Investigation of the DNA strand separation step by DNA methyltransferase Caulobacter Crescentus

Abstract

Epigenetic DNA methylation by CcrM C. crescentus is an important regulator of gene expression. CcrM methylates adenine bases on hemimethylated double stranded DNA substrates containing 5′‐GANTC‐3′ sites to regulate gene expression. The recent cocrystal structure shows that CcrM uses a unique DNA recognition mechanism by which four of the five basepairs are completely disrupted. The target strand interacts in base‐specific ways whereas the non‐target strand is largely unperturbed, and does not involve any base‐specific interactions. The dimeric enzyme’s methyltransferase domain and c‐terminal domain interact with the DNA target strand and non‐target strand respectively. Here we present a novel assay that tracks the DNA strand separation step by using a fluorescent probe (Pyrollo‐dC). Placement of Pyrollo‐dC within double stranded DNA at the N position of the CcrM recognition site results in a dramatic increase in fluorescence when CcrM binds; this only occurs when the analog is positioned within the target strand, with much smaller effects involving the non‐target strand. Serine 315 and Histidine 317, which reside in the highly conserved 80‐residue C‐terminal tail, interact with phosphates within the non‐target strand and mutation to alanine results in significant losses of activity and a loss of strand separation, as determine by the Pyrollo‐dC assay. Residue G305A does not interact with the phosphate backbone of the non‐target strand and does show fluorescent signal, suggesting that this mutant is able to strand separate. This mechanism appears to be widespread since other enzymes (e.g., HinfI and an ortholog of CcrM, BabI) exhibit fluorescent signals similar to CcrM.Support or Funding InformationNIH Grant