Although the polymerase reaction that is involved in DNA replication is the same, mechanistically, as the one that we saw in the transcription of structural genes on DNA onto complementary mRNA strands, the overall process of DNA replication is much more complicated. We will look first at replication in prokaryotes and then at replication in eukaryotes. Then, we will look at DNA modifications that occur in cellular organisms, specifically methylation reactions.
There's a lot of interesting chemistry here! We have seen some of it before, and I hope to tie it all together by the time we are through. Methylation of the 5'-carbon of the base cytosine is particularly important and the resulting methylation pattern of a gene, along with the gene itself, constitute the "epigenome". Indeed, 5-methylcytosine is now being referred to as "the fifth base". Methylation is a process that is used to control gene expression, and it is what determines the timing of gene expression (as in embryologic development, in which genes are turned on and off in a sequential fashion), inactivation of an X-chromosome in a female ("Lyonization"), and, in mammals, differential expression of certain genes depending upon whether they are maternally- or paternally-derived ("genomic imprinting"). Defects in methylation are responsible for many interesting , and previously unexplainable genetics diseases, and we will consider some of these. Epigenetic phenomena are part of the "new genetics" (non-Mendelian genetics), as are mitochondrial inheritance patterns and trinucleotide repeat expansions and their consequences. We will also consider these latter mutations and some of the diseases that they are associated with.
We will not study DNA Repair, Recombination, or Mobile Elements but these are covered well in your text as well as in other courses that you are taking. At the end of this section, we will have come in full circle back to transcription regulation by the epigenome.
Highlights of the Replication Process:
"Semiconservative" : See the Messelson and Stahl Experiment
"Replication Forks" : Can be uni- or bi-directional
"Semidiscontinuous" : In dsDNAs, the "leading strand" is synthesized in the 5'-->3' direction as a single, continuous strand, using the parent 3'-5' strand as its template. The "lagging strand", which uses the parental 5'-3' strand as its template, is synthesized discontinuously, but also in the 5'-->3' direction. These fragments that make up the lagging strand are called "Okazaki fragments".
RNA Primers: These are short fragments of RNA that initiate the 5'-end of both the leading strand and each of the Okazaki fragments. They will ultimately be removed and the ends of fragments will be joined.
The Enzymes Involved:
"Helicases" : These enzymes separate dsDNA at its replication fork.
DNA Gyrase: Introduces negative supercoils to compensate for the strain that results from positive supercoiling at the replication fork in prokaryotes. It is a Type IIB topoisomerase.
RNA Polymerase: Catalyzes the formation of RNA primers in the leading strand. This is the same polymerase that catalyzes the formation of RNA transcripts.
Primase: Initiates Okazaki fragment primers. Can also initiate leading strand synthesis.
DNA Polymerases: These enzymes catalyze synthesis of complementary strands of DNA from parent strands, but only in the 5'->3' direction.
DNA Ligase: Catalyzes the joining together of Okazaki fragments.
We will now look at the chemistry of these reactions in depth, first for prokaryotic replication (specifically in bacteriophages and E.Coli) and then for eukaryotic replication.
See class notes for this material. You can link to these below.
There is a "Short" version, as well as a longer version
Intracellular Chemical Modifications of DNA
A and C residues can be modified by methylation reactions in which the methyl groups are contributed by S-adenosylmethionine (SAM) and the the reactions are catalyzed by "methyltransferases". Methyl groups are added to nitrogen (N) or carbon (C) atoms as follows:
The mechanism of methyl transfer is similar to that seen in the thymidylate synthase reaction, in which dTMP is synthesized from dUMP and N5,N10 -methylene-THF is the methyl donor.
A pyrimidine C6 is nucleophilically attacked by a Cys thiolate ( -S-E )
The pyrimidine C5 is activated as a resonance-stabilized carbanion, which attacks the methyl group of SAM (or of N5,N10 -methylene-THF )
A base on the enzyme abstracts the proton on C5 of the covalent intermediate, releasing S-Adenosylhomocysteine as well as the enzyme itself and the m5 C residue.
5-Fluorocytosine is a "suicide substrate" for the m5C methyltransferases
Methylation sites in eukaryotes are palindromic.
When DNA replicates, the "methylation pattern" is thereby maintained:
The methyltransferase DNMT1 catalyzes maintenance methylation in mammals. It differentiates between hemimethylated and fully methylated DNAs and strongly prefers the hemimethylated form. The strand complementary to the methylated parent strand is not methylated after the replication process, but becomes so afterward.
This maintains the methylation pattern in a cell line, and thus is a form of stable inheritance. Methylation thus adds additional information to the genome that is not otherwise available only by knowing the sequences of bases in a gene. These changes are known as "epigenetic" changes.
Epigenetic characteristics do not follow Mendelian ineritance.
Link here to learn more about epigenetic phenomena.
Link not yet established.
The DNA methylation patterns in mammalian gametes are mostly erased at the blastocyst stage of embryonic development. They then rise until the gastrula stage is reached, at which time adult levels are reached and are then maintained. The new methylation that occurs after the erasure of the original mathylation patterns is called "de novo" methylation and is mediated by the methyltransferases DNMT3a and DNMT3b.
In mammals, some maternal and paternal genes are differentially expressed and this is known as "genomic imprinting". Methylation patterns of these genes during gametogenesis are different, and these are not demethylated during blastocyst formation, nor are they subject to de novo methylation.
Link here to learn more about genomic imprinting.
Link not yet established.
How does DNA methylation prevent gene expression?
DNA methylation is often recognized by proteins that contain a conserved methyl-CpG binding domain (MBD). MBDs can bind to 5-methylated cytosine residues because these project into the major groove of DNA where DNA-protein interactions can occur without disrupting the double helical structure. When an MBD-containing protein binds to m5Cs in the promoter region of a gene, inhibition of transcription can occur.
EXERCISE: MeCP2 is a mammalian protein that binds to mCpG sequences in dsDNA and it represses transcription by recruiting histone deacetylases. It is also essential for embryologic development and, when defective, can result in a condition known as "Rett Syndrome" which is usually seen in girls. The solution structure of the domain from MeCP2 that binds o methylated DNA was described in 1999 by Wakefield et.al. and its atomic coordinatesin the Protein Data Bank are 1qk9. Look at its structure and answer the following questions:
Note to Students in Spring 2004 Course: This is a "work-in progress". You are not responsible for it for your course.
Link here for "Short Version" class notes:
Link here for longer version of class notes: