Molecular genetics - DNA is semiconservative because from 1 parental...

Molecular genetics

Lecture 1 Genome replication:

DNA is semiconservative because from 1 parental strand 2 daughter strands are made that both contain half of the parental strand. Therefore, the parental strand is semi conserved in the daughter strands.By overturning DNA, it can become either overwound or underwound. Resulting in either a length of less than 10.5 bp per full turn or more than 10.5 bp per full turn.By introducing negative supercoils you can relax DNA, and by introducing positive supercoils you can cause more strain in the DNA.Topoisomerases can change the winding of the DNA by cutting within the DNA and rewinding it. Type I topoisomerases are for single stranded breaks and type II topoisomerases (Gyrases) are for double stranded breaks. To unwind the DNA a Helicase can be used.

For genome replication to occur a replication bubble needs to appear, this contains two replication forks where the replication can begin. The two forks will start replication in different directions, one to the right and one to the left. This means that the replication of the genome is bidirectional.

A replicon is a section of the sequence where replication can start independently from the origin of replication (ORI) (Where the original replication bubble is located). Circular chromosomes only have a single ORI and linear chromosomes have multiple ORI (the human chromosome has between 30000 and 50000 ORI)

The initiation of replication can for instance by regulated through methylation. When the parental helix is double methylated (on both strands) the separation of the strands can begin in the ORI and replication can begin. This will result in the daughter helixes to be singly methylated, meaning replication will not yet begin again, until methylated again.

In the replication process of E. coli there are 6 different proteins involved to form the

replication forks and the initiation of the replication:

DnaA: The initiator protein. It will melt the helix with the help of HU (Histone like proteins)

DnaB/DnaC: DnaB is a helicase that breaks the bonds between the nucleotides and

DnaC is a chaperone, which form the two replication forks

Gyrase: Unwinds the DNA by relaxing supercoils

SSB: Single strand binding protein stabilizes DNA, keeps the replication bubble open and protects from degradation. 1 / 3

The replication itself is done by the DNA polymerase from the 5’ to the 3’ direction. It needs a primer to function and a proofreading error control system (exonuclease activity) in the 3’ to 5’ direction.DNA polymerase I contain both 3’ to 5’ as 5’ to 3’ exonuclease activity

DNA polymerase cannot synthesize DNA out of nothing. Therefore, a primase is needed. This is a DNA-dependent RNA polymerase that synthesizes RNA primers of around 10 bp long.DNA replication is semi discontinuous since one strand can just freely be synthesized while the other has to replicate in Okazaki fragments. Both the strands are synthesized by DNA polymerase III. The primers that are incorporated into the DNA in the lagging strand (for creating the Okazaki fragments) are cut out by DNA polymerase I (exonuclease), then the right DNA is synthesized in and then everything is ligated together with a ligase.

The replication fork is maintained by enzymes such as Gyrase, Helicase, SSB, Primase, DNA polymerase III and I and a Ligase Helicase is the enzyme that cuts open the helix so that the fork can move through.The synthesis of both the leading and lagging strand happens at the same time and at the same speed since the DNA polymerases that are synthesizing the strands are both connected to the replisome (A complex structure that facilitates the entire replication process). The fork can move in a single direction by looping the lagging strand so that the strands move towards the same direction. 2 / 3

The replication fork trap is where the replication fork is stopped, meaning the replication will also stop at this point. The place of this replication fork trap is marked by Ter sites and recognized by TUS (Terminator Utilization Substance) proteins. TUS then binds in a specific orientation to the DNA which stops the replication fork.In eukaryotes the replication forks only stop when the collide with each other.After each replication cycle the chromosomes could become slightly shorter. This is because the last RNA primer used for the Okazaki fragments cannot be removed, causing an overhang that is eventually removed. To avoid this shortening to form a problem there are area's called Telomeres at the ends of chromosomes. These are Guanine rich sequences that don't really encode for anything. The Telomeres also lead to a higher stability within the chromosome by forming something called a T loop.Telomerases are a reverse transcriptase enzyme that synthesizes DNA from an RNA template and extend the telomeres in dividing cells again when they get too short.

Lecture 2 Mutations and repair:

Many different mutations can occur during the replication process, either spontaneous or caused by a mutagen.The types of Spontaneous mutations are Point mutations

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