N6-methyl-adenine: an epigenetic signal for DNA-protein interactions

Abstract

N(6)-methyl-adenine is found in the genomes of bacteria, archaea, protists and fungi. Most bacterial DNA adenine methyltransferases are part of restriction-modification systems. Certain groups of Proteobacteria also harbour solitary DNA adenine methyltransferases that provide signals for DNA-protein interactions. In gamma-proteobacteria, Dam methylation regulates chromosome replication, nucleoid segregation, DNA repair, transposition of insertion elements and transcription of specific genes. In Salmonella, Haemophilus, Yersinia and Vibrio species and in pathogenic Escherichia coli, Dam methylation is required for virulence. In alpha-proteobacteria, CcrM methylation regulates the cell cycle in Caulobacter, Rhizobium and Agrobacterium, and has a role in Brucella abortus infection.

Figure 1

Overview of the roles of m6A in enteric bacteria: (i) defense against phages and transposons; (ii) regulation of chromosome replication, chromosome segregation, and reorganization of the nucleoid after DNA replication; (iii) DNA strand discrimination for mismatch repair: (iv) regulation of conjugal transfer of plasmids; (v) packaging of phage DNA into capsids; and (vi) transcriptional regulation of fimbrial operons and other virulence genes. When known, the methylation-sensitive DNA-binding proteins involved in each process are also indicated.

Figure 2

Dam-directed mismatch repair. Assembly of the MutHLS complex at a mismatch is followed by MutH-mediated cleavage of the newly synthesized strand (shown in yellow) at the nearest GATC. Transient GATC hemimethylation in the newly synthesized strand provides the signal for strand discrimination. Depending on the distance, cleavage may require DNA looping (not drawn for simplicity).

Figure 3

Regulation of the pap operon of uropathogenic E. coli (reproduced from ref. 52). Top panel: DNA adenine methylation states of the pap regulatory region. In the ON state, binding of Lrp to sites 4, 5, and 6 prevents methylation of GATC^dist, while the unbound GATC^prox is methylated. In the OFF state, Lrp binding to sites 1, 2, and 3 prevents methylation of GATC^prox, while the unbound GATC^dist is methylated. Bottom panel: Model for switching from phase OFF to phase ON. (A) In the OFF state, Lrp binds to sites 1–3, and prevents methylation of GATC^prox. Non-methylated GATCs are shown as open circles, and methylated GATCs are shown as closed circles. Binding of Lrp to sites 1–3 reduces the affinity of Lrp for sites 4–6. (B) Every replication round offers an opportunity for switching. (C) In the presence of PapI, Lrp will translocate from sites 1–3 to sites 4–6. Lrp binding to GATC^dist will prevent methylation of the nascent DNA strand, while the unbound GATC^prox will undergo methylation. (D, E). If the intracellular concentration of cAMP is high, CRP will activate papI transcription. (F) PapB will stimulate papI transcription, creating a positive feedback loop that will perpetuate the ON state.

Figure 4

(Adapted from ref. 60). Model for switching in the agn43 gene. Methylation of GATC sites in the agn43 operator prevents binding of the OxyR repressor, and defines the ON state. After DNA replication, SeqA and OxyR compete for binding to the hemimethylated operator. SeqA binding is only transient and permits GATC methylation, thus propagating the ON state. In contrast, OxyR binding hinders GATC methylation, and agn43 is switched OFF in the following replication round. Switching to the ON state occurs when OxyR leaves the operator, permitting GATC methylation.

Figure 5

Model for regulation of traJ transcription in the Salmonella virulence plasmid. The transcriptional activator Lrp binds to two cognate sites in the traJ UAS. Lrp binding to the downstream site is inhibited by GATC methylation; as a consequence, traJ is not transcribed in a non-replicating plasmid. Passage of the replication fork leaves the traJ UAS hemimethylated. However, activation of transcription occurs only in one daughter plasmid DNA molecule, because methylation of the coding strain does not permit formation of the Lrp-DNA activating complex.

Figure 6

(Adapted from ref. 5). In Caulobacter crescentus, asymmetric cell division produces a non-replicating swarmer cell and a replicating stalked cell. In the swarmer cell, CtrA binding to the chromosomal origin of replication (Cori) inhibits DNA replication initiation, whereas CtrA proteolysis allows replication to occur in the stalked cell. Hence, DNA replication is only initiated on the methylated Cori of the stalked cell. Because CcrM methylase is degraded after cell division, passage of the replication fork is not followed by methylation of the daughter DNA strands. The extent of hemimethylation indicates the distance covered by the replication machinery, and can be viewed as a measure of time. The existence of DNA-binding proteins (e. g., transcription factors) that recognize hemimethylated sites could thus permit an ordered sequence of events occuring during DNA synthesis.