E Coli Replication

Explain how the OriC region of DNA on an E Coli is opened for replication

The OriC region of DNA, which is 250 base pairs long, has a region of A-T rich DNA base pairing as well as site specific sites for pairing with DnaA-ATP. Once DnaA-ATP is formed, it attaches to the site specific 9-mer region, which causes it to make a protein-DNA complex that causes negative supercoiling and takes away from the A-T rich 13-mer region of the DNA. Once this region is melted, DnaC loads Helicase onto the DNA and starts a replication fork. Helicase then loads primers that call for the loading of DNA pol III. The primers for the leading strands in this process are also the first Okazaki fragments for the lagging stand going the opposite direction, and are laid by the Helicase for the lagging strand on the same strand going the opposite direction.

Explain how DNA in E. Coli is methylated, and what this process means for the DNA

After DNA is replicated, it has specific sites every 256 base pairs that this enzyme called dam adds a methyl group to (the A nucleotide on GATC). Once a methyl group is added, the cell knows that this strand isn’t newly replicated since methylation takes a lot longer than replication. When there is a new strand of DNA and and old strand (semi-conservative), this is known as hemi-methylated dsDNA. This can help control DNA replication because a protein called SeqA will bind to unmethylated DNA and slow down the binding of DnaA and Dam. DnaA binding is important because it is rate limiting in bacterial DNA replication. When the dsDNA is fully methylated, SeqA cannot bind anymore and dam and DnaA will be allowed to do its thing and let it replicate again.

What are the topoisomerases used in DNA replication

Gyrase is used to relieve positive supercoiling and topoIV is sometimes used if cantanaes form

How many base pairs does E Coli have?

4.5 million

In what ways is Eukaryotic replication different from prokaryotic replication?

In eukaryotes, there is 1000x more DNA

• There are origin sites every 30 kb apart, and there could be thousands of these in large mammals, but not all are used/activated

• During S phase, DNA must be replicated once and only once, to prevent extra chromosomes and chromosomal breaks, so regulation is very important

• The Helicase is already loaded onto the DNA waiting for replication and has been there since the G1 phase (CDK in S phase gives it the green light)

What happens to histones during DNA replication?

Histones must be removed and then put back after replication has taken place— when they are put back, the NTDs have to be oriented to promote gene expression.

What is the end-replication problem?

Basically after the DNA is done replicating, the DNA Pol I attaches to replace the RNA primers with DNA. However, to do this it usually attaches to the OH-3’ group of the end of the previous Okazaki fragment since this exonuclease runs 5–>3. So instead, it has to attach to the OH group at the front of the primer, and that primer isn’t replace and later falls off. As time goes on, the chromosome will get shorter and shorter until it eventually doesn’t exist anymore. This is prevented by telomeres, that create extended TG sequences that extend DNA.

What is telomerase

Telomerase is a ribonucleoprotein that contains both telomerase RNA as a template to create telomeric DNA and TERT, a reverse transcriptase. The RNA template is added to the other strand at the 3’ end and TERT uses it to extend the end of the chromosome, synthesizing additional telomeric DNA.