The Highly Specific, Cell Cycle-regulated Methyltransferase from Relies on a Novel DNA Recognition Mechanism

Overview

Journal J Biol Chem

Specialty Biochemistry

Date 2018 Oct 17

PMID 30323065

Citations 7

Authors

Norbert O Reich

Eric Dang

Martin Kurnik

Sarath Pathuri

Clayton B Woodcock

Affiliations

Soon will be listed here.

Abstract

Two DNA methyltransferases, Dam and β-class cell cycle-regulated DNA methyltransferase (CcrM), are key mediators of bacterial epigenetics. CcrM from the bacterium (CcrM , methylates adenine at 5'-GANTC-3') displays 10-10-fold sequence discrimination against noncognate sequences. However, the underlying recognition mechanism is unclear. Here, CcrM activity was either improved or mildly attenuated with substrates having one to three mismatched bp within or adjacent to the recognition site, but only if the strand undergoing methylation is left unchanged. By comparison, single-mismatched substrates resulted in up to 10-fold losses of activity with α (Dam) and γ-class (M.HhaI) DNA methyltransferases. We found that CcrM has a greatly expanded DNA-interaction surface, covering six nucleotides on the 5' side and eight nucleotides on the 3' side of its recognition site. Such a large interface may contribute to the enzyme's high sequence fidelity. CcrM displayed the same sequence discrimination with single-stranded substrates, and a surprisingly large (>10-fold) discrimination against ssRNA was largely due to the presence of two or more riboses within the cognate (DNA) site but not outside the site. Results from C-terminal truncations and point mutants supported our hypothesis that the recently identified C-terminal, 80-residue segment is essential for dsDNA recognition but is not required for single-stranded substrates. CcrM orthologs from and share some of these newly discovered features of the enzyme, suggesting that the recognition mechanism is conserved. In summary, CcrM uses a previously unknown DNA recognition mechanism.

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References

Shieh F, Youngblood B, Reich N . The role of Arg165 towards base flipping, base stabilization and catalysis in M.HhaI. J Mol Biol. 2006; 362(3):516-27. DOI: 10.1016/j.jmb.2006.07.030. View

Adhikari S, Curtis P . DNA methyltransferases and epigenetic regulation in bacteria. FEMS Microbiol Rev. 2016; 40(5):575-91. DOI: 10.1093/femsre/fuw023. View

Estabrook R, Nguyen T, Fera N, Reich N . Coupling sequence-specific recognition to DNA modification. J Biol Chem. 2009; 284(34):22690-6. PMC: 2755677. DOI: 10.1074/jbc.M109.015966. View

Cheng X, Roberts R . AdoMet-dependent methylation, DNA methyltransferases and base flipping. Nucleic Acids Res. 2001; 29(18):3784-95. PMC: 55914. DOI: 10.1093/nar/29.18.3784. View

Wright R, Stephens C, Shapiro L . The CcrM DNA methyltransferase is widespread in the alpha subdivision of proteobacteria, and its essential functions are conserved in Rhizobium meliloti and Caulobacter crescentus. J Bacteriol. 1997; 179(18):5869-77. PMC: 179479. DOI: 10.1128/jb.179.18.5869-5877.1997. View

Darbinian N, Gallia G, Khalili K . Helix-destabilizing properties of the human single-stranded DNA- and RNA-binding protein Puralpha. J Cell Biochem. 2001; 80(4):589-95. View

Lu D, Searles M, Klug A . Crystal structure of a zinc-finger-RNA complex reveals two modes of molecular recognition. Nature. 2003; 426(6962):96-100. DOI: 10.1038/nature02088. View

Wortman M, Johnson E, Bergemann A . Mechanism of DNA binding and localized strand separation by Pur alpha and comparison with Pur family member, Pur beta. Biochim Biophys Acta. 2005; 1743(1-2):64-78. DOI: 10.1016/j.bbamcr.2004.08.010. View

Kozdon J, Melfi M, Luong K, Clark T, Boitano M, Wang S . Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle. Proc Natl Acad Sci U S A. 2013; 110(48):E4658-67. PMC: 3845142. DOI: 10.1073/pnas.1319315110. View

10.

Bassing C, Kim Y, Li L, Chandrasegaran S . Overproduction, purification and characterization of M.HinfI methyltransferase and its deletion mutant. Gene. 1992; 113(1):83-8. DOI: 10.1016/0378-1119(92)90672-c. View

11.

Youngblood B, Bonnist E, Dryden D, Jones A, Reich N . Differential stabilization of reaction intermediates: specificity checkpoints for M.EcoRI revealed by transient fluorescence and fluorescence lifetime studies. Nucleic Acids Res. 2008; 36(9):2917-25. PMC: 2396439. DOI: 10.1093/nar/gkn131. View

12.

Woodcock C, Yakubov A, Reich N . Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition. Biochemistry. 2017; 56(30):3913-3922. DOI: 10.1021/acs.biochem.7b00378. View

13.

Robertson G, Reisenauer A, Wright R, Jensen R, Jensen A, Shapiro L . The Brucella abortus CcrM DNA methyltransferase is essential for viability, and its overexpression attenuates intracellular replication in murine macrophages. J Bacteriol. 2000; 182(12):3482-9. PMC: 101938. DOI: 10.1128/JB.182.12.3482-3489.2000. View

14.

Rohs R, Jin X, West S, Joshi R, Honig B, Mann R . Origins of specificity in protein-DNA recognition. Annu Rev Biochem. 2010; 79:233-69. PMC: 3285485. DOI: 10.1146/annurev-biochem-060408-091030. View

15.

Graebsch A, Roche S, Niessing D . X-ray structure of Pur-alpha reveals a Whirly-like fold and an unusual nucleic-acid binding surface. Proc Natl Acad Sci U S A. 2009; 106(44):18521-6. PMC: 2765457. DOI: 10.1073/pnas.0907990106. View

16.

Roberts R, Cheng X . Base flipping. Annu Rev Biochem. 1998; 67:181-98. DOI: 10.1146/annurev.biochem.67.1.181. View

17.

Zhou B, Schrader J, Kalogeraki V, Abeliuk E, Dinh C, Pham J . The global regulatory architecture of transcription during the Caulobacter cell cycle. PLoS Genet. 2015; 11(1):e1004831. PMC: 4287350. DOI: 10.1371/journal.pgen.1004831. View

18.

Weber J, Bao H, Hartlmuller C, Wang Z, Windhager A, Janowski R . Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha. Elife. 2016; 5. PMC: 4764581. DOI: 10.7554/eLife.11297. View

19.

Klimasauskas S, Kumar S, Roberts R, Cheng X . HhaI methyltransferase flips its target base out of the DNA helix. Cell. 1994; 76(2):357-69. DOI: 10.1016/0092-8674(94)90342-5. View

20.

Cheng X . Structural and functional coordination of DNA and histone methylation. Cold Spring Harb Perspect Biol. 2014; 6(8). PMC: 4107986. DOI: 10.1101/cshperspect.a018747. View