saccharomyces cerevisiae Dna2 can function as a sole nuclease in the processing of Okazaki fragments in DNA replication. Nucleic Acids Res 43(16):7888–7897. PubMedPubMedCentralCrossRefGoogle Scholar
  59. Levin DS, McKenna AE, Motycka TA, Matsumoto Y, Tomkinson AE (2000) Interaction between PCNA and DNA ligase I is critical for joining of Okazaki fragments and long-patch base-excision repair. Curr Biol 10(15):919–922PubMedCrossRefGoogle Scholar
  60. Lin SH, Wang X, Zhang S, Zhang Z, Lee EY, Lee MY (2013) Dynamics of enzymatic interactions during short flap human Okazaki fragment processing by two forms of human DNA polymerase delta. DNA Repair (Amst) 12(11):922–935. CrossRefGoogle Scholar
  61. Liu S, Lu G, Ali S, Liu W, Zheng L, Dai H, Li H, Xu H, Hua Y, Zhou Y, Ortega J, Li GM, Kunkel TA, Shen B (2015) Okazaki fragment maturation involves alpha-segment error editing by the mammalian FEN1/MutS alpha functional complex. EMBO J 34(13):1829–1843. 10.15252/embj.201489865 PubMedPubMedCentralCrossRefGoogle Scholar
  62. Liu B, Hu J, Wang J, Kong D (2017) Direct visualization of RNA-DNA primer removal from Okazaki fragments provides support for flap cleavage and exonucleolytic pathways in eukaryotic cells. J Biol Chem.
  63. Lujan SA, Clausen AR, Clark AB, MacAlpine HK, MacAlpine DM, Malc EP, Mieczkowski PA, Burkholder AB, Fargo DC, Gordenin DA, Kunkel TA (2014) Heterogeneous polymerase fidelity and mismatch repair bias genome variation and composition. Genome Res 24(11):1751–1764. PubMedPubMedCentralCrossRefGoogle Scholar
  64. Maga G, Stucki M, Spadari S, Hubscher U (2000) DNA polymerase switching: I. Replication factor C displaces DNA polymerase alpha prior to PCNA loading. J Mol Biol 295(4):791–801PubMedCrossRefGoogle Scholar
  65. Mikhailov VS, Bogenhagen DF (1996) Termination within oligo(dT) tracts in template DNA by DNA polymerase gamma occurs with formation of a DNA triplex structure and is relieved by mitochondrial single-stranded DNA-binding protein. J Biol Chem 271(48):30774–30780PubMedCrossRefGoogle Scholar
  66. Miyabe I, Kunkel TA, Carr AM (2011) The major roles of DNA polymerases epsilon and delta at the eukaryotic replication fork are evolutionarily conserved. PLoS Genet 7(12):e1002407. PGENETICS-D-11-01459 [pii]PubMedPubMedCentralCrossRefGoogle Scholar
  67. Montecucco A, Rossi R, Levin DS, Gary R, Park MS, Motycka TA, Ciarrocchi G, Villa A, Biamonti G, Tomkinson AE (1998) 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 17(13):3786–3795PubMedPubMedCentralCrossRefGoogle Scholar
  68. Mossi R, Keller RC, Ferrari E, Hubscher U (2000) DNA polymerase switching: II. Replication factor C abrogates primer synthesis by DNA polymerase alpha at a critical length. J Mol Biol 295(4):803–814PubMedCrossRefGoogle Scholar
  69. Murakami Y, Hurwitz J (1993) Functional interactions between SV40 T antigen and other replication proteins at the replication fork. J Biol Chem 268(15):11008–11017PubMedGoogle Scholar
  70. Murante RS, Rust L, Bambara RA (1995) Calf 5′ to 3′ exo/endonuclease must slide from a 5′ end of the substrate to perform structure-specific cleavage. J Biol Chem 270(51):30377–30383PubMedCrossRefGoogle Scholar
  71. Nick McElhinny SA, Gordenin DA, Stith CM, Burgers PM, Kunkel TA (2008) Division of labor at the eukaryotic replication fork. Mol Cell 30(2):137–144PubMedPubMedCentralCrossRefGoogle Scholar
  72. Nick McElhinny SA, Watts BE, Kumar D, Watt DL, Lundstrom EB, Burgers PM, Johansson E, Chabes A, Kunkel TA (2010a) Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases. Proc Natl Acad Sci U S A 107(11):4949–4954. 0914857107 [pii] 10.1073/pnas.0914857107 PubMedPubMedCentralCrossRefGoogle Scholar
  73. Nick McElhinny SA, Kissling GE, Kunkel TA (2010b) Differential correction of lagging-strand replication errors made by DNA polymerases {alpha} and {delta}. Proc Natl Acad Sci U S A 107(49):21070–21075. PubMedPubMedCentralCrossRefGoogle Scholar
  74. Nunez-Ramirez R, Klinge S, Sauguet L, Melero R, Recuero-Checa MA, Kilkenny M, Perera RL, Garcia-Alvarez B, Hall RJ, Nogales E, Pellegrini L, Llorca O (2011) Flexible tethering of primase and DNA Pol alpha in the eukaryotic primosome. Nucleic Acids Res 39(18):8187–8199. PubMedPubMedCentralCrossRefGoogle Scholar
  75. Perera RL, Torella R, Klinge S, Kilkenny ML, Maman JD, Pellegrini L (2013) Mechanism for priming DNA synthesis by yeast DNA polymerase alpha. Elife 2:e00482. [pii]PubMedPubMedCentralCrossRefGoogle Scholar
  76. Pursell ZF, Isoz I, Lundstrom EB, Johansson E, Kunkel TA (2007) Yeast DNA polymerase epsilon participates in leading-strand DNA replication. Science 317(5834):127–130PubMedPubMedCentralCrossRefGoogle Scholar
  77. Reijns MA, Kemp H, Ding J, de Proce SM, Jackson AP, Taylor MS (2015) Lagging-strand replication shapes the mutational landscape of the genome. Nature 518(7540):502–506. nature14183 [pii] 10.1038/nature14183 PubMedPubMedCentralCrossRefGoogle Scholar
  78. Rossi ML, Bambara RA (2006) Reconstituted Okazaki fragment processing indicates two pathways of primer removal. J Biol Chem 281(36):26051–26061. PubMedCrossRefGoogle Scholar
  79. Rossi ML, Pike JE, Wang W, Burgers PM, Campbell JL, Bambara RA (2008) Pif1 helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavage pathway for primer removal. J Biol Chem 283(41):27483–27493. M804550200 [pii] 10.1074/jbc.M804550200 PubMedPubMedCentralCrossRefGoogle Scholar
  80. Sauguet L, Klinge S, Perera RL, Maman JD, Pellegrini L (2010) Shared active site architecture between the large subunit of eukaryotic primase and DNA photolyase. PLoS One 5(4):e10083. PubMedPubMedCentralCrossRefGoogle Scholar
  81. Schauer GD, O’Donnell ME (2017) Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork. Proc Natl Acad Sci U S A 114(4):675–680. PubMedPubMedCentralCrossRefGoogle Scholar
  82. Singh H, Brooke RG, Pausch MH, Williams GT, Trainor C, Dumas LB (1986) Yeast DNA primase and DNA polymerase activities. An analysis of RNA priming and its coupling to DNA synthesis. J Biol Chem 261(18):8564–8569PubMedGoogle Scholar
  83. Smith DJ, Whitehouse I (2012) Intrinsic coupling of lagging-strand synthesis to chromatin assembly. Nature 483(7390):434–438. PubMedPubMedCentralCrossRefGoogle Scholar
  84. Smith DJ, Yadav T, Whitehouse I (2015) Detection and sequencing of Okazaki fragments in S. cerevisiae. Methods Mol Biol 1300:141–153. PubMedPubMedCentralCrossRefGoogle Scholar
  85. Sparks JL, Chon H, Cerritelli SM, Kunkel TA, Johansson E, Crouch RJ, Burgers PM (2012) RNase H2-initiated ribonucleotide excision repair. Mol Cell 47(6):980–986. S1097-2765(12)00599-0 [pii] 10.1016/j.molcel.2012.06.035 PubMedPubMedCentralCrossRefGoogle Scholar
  86. Stewart JA, Campbell JL, Bambara RA (2010) Dna2 is a structure-specific nuclease, with affinity for 5′-flap intermediates. Nucleic Acids Res 38(3):920–930. PubMedCrossRefGoogle Scholar
  87. Stillman B (2015) Reconsidering DNA polymerases at the replication fork in eukaryotes. Mol Cell 59(2):139–141. S1097-2765(15)00535-3 [pii] 10.1016/j.molcel.2015.07.004 PubMedPubMedCentralCrossRefGoogle Scholar
  88. Stith CM, Sterling J, Resnick MA, Gordenin DA, Burgers PM (2008) Flexibility of eukaryotic Okazaki fragment maturation through regulated strand displacement synthesis. J Biol Chem 283(49):34129–34140PubMedPubMedCentralCrossRefGoogle Scholar
  89. Stodola JL, Burgers PM (2016) Resolving individual steps of Okazaki-fragment maturation at a millisecond timescale. Nat Struct Mol Biol 23(5):402–408. PubMedPubMedCentralCrossRefGoogle Scholar
  90. Suwa Y, Gu J, Baranovskiy AG, Babayeva ND, Pavlov YI, Tahirov TH (2015) Crystal structure of the human pol alpha B subunit in complex with the C-terminal domain of the catalytic subunit. J Biol Chem 290(23):14328–14337. PubMedPubMedCentralCrossRefGoogle Scholar
  91. Thangavel S, Berti M, Levikova M, Pinto C, Gomathinayagam S, Vujanovic M, Zellweger R, Moore H, Lee EH, Hendrickson EA, Cejka P, Stewart S, Lopes M, Vindigni A (2015) DNA2 drives processing and restart of reversed replication forks in human cells. J Cell Biol 208(5):545–562. jcb.201406100 [pii] 10.1083/jcb.201406100 PubMedPubMedCentralCrossRefGoogle Scholar
  92. Tishkoff DX, Filosi N, Gaida GM, Kolodner RD (1997a) A novel mutation avoidance mechanism dependent on S. cerevisiae RAD27 is distinct from DNA mismatch repair [see comments]. Cell 88(2):253–263PubMedCrossRefGoogle Scholar
  93. Tishkoff DX, Boerger AL, Bertrand P, Filosi N, Gaida GM, Kane MF, Kolodner RD (1997b) Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2. Proc Natl Acad Sci U S A 94(14):7487–7492PubMedPubMedCentralCrossRefGoogle Scholar
  94. Tom S, Henricksen LA, Park MS, Bambara RA (2001) DNA ligase I and proliferating cell nuclear antigen form a functional complex. J Biol Chem 276(27):24817–24825PubMedCrossRefGoogle Scholar
  95. Tran PT, Simon JA, Liskay RM (2001) Interactions of Exo1p with components of MutLalpha in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 98(17):9760–9765. PubMedPubMedCentralCrossRefGoogle Scholar
  96. Tran PT, Erdeniz N, Dudley S, Liskay RM (2002) Characterization of nuclease-dependent functions of Exo1p in Saccharomyces cerevisiae. DNA Repair (Amst) 1(11):895–912CrossRefGoogle Scholar
  97. Tsurimoto T, Stillman B (1991) Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis. J Biol Chem 266(3):1961–1968PubMedGoogle Scholar
  98. Tsutakawa SE, Classen S, Chapados BR, Arvai AS, Finger LD, Guenther G, Tomlinson CG, Thompson P, Sarker AH, Shen B, Cooper PK, Grasby JA, Tainer JA (2011) Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily. Cell 145(2):198–211. S0092-8674(11)00241-8 [pii] 10.1016/j.cell.2011.03.004 PubMedPubMedCentralCrossRefGoogle Scholar
  99. Tsutakawa SE, Lafrance-Vanasse J, Tainer JA (2014) The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once. DNA Repair (Amst) 19:95–107. CrossRefGoogle Scholar
  100. Vaithiyalingam S, Arnett DR, Aggarwal A, Eichman BF, Fanning E, Chazin WJ (2014) Insights into eukaryotic primer synthesis from structures of the p48 subunit of human DNA primase. J Mol Biol 426(3):558–569. PubMedCrossRefGoogle Scholar
  101. Vijayakumar S, Chapados BR, Schmidt KH, Kolodner RD, Tainer JA, Tomkinson AE (2007) The C-terminal domain of yeast PCNA is required for physical and functional interactions with Cdc9 DNA ligase. Nucleic Acids Res 35(5):1624–1637. gkm006 [pii] 10.1093/nar/gkm006 PubMedPubMedCentralCrossRefGoogle Scholar
  102. Xie Y, Liu Y, Argueso JL, Henricksen LA, Kao HI, Bambara RA, Alani E (2001) Identification of rad27 mutations that confer differential defects in mutation avoidance, repeat tract instability, and flap cleavage. Mol Cell Biol 21(15):4889–4899PubMedPubMedCentralCrossRefGoogle Scholar
  103. Yeeles JT, Janska A, Early A, Diffley JF (2017) How the eukaryotic replisome achieves rapid and efficient DNA replication. Mol Cell 65(1):105–116. PubMedPubMedCentralCrossRefGoogle Scholar
  104. Yu C, Gan H, Han J, Zhou ZX, Jia S, Chabes A, Farrugia G, Ordog T, Zhang Z (2014) Strand-specific analysis shows protein binding at replication forks and PCNA unloading from lagging strands when forks stall. Mol Cell 56(4):551–563. PubMedPubMedCentralCrossRefGoogle Scholar
  105. Yuzhakov A, Kelman Z, Hurwitz J, O’Donnell M (1999) Multiple competition reactions for RPA order the assembly of the DNA polymerase delta holoenzyme. EMBO J 18(21):6189–6199PubMedPubMedCentralCrossRefGoogle Scholar
  106. Zerbe LK, Kuchta RD (2002) The p58 subunit of human DNA primase is important for primer initiation, elongation, and counting. Biochemistry 41(15):4891–4900PubMedCrossRefGoogle Scholar
  107. Zhang Y, Baranovskiy AG, Tahirov ET, Tahirov TH, Pavlov YI (2016) Divalent ions attenuate DNA synthesis by human DNA polymerase alpha by changing the structure of the template/primer or by perturbing the polymerase reaction. DNA Repair (Amst) 43:24–33. CrossRefGoogle Scholar
  108. Zhou C, Pourmal S, Pavletich NP (2015) Dna2 nuclease-helicase structure, mechanism and regulation by Rpa. Elife 4.

Copyright information

© Springer Nature Singapore Pte Ltd. 2017

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiophysicsWashington University School of MedicineSaint LouisUSA

Personalised recommendations