Landsberger: Lecture XXI Flashcards
Enzymes Involved in Methylation Patterns
Describe moments where our methylation pattern is established:
gametes contain high sperm count and moderate DNA methylation
they generate the zygote which has a high or medium level of methylation
DNA methylation level drops in the 1st division up to the blastocyst stage, where it is widely demethylated
What 3 actors does epigenetics rely on?
writers, readers, and erasers (editors)
Describe DNA methylation in the zygote:
DNa (methylated on both sides) go through replication during the 1st cycle of cell division
after the 1st division, we will have hemimethylated DNA (1 strand is methylated: paternal strand & the other is not)
*methylation occurs only after DNA replication
Describe what happens if a cell goes through replication without having any enzymes methylating the strand that has been formed:
after the 1st division → 2 molecules of DNA (Both hemi-methylated)
after the 2nd division → 4 molecules of DNA (2 hemimethylated and 2 not methylated)
after the 3rd division → 8 molecules of DNA (2 hemimethylated and 6 not methylated)
after the 4th division → 16 molecules of DNA (2 hemimethylated and 14 not methylated)
*point is DNA methylation is being diluted due to a passive mechanism, so we should not look for an enzyme
Describe the sceneario where the hemimethylated DNA is recognized by an enzyme (a DNA methyltransferase) that restores the methylation pattern:
maintenance DNA methyltransferase activity maintains the already present methylation pattern
Describe demethylation without DNA replication:
DNA is fully methylated and then becomes not methylated because an enzyme is removing the methyl groups, which means we have an active demethylation pattern
*assumes demethylating enzymes exist
Describe the scenario where we have a DNA that is not methylated and then becomes methylated due to DNA methyltransferase:
we have de novo activity of DNA methyltransferase due to an enzyme
What kindo f mechanism is involved in zygotes in regards to methylation?
both the passive and active mechanism
What is seen when observing demthylases?
loss of DNA methylation in our cells often occurs without replication, so there is an active mechanism of demethylation
What are the 2 possibilites of methylase activity?
maintenance and de novo enzyme
What activity should we look for in methylase activity?
both maintenance and de novo
What 3 processes is required for the methylation patterns in our cells?
maintenance of methylase activity
de novo methylase activity
demethylase activity
What does a valid assay allow us to understand?
if DNA methyltransferase activity is present or not in our fraction
if there is maintenance of de novo activity
What do we use nowadays as a replacement for radioactive molecules?
S-Adenosyl methionine (SAM)
How can we distinguish between de novo and maintenance activity?
we have to provide a non-methylated DNA if we want to see if the enzyme has a de novo activity
we have to provide a hemimehtylated DNA if we want to see if the enzyme has maintenance activity
What are the limitations to using the assay to identify DNA methyltransferase?
this is done in vitro, so there are no competitors
experiment does not explain if our DNA methyltransferase has a high activity on one of the substrates (not methylated/hemimethylated) in respect to the other
When was the 1st DNA methyltransferase isolated?
1988
What 2 parts make-up DNA methyltransferase 1?
long N-terminal region that does not contain catalytic activity
C-terminal region that contains catalytic activity
What is the N-terminal important for?
regulate the activity of the enzyme
What in PCNA?
proliferating cell nuclear antigen
*binds with a region of DNA important for replication (aka replication fork targeting sequence)
What kind of activity does DNA methyltransferase 1 have?
maintenance activity
What 2 kinds of domains does methyltransferase 1 have?
domain that interacts with transcriptional factors or chromatin remodelling activity
domain that binds to transcriptional factors that interact directly with DNA and bring the enzyme to the DNA
DNA methyltransferase 1 is unable to ______.
methylate C followed by any nucleotide other than G because then it would be a palindromic sequence
What is the role of DNA methyltransferase 1?
maintnenance enzyme activity and can also generate de novo activity
