Eukaryotic DNA Replication
Eukaryotic Cells
Initiation of replication Cdk - cyclin-dependent kinase is utilized to start the DNA replication in the S phase Phosphorylates the prereplicative complex Note phases of the cell cycle
DNA polymerase Inhibitors
Acyclovir represents a class of inhibitors used against viral infections Used against Herpes and Varicella Zoster virus Acyclovir is an analog of guanosine, but the carbohydrate ring is acyclic To be active, acyclovir must be phosphorylated inside the host cell
Histones
Basic proteins that neutralize the acidic nature of DNA Removed during replication, then quickly replaced Different types of histones H1, H2A, H2B, H3, H4 H2A, H2B, H3, and H4 form the core Are covalently modified Methylations, phosphorylations, acetylations, etc.
Initiation of Replication
Eukaryotic cells employ multiple sites along their large chromosomes to start replication There are many chromosomes with multiple sites There may be different times for the replication of different segments of DNA (it is not sequential)
Termination of Replication
Eukaryotic chromosomes are linear The ends of the chromosome (telomeres) pose a special problem for the cell DNA-protein complexes at the ends stabilize the strands
Tautomers
Every now and then, the structure will be in the unfavored structure You will incorporate the wrong base into the structure because the structure looks different than it should In 1/1000th of a second this occurs
Eukaryotic Replication
For the most part, it is not very different from the bacterial replication Contains DNA polymerase, replication forks, topoisomerase I, etc. One of the big differences is the use of Histones DNA is wrapped around Histones - proteins DNA is slightly acidic - deoxyribonucleic acid The DNA is wrapped around histones, which neutralize the charge
Histones
H1 is a histone that acts in between the individual nucleosomes Responsible for making sure the nucleosomes are situated correctly with respect to one another Positioned the nucleosomes so that they are packed correctly in the cell Histones are heavily modified Based on the pattern it sees, it will know where it sits and will be able to determine where the next nucleosome sits Typically modified on the N-terminal tail of the histone
DNA polymerase inhibitors
It then selects the viral DNA polymerase and competes with dGTP for incorporation into DNA Acyclovir lacks a 3'-OH group, so if it is incorporated the DNA chain cannot be extended beyond this residue This causes premature chain termination during DNA replication, and so blocks viral replication
Eukaryotic cells
Most of the cells in the body are not thinking about replication All are ready to go When they start the S phase, they will use the prereplicative complex and replicate
Yeast Replication Origin
ORC - origin recognition complex Unwinding - AT-rich region Not where the replication actually starts Abf-1 - accessory protein (variable) There are many sites that are poorly undefined
Termination of Replication
One strand in the telomere is longer than the other, and it is richer in guanine nucleotides This strand loops back and pairs up with preceding DNA sequences in the same strand, forming stacked G-quadruplexes (four coplanar guanine bases, linked by hydrogen bonds)
Initiation of Replication
Selection of a particular site for starting replication depends on a number of factors: The chromatin structure and histone acetylation Local supercoiling of the DNA Positioning of the DNA segment within the nucleus Transcription of nearby genes
Telomerase
Since the two strands are different lengths, and the lagging strand has difficulty in finishing its strand, the telomerase is needed A protein-RNA complex with reverse transcriptase activity Takes a piece of RNA and back copy it to a piece of DNA Recognizes GGGTTA sequence in human DNA, with a thousand copies
Telomerase
Some cells (fibroblasts) have poor telomerase activity This is a bad thing! Every time the skin cells must repair damages from the sun, there is no telomerase activity At a certain point, you upset the 2-dimensionality of DNA Aging is also affected by telomerase activity If telomerase activity is good - you will live a longer life
Eukaryotic Replication
The histones are then in nucleosome fibers that twist up into a chromosome Nucleosome - DNA and Histones
Initiation of Replication
The origins for DNA replication are not as well defined as they are for prokaryotes They are still A-T rich In prokaryotes, the sites are very well defined and can be spotted right away They are all in a very small space In eukaryotes, the sites are not well defined at all They are spread out in a very large area
Histones
The replication fork and DNA polymerase causes the histones to be knocked off of the DNA for a while However, they are attracted to the DNA Jump right back on to the DNA after they are knocked off When the histones roll up again, they roll up the DNA typically about two times around the histone body
Histones
There are two copies of H2A, H2B, H3, and H4 in the parental chromatin An octomer This is not a random process!! Other things in the body must help the histones to wind up If they are not put back in the correct formation, genes will be turned on when they shouldn't be
Histones
There is a piece of linker DNA between the histones About 150 base pairs of DNA are wrapped around the nucleosome The linker DNA is probably about 50-60 bases If you have chromosomes with billions of base pairs, that chromosome has thousands of histones
DNA Errors
This happens so fast that the enzyme is able to find the error right away/proofread Can determine that the wrong nucleotide was placed in the DNA The enzyme pauses and cuts the phosphodiester bond that it just made Had already added the base to the growing end of the DNA Throws the wrong base out Replaces the base with the right one
DNA Errors
This proofreading activity occurs because the enzyme has exonuclease activity DNA polymerase III has 3' to 5' exonucleolytic proofreading Can only go back one DNA base pair Cannot back up more than one DNA base pair to fix it DNA polymerase I can go 5' to 3' exonuclease activity Replaces the RNA primer with DNA Moves at about 1000 nucleotides per second and can find an error in the sequence Stops to fix that error and then continues on
Histone H1
a family of related histones which bind the nucleosome and affect the direction of the DNA strand One large H1 per nucleosome The nucleosome is not a tight complex; it unfolds ("loosens") frequently Exposes different genes at various times The ATP-dependent chromatin remodeling complex is used for DNA sliding
Tautomers
constitutional isomers of organic compounds that readily interconvert by a chemical reaction called tautomerization Every now and then, mistakes are made because the bonds move around The electrons/bonds move around in the DNA This happens all the time One form of the structure will be favored over the other structures
G-quadruplexes
four coplanar guanine bases, linked by hydrogen bonds
Eukaryotic cells
have a cell cycle DNA synthesis occurs only during S phase of the cell cycle Everything will be primed for replication Make a prereplicative complex Then, it stops This is made within the G1 phase of the cell cycle