Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1998 Mar 17;95(6):2874-9.
doi: 10.1073/pnas.95.6.2874.

A cell cycle-regulated adenine DNA methyltransferase from Caulobacter crescentus processively methylates GANTC sites on hemimethylated DNA

A J Berdis  1 , I Lee, J K Coward, C Stephens, R Wright, L Shapiro, S J Benkovic
Affiliations

A cell cycle-regulated adenine DNA methyltransferase from Caulobacter crescentus processively methylates GANTC sites on hemimethylated DNA

A J Berdis et al. Proc Natl Acad Sci U S A. .

Abstract

The kinetic properties of an adenine DNA methyltransferase involved in cell cycle regulation of Caulobacter crescentus have been elucidated by using defined unmethylated or hemimethylated DNA (DNAHM) substrates. Catalytic efficiency is significantly enhanced with a DNAHM substrate. Biphasic kinetic behavior during methyl incorporation is observed when unmethylated or DNAHM substrates are used, indicating that a step after chemistry limits enzyme turnover and is most likely the release of enzyme from methylated DNA product. The enzyme is thermally inactivated at 30 degrees C within 20 min; this process is substantially decreased in the presence of saturating concentrations of DNAHM, suggesting that the enzyme preferentially binds DNA before S-adenosylmethionine. The activity of the enzyme shows an unusual sensitivity to salt levels, apparently dissociating more rapidly from methylated DNA product as the salt level is decreased. The enzyme acts processively during methylation of specific DNA sequences, indicating a preferred order of product release in which S-adenosylhomocysteine is released from enzyme before fully methylated DNA. The kinetic behavior and activity of the enzyme are consistent with the temporal constraints during the cell cycle-regulated methylation of newly replicated chromosomal DNA.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Defined DNA substrates used to measure CcrM activity in vitro. For convenience, only DNAHM substrates are depicted with the target DNA methylation in bold. Unmethylated DNA substrates contain identical sequences except that the target adenine residue is not methylated at the N6 position.
Figure 2
Figure 2
Preference for DNAHM vs. unmethylated DNA. Reaction mixtures of 50 μl contained 250 nM CcrM preincubated with 5 μM unmethylated or DNAHM and initiated by the addition of 50 μM AdoMet. Data with DNAHM were fit to the equation, y = Ae−kt + Bx + C, where A is the burst amplitude, k is the observed rate constant for the burst amplitude, B is the steady state rate, and C is a defined constant. Data with unmethylated DNA were fit to the equation, y = Ae−kt + C, where A is the burst amplitude, k is the observed rate constant for the burst amplitude, and C is a defined constant.
Figure 3
Figure 3
(A) Time-dependent, thermal inactivation of CcrM. Reaction mixtures of 50 μl contained 250 nM CcrM incubated for variable time and initiated with the simultaneous addition of 5 μM N623/30-mer and 50 μM AdoMet. Data were fit as previously described in Fig. 2. (B) Effect of increasing potassium acetate concentrations on CcrM activity. Reaction mixtures of 50 μl contained 250 nM CcrM preincubated with 5 μM N645/50-mer and variable concentrations of potassium acetate before initiation by the addition of 50 μM AdoMet. The burst amplitudes and the observed rate constant for initial product formation are independent of varying salt concentration, whereas the steady-state rate decreases as the salt concentration is increased.
Figure 4
Figure 4
(A) Time course for product formation with N660/66-mer and N645/50-mer DNA as substrates. One mole equivalent of [3H]CH3 is incorporated into N645/50-mer, whereas 2 mol are incorporated into N660/66-mer, suggesting that CcrM processively methylates DNA. (B) Time course for product formation with N660/66-mer and 60/66-mer DNA as substrates. One mole equivalent of [3H]CH3 is incorporated into 60/66-mer, whereas 2 mol are incorporated into N660/66-mer, indicating that CcrM is capable of processively methylating DNAHM but not unmethylated DNA.
Figure 5
Figure 5
Visualization of processive DNA methylation by CcrM. (A) The increase in full-length N660/66-mer indicates that both sites are resistant to HindII digestion as a result of processive methylation. The majority of N660/66-mer is not utilized by CcrM and is therefore susceptible to restriction digestion by HindII. Unreacted N660/66-mer is cleaved into several distinct smaller DNA fragments, although only the largest of these fragments (≈52-mer) is shown for convenience. (B) Addition of a molar excess (10 μM) of N623/30AP-mer does not alter the production of full-length N660/66-mer, indicating that CcrM processively methylates DNA under physiological conditions.
Figure 6
Figure 6
Proposed minimal kinetic mechanism for CcrM. CcrM preferentially binds to duplex DNA followed by the binding of AdoMet. CcrM in the absence of DNA is thermally inactivated at a rate of 0.14 min−1.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Cheng X. Annu Rev Biophys Biomol Struct. 1995;24:293–318. - PubMed
    1. Braaten B, Nou X, Koltenbach L, Low D. Cell. 1994;76:577–588. - PubMed
    1. Gartler S M, Riggs A D. Annu Rev Genet. 1983;17:155–190. - PubMed
    1. Swain J L, Stewart T A, Leder P. Cell. 1987;50:719–727. - PubMed
    1. Li E, Bestor T, Jaenisch R. Cell. 1992;69:915–926. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources