In vivo investigation of interactions between replisome components in Escherichia coli: An expanded model for the processivity switch

Atif A. Patoli, Bushra B. Patoli


Background: Protein interactions within the replisome (a highly coordinated protein complex) are crucial to maintain temporal and spatial regulation for high fidelity DNA synthesis in Escherichia coli (E. coli). A key component of these interactions is the processivity switch, ensuring smooth transition of the replicative DNA polymerase III (Pol III) between Okazaki fragments on the lagging strand. Multiple interaction studies between replisome components have been performed to indicate the essential roles of Pol III (DnaE), β-clamp, DnaB helicase, DNA and the t (DnaX) subunit for this switch.

Methods: Known interacting regions of both DnaE and various truncated versions of t were chosen for co-expression in E. coli. Differences in the growth pattern of cells co-expressing various truncated versions of DnaX and DnaE, on liquid and solid media were subsequently analyzed. Based on in vivo analyses to explore the interactions between these components, an expanded model for the processivity switch is presented here.

Results: The analyses suggest that residues 481-643 of t are sufficient to establish a functional interaction with the DnaB helicase and DnaE during replication, while residues 461-480 of t interact with the C-terminal tail of DnaE to disengage Pol III from the β-clamp during processivity switching. We also propose that residues 430-460 of t are involved in sensing the DNA structure required for the processivity switch.

Conclusion: These observations expand the current understanding of processivity switching and help dissect the regions of t utilized for binding to different replisome components such as DnaB helicase, polymerase and DNA.

Keywords: Processivity Switch; Clamp Loader; DnaE; DnaX; DnaB Helicase

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Kim DR, McHenry CS. Identification of the beta-binding domain of the alpha subunit of Escherichia coli polymerase III holoenzyme. Journal of Biological Chemistry, (1996); 271(34): 20699-20704.

Rothwell PJ, Waksman G. Structure and mechanism of DNA polymerases. Advances in Protein Chemistry (2005); 71401-440.

Robinson A, van Oijen AM. Bacterial replication, transcription and translation: mechanistic insights from single-molecule biochemical studies. Nature Reviews Microbiology, (2013); 11(5): 303-315.

Lewis JS, Spenkelink LM, Jergic S, Wood EA, Monachino E, et al. Single-molecule visualization of fast polymerase turnover in the bacterial replisome. Elife, (2017); 6.

Lewis JS, Jergic S, Dixon NE. The E. coli DNA Replication Fork. Enzymes, (2016); 3931-88.

McHenry CS, Crow W. DNA polymerase III of Escherichia coli. Purification and identification of subunits. Journal of Biological Chemistry, (1979); 254(5): 1748-1753.

Johnson A, O'Donnell M. Cellular DNA replicases: components and dynamics at the replication fork. Annual Review of Biochemistry, (2005); 74283-315.

Welch MM, McHenry CS. Cloning and identification of the product of the dnaE gene of Escherichia coli. Journal of Bacteriology, (1982); 152(1): 351-356.

Lamers MH, Georgescu RE, Lee SG, O'Donnell M, Kuriyan J. Crystal structure of the catalytic alpha subunit of E. coli replicative DNA polymerase III. Cell, (2006); 126(5): 881-892.

Dohrmann PR, McHenry CS. A bipartite polymerase-processivity factor interaction: only the internal beta binding site of the alpha subunit is required for processive replication by the DNA polymerase III holoenzyme. Journal of Molecular Biology, (2005); 350(2): 228-239.

Dohrmann PR, Correa R, Frisch RL, Rosenberg SM, McHenry CS. The DNA polymerase III holoenzyme contains gamma and is not a trimeric polymerase. Nucleic Acids Research, (2016); 44(3): 1285-1297.

Flower AM, McHenry CS. The gamma subunit of DNA polymerase III holoenzyme of Escherichia coli is produced by ribosomal frameshifting. Proceedings of the National Academy of Sciences of the United States of America, (1990); 87(10): 3713-3717.

Yuan Q, Dohrmann PR, Sutton MD, McHenry CS. DNA Polymerase III, but Not Polymerase IV, Must Be Bound to a t-Containing DnaX Complex to Enable Exchange into Replication Forks. The Journal of Biological Chemistry, (2016); 291(22): 11727-11735.

Georgescu RE, Kurth I, Yao NY, Stewart J, Yurieva O, et al. Mechanism of polymerase collision release from sliding clamps on the lagging strand. EMBO Journal, (2009); 28(19): 2981-2991.

Dohrmann PR, Manhart CM, Downey CD, McHenry CS. The rate of polymerase release upon filling the gap between Okazaki fragments is inadequate to support cycling during lagging strand synthesis. Journal of Molecular Biology, (2011); 414(1): 15-27.

Leu FP, Georgescu R, O'Donnell M. Mechanism of the E. coli tau processivity switch during lagging-strand synthesis. Molecular Celll, (2003); 11(2): 315-327.

Lopez de Saro FJ, Georgescu RE, O'Donnell M. A peptide switch regulates DNA polymerase processivity. Proceedings of the National Academy of Sciences USA, (2003); 100(25): 14689-14694.

O'Donnell M. Replisome architecture and dynamics in Escherichia coli. Journal of Biological Chemistry, (2006); 281(16): 10653-10656.

Su XC, Jergic S, Keniry MA, Dixon NE, Otting G. Solution structure of Domains IVa and V of the tau subunit of Escherichia coli DNA polymerase III and interaction with the alpha subunit. Nucleic Acids Research, (2007); 35(9): 2825-2832.

Gao D, McHenry CS. t Binds and Organizes Escherichia coli Replication Proteins through Distinct Domains: PARTIAL PROTEOLYSIS OF TERMINALLY TAGGED t TO DETERMINE CANDIDATE DOMAINS AND TO ASSIGN DOMAIN V AS THE α BINDING DOMAIN. Journal of Biological Chemistry, (2001); 276(6): 4433-4440.

Jergic S, Ozawa K, Williams NK, Su XC, Scott DD, et al. The unstructured C-terminus of the tau subunit of Escherichia coli DNA polymerase III holoenzyme is the site of interaction with the alpha subunit. Nucleic Acids Research, (2007); 35(9): 2813-2824.

Fernandez-Leiro R, Conrad J, Scheres SH, Lamers MH. cryo-EM structures of the E. coli replicative DNA polymerase reveal its dynamic interactions with the DNA sliding clamp, exonuclease and tau. Elife, (2015); 4.

Kim S, Dallmann HG, McHenry CS, Marians KJ. Coupling of a replicative polymerase and helicase: a tau-DnaB interaction mediates rapid replication fork movement. Cell, (1996); 84(4): 643-650.

Dallmann HG, Kim S, Pritchard AE, Marians KJ, McHenry CS. Characterization of the Unique C Terminus of theEscherichia coli t DnaX Protein: MONOMERIC C-t BINDS α AND DnaB AND CAN PARTIALLY REPLACE t IN RECONSTITUTED REPLICATION FORKS. Journal of Biological Chemistry, (2000); 275(20): 15512-15519.

Liu B, Lin J, Steitz TA. Structure of the PolIIIalpha-tauc-DNA complex suggests an atomic model of the replisome. Structure, (2013); 21(4): 658-664.


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