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Volume 170, Issue 7

Mapping the DnaA-binding landscape reveals a preference for binding pairs of closely spaced DNA sites Open Access

Abstract

DnaA is a widely conserved DNA-binding protein that is essential for the initiation of DNA replication in many bacterial species, including . Cooperative binding of ATP-bound DnaA to multiple 9mer sites (‘DnaA boxes’) at the origin of replication results in local unwinding of the DNA and recruitment of the replication machinery. DnaA also functions as a transcription regulator by binding to DNA sites upstream of target genes. Previous studies have identified many sites of direct positive and negative regulation by DnaA. Here, we use a ChIP-seq to map the DnaA-binding landscape. Our data reveal a compact regulon for DnaA that coordinates the initiation of DNA replication with expression of genes associated with nucleotide synthesis, replication, DNA repair and RNA metabolism. We also show that DnaA binds preferentially to pairs of DnaA boxes spaced 2 or 3 bp apart. Mutation of either the upstream or downstream site in a pair disrupts DnaA binding, as does altering the spacing between sites. We conclude that binding of DnaA at almost all target sites requires a dimer of DnaA, with each subunit making critical contacts with a DnaA box.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): ChIP-seq , DnaA and DnaA box
Funding
This study was supported by the:
  • National Institute of General Medical Sciences (Award R35GM144328)
    • Principle Award Recipient: JosephWade
  • NIH Office of the Director (Award DP2OD007188)
    • Principle Award Recipient: JosephWade
© 2024 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2024-07-16
2025-07-16
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References

  1. Skarstad K, Boye E. The initiator protein DnaA: evolution, properties and function. Biochim Biophys Acta 1994; 1217:111–130 [View Article] [PubMed]
     [Google Scholar]
  2. Leonard AC, Grimwade JE. Regulation of DnaA assembly and activity: taking directions from the genome. Annu Rev Microbiol 2011; 65:19–35 [View Article] [PubMed]
     [Google Scholar]
  3. Schaper S, Messer W. Interaction of the initiator protein DnaA of Escherichia coli with its DNA target. J Biol Chem 1995; 270:17622–17626 [View Article] [PubMed]
     [Google Scholar]
  4. Miller DT, Grimwade JE, Betteridge T, Rozgaja T, Torgue J-C et al. Bacterial origin recognition complexes direct assembly of higher-order DnaA oligomeric structures. Proc Natl Acad Sci U S A 2009; 106:18479–18484 [View Article] [PubMed]
     [Google Scholar]
  5. Felczak MM, Kaguni JM. The box VII motif of Escherichia coli DnaA protein is required for DnaA oligomerization at the E. coli replication origin. J Biol Chem 2004; 279:51156–51162 [View Article] [PubMed]
     [Google Scholar]
  6. Kato J, Katayama T. Hda, a novel DnaA-related protein, regulates the replication cycle in Escherichia coli. EMBO J 2001; 20:4253–4262 [View Article] [PubMed]
     [Google Scholar]
  7. Nievera C, Torgue J-C, Grimwade JE, Leonard AC. SeqA blocking of DnaA-oriC interactions ensures staged assembly of the E. coli pre-RC. Mol Cell 2006; 24:581–592 [View Article] [PubMed]
     [Google Scholar]
  8. Campbell JL, Kleckner N. E. coli oriC and the dnaA gene promoter are sequestered from dam methyltransferase following the passage of the chromosomal replication fork. Cell 1990; 62:967–979 [View Article] [PubMed]
     [Google Scholar]
  9. Kitagawa R, Mitsuki H, Okazaki T, Ogawa T. A novel DnaA protein-binding site at 94.7 min on the Escherichia coli chromosome. Mol Microbiol 1996; 19:1137–1147 [View Article] [PubMed]
     [Google Scholar]
  10. Kitagawa R, Ozaki T, Moriya S, Ogawa T. Negative control of replication initiation by a novel chromosomal locus exhibiting exceptional affinity for Escherichia coli DnaA protein. Genes Dev 1998; 12:3032–3043 [View Article] [PubMed]
     [Google Scholar]
  11. Kasho K, Katayama T. DnaA binding locusdatApromotes DnaA-ATP hydrolysis to enable cell cycle-coordinated replication initiation. Proc Natl Acad Sci USA 2013; 110:936–941 [View Article]
     [Google Scholar]
  12. Fujimitsu K, Senriuchi T, Katayama T. Specific genomic sequences of E. coli promote replicational initiation by directly reactivating ADP-DnaA. Genes Dev 2009; 23:1221–1233 [View Article] [PubMed]
     [Google Scholar]
  13. Saxena R, Fingland N, Patil D, Sharma AK, Crooke E. Crosstalk between DnaA protein, the initiator of Escherichia coli chromosomal replication, and acidic phospholipids present in bacterial membranes. Int J Mol Sci 2013; 14:8517–8537 [View Article] [PubMed]
     [Google Scholar]
  14. Messer W, Weigel C. DnaA initiator--also a transcription factor. Mol Microbiol 1997; 24:1–6 [View Article] [PubMed]
     [Google Scholar]
  15. Menikpurage IP, Woo K, Mera PE. Transcriptional activity of the bacterial replication initiator DnaA. Front Microbiol 2021; 12:662317 [View Article] [PubMed]
     [Google Scholar]
  16. Quiñones A, Wandt G, Kleinstäuber S, Messer W. DnaA protein stimulates polA gene expression in Escherichia coli. Mol Microbiol 1997; 23:1193–1202 [View Article] [PubMed]
     [Google Scholar]
  17. Augustin LB, Jacobson BA, Fuchs JA. Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion. J Bacteriol 1994; 176:378–387 [View Article] [PubMed]
     [Google Scholar]
  18. Mizushima T, Tomura A, Shinpuku T, Miki T, Sekimizu K. Loss of flagellation in dnaA mutants of Escherichia coli. J Bacteriol 1994; 176:5544–5546 [View Article] [PubMed]
     [Google Scholar]
  19. Sass TH, Ferrazzoli AE, Lovett ST. DnaA and SspA regulation of the iraD gene of Escherichia coli: an alternative DNA damage response independent of LexA/RecA. Genetics 2022; 221:iyac062 [View Article] [PubMed]
     [Google Scholar]
  20. Wurihan W, Gezi B, Brambilla E, Wang S, Sun H et al. DnaA and LexA proteins regulate transcription of the uvrB gene in Escherichia coli: the role of DnaA in the control of the SOS regulon. Front Microbiol 2018; 9:1212 [View Article] [PubMed]
     [Google Scholar]
  21. Løbner-Olesen A, Atlung T, Rasmussen KV. Stability and replication control of Escherichia coli minichromosomes. J Bacteriol 1987; 169:2835–2842 [View Article] [PubMed]
     [Google Scholar]
  22. van den Berg EA, Geerse RH, Memelink J, Bovenberg RA, Magnée FA et al. Analysis of regulatory sequences upstream of the E. coli uvrB gene; involvement of the DnaA protein. Nucleic Acids Res 1985; 13:1829–1840 [View Article] [PubMed]
     [Google Scholar]
  23. Ozaki T, Kumaki Y, Kitagawa R, Ogawa T. Anomalous DnaA protein binding to the regulatory region of the Escherichia coli aldA gene. Microbiology (Reading) 2001; 147:153–159 [View Article] [PubMed]
     [Google Scholar]
  24. Tesfa-Selase F, Drabble WT. Regulation of the gua operon of Escherichia coli by the DnaA protein. Mol Gen Genet 1992; 231:256–264 [View Article] [PubMed]
     [Google Scholar]
  25. Zhou Z, Syvanen M. Identification and sequence of the drpA gene from Escherichia coli. J Bacteriol 1990; 172:281–286 [View Article] [PubMed]
     [Google Scholar]
  26. Wang QP, Kaguni JM. dnaA protein regulates transcriptions of the rpoH gene of Escherichia coli. J Biol Chem 1989; 264:7338–7344 [PubMed]
     [Google Scholar]
  27. Gielow A, Kücherer C, Kölling R, Messer W. Transcription in the region of the replication origin, oriC, of Escherichia coli: termination of asnC transcripts. Mol Gen Genet 1988; 214:474–481 [View Article] [PubMed]
     [Google Scholar]
  28. Schaefer C, Messer W. Termination of the Escherichia coli asnC transcript. The DnaA protein/dnaA box complex blocks transcribing RNA polymerase. Gene 1988; 73:347–354 [View Article] [PubMed]
     [Google Scholar]
  29. Tesfa-Selase F, Drabble WT. Specific binding of DnaA protein to a DnaA box in the guaB gene of Escherichia coli K12. Eur J Biochem 1996; 241:411–416 [View Article] [PubMed]
     [Google Scholar]
  30. Zeghouf M, Li J, Butland G, Borkowska A, Canadien V et al. Sequential Peptide Affinity (SPA) system for the identification of mammalian and bacterial protein complexes. J Proteome Res 2004; 3:463–468 [View Article] [PubMed]
     [Google Scholar]
  31. Takiff HE, Baker T, Copeland T, Chen SM, Court DL. Locating essential Escherichia coli genes by using mini-Tn10 transposons: the pdxJ operon. J Bacteriol 1992; 174:1544–1553 [View Article] [PubMed]
     [Google Scholar]
  32. Lambalot RH, Walsh CT. Cloning, overproduction, and characterization of the Escherichia coli holo-acyl carrier protein synthase. J Biol Chem 1995; 270:24658–24661 [View Article] [PubMed]
     [Google Scholar]
  33. Torrents E, Grinberg I, Gorovitz-Harris B, Lundström H, Borovok I et al. NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes. J Bacteriol 2007; 189:5012–5021 [View Article] [PubMed]
     [Google Scholar]
  34. Mackie GA. RNase E: at the interface of bacterial RNA processing and decay. Nat Rev Microbiol 2013; 11:45–57 [View Article] [PubMed]
     [Google Scholar]
  35. Yajnik V, Godson GN. Selective decay of Escherichia coli dnaG messenger RNA is initiated by RNase E. J Biol Chem 1993; 268:13253–13260 [PubMed]
     [Google Scholar]
  36. Erzberger JP, Mott ML, Berger JM. Structural basis for ATP-dependent DnaA assembly and replication-origin remodeling. Nat Struct Mol Biol 2006; 13:676–683 [View Article] [PubMed]
     [Google Scholar]
  37. Weigel C, Schmidt A, Seitz H, Tüngler D, Welzeck M et al. The N-terminus promotes oligomerization of the Escherichia coli initiator protein DnaA. Mol Microbiol 1999; 34:53–66 [View Article] [PubMed]
     [Google Scholar]
  38. Rozgaja TA, Grimwade JE, Iqbal M, Czerwonka C, Vora M et al. Two oppositely oriented arrays of low-affinity recognition sites in oriC guide progressive binding of DnaA during Escherichia coli pre-RC assembly. Mol Microbiol 2011; 82:475–488 [View Article] [PubMed]
     [Google Scholar]
  39. Keyamura K, Fujikawa N, Ishida T, Ozaki S, Su’etsugu M et al. The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP DnaA-specific initiation complexes. Genes Dev 2007; 21:2083–2099 [View Article] [PubMed]
     [Google Scholar]
  40. Olliver A, Saggioro C, Herrick J, Sclavi B. DnaA-ATP acts as a molecular switch to control levels of ribonucleotide reductase expression in Escherichia coli. Mol Microbiol 2010; 76:1555–1571 [View Article] [PubMed]
     [Google Scholar]
  41. Doran KS, Helinski DR, Konieczny I. A critical DnaA box directs the cooperative binding of the Escherichia coli DnaA protein to the plasmid RK2 replication origin. J Biol Chem 1999; 274:17918–17923 [View Article] [PubMed]
     [Google Scholar]
  42. Hansen FG, Christensen BB, Atlung T. Sequence characteristics required for cooperative binding and efficient in vivo titration of the replication initiator protein DnaA in E. coli. J Mol Biol 2007; 367:942–952 [View Article] [PubMed]
     [Google Scholar]
  43. Butland G, Peregrín-Alvarez JM, Li J, Yang W, Yang X et al. Interaction network containing conserved and essential protein complexes in Escherichia coli. Nature 2005; 433:531–537 [View Article] [PubMed]
     [Google Scholar]
  44. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 2000; 97:6640–6645 [View Article]
     [Google Scholar]
  45. Stringer AM, Singh N, Yermakova A, Petrone BL, Amarasinghe JJ et al. FRUIT, a scar-free system for targeted chromosomal mutagenesis, epitope tagging, and promoter replacement in Escherichia coli and Salmonella enterica. PLoS One 2012; 7:e44841 [View Article] [PubMed]
     [Google Scholar]
  46. Horton RM, Cai ZL, Ho SN, Pease LR. Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques 1990; 8:528–535 [PubMed]
     [Google Scholar]
  47. Stringer AM, Currenti S, Bonocora RP, Baranowski C, Petrone BL et al. Genome-scale analyses of Escherichia coli and Salmonella enterica AraC reveal noncanonical targets and an expanded core regulon. J Bacteriol 2014; 196:660–671 [View Article] [PubMed]
     [Google Scholar]
  48. McClure R, Balasubramanian D, Sun Y, Bobrovskyy M, Sumby P et al. Computational analysis of bacterial RNA-Seq data. Nucleic Acids Res 2013; 41:e140 [View Article] [PubMed]
     [Google Scholar]
  49. Fitzgerald DM, Bonocora RP, Wade JT. Comprehensive mapping of the Escherichia coli flagellar regulatory network. PLoS Genet 2014; 10:e1004649 [View Article] [PubMed]
     [Google Scholar]
  50. Bonocora RP, Fitzgerald DM, Stringer AM, Wade JT. Non-canonical protein-DNA interactions identified by ChIP are not artifacts. BMC Genomics 2013; 14:254 [View Article] [PubMed]
     [Google Scholar]
  51. Wade JT, Reppas NB, Church GM, Struhl K. Genomic analysis of LexA binding reveals the permissive nature of the Escherichia coli genome and identifies unconventional target sites. Genes Dev 2005; 19:2619–2630 [View Article] [PubMed]
     [Google Scholar]
  52. Bailey TL, Elkan C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol 1994; 2:28–36 [PubMed]
     [Google Scholar]
  53. Grant CE, Bailey TL, Noble WS. FIMO: scanning for occurrences of a given motif. Bioinformatics 2011; 27:1017–1018 [View Article] [PubMed]
     [Google Scholar]
/content/journal/micro/10.1099/mic.0.001474
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Mapping the Escherichia coli DnaA-binding landscape reveals a preference for binding pairs of closely spaced DNA sites
Microbiology 170, 001474 (2024); https://doi.org/10.1099/mic.0.001474
/content/journal/micro/10.1099/mic.0.001474
/content/journal/micro/10.1099/mic.0.001474
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