DNA replication and repair Flashcards
What are initiator proteins?
DNA binding proteins that initiate the assembly of replication machinery (e.g. the recruitment of helicase) by binding to the initiator sequence on DNA. In eukaryotes -> orc/cdc6 protein. In prokaryotes dnaA protein which causes some unwinding as well.
How does initiation work in bacteria?
Bacteria have a single start site (oriC) followed by a consensus sequence of DnaA binding sites. Binding of DnaA triggers the process, promoting unwinding of DNA. There are at least 10 proteins involved in bacterial DNA replication and the timing of the process is regulated by DNA methylation ( replication will not start until oriC is methylated).
How is eukaryotic DNA replication regulated?
By cell cycle checkpoints (prokaryotes can replicate DNA continuously even before cell division is complete). Sequence specificity varies widely between eukaryotes some organisms have consensus sequences while others don’t but have the same set of chromatin modifications to initiate replication.
DNA replication is semi conservative how was this discovered?
Two groups of bacteria where grown, one in normal conditions the other in the presence of heavier carbon isotopes which created heavier DNA molecules. When the 2 samples where mixed, their DNA weight was measured which showed that the heavy and light DNA had combinedinto one helix: semi conservative. When these DNA molecules where denatured it was shown that one strand was heavy the other light not each strand being a mix.
What is dNTP and what does it do?
Deoxynucleotide triphosphate.
Substrate for genome replication. The phosphate group binds to the OH group on the strand being constructed creating a new phosphodiester bond (DNA polymerase catalyses the hydrolysis, removing 2x Pi from the dNTp) and the base hydrogen bonds to the base on the template strand. dNTP is called something different dependent on the base it carries, e.g. guanine = dGTP.
How is a new DNA chain initiated?
By the synthesis of a short RNA primer on the template strand (catalysed by primase) that is then extended by DNA polymerase/dNTPs. RNA primers are then removed.
Why is DNA synthesis always 5’-3’?
Most likely to make proofreading easier.
How does DNA polymerase stop rNTP binding in the place of dNTP?
Discriminator amino acids in DNA polymerase prevent rNTP binding. rNTP is incompatible with the active site because of its hydroxyl group whereas dNTPs have a hydrogen which can bind with the active site.
How is DNA polymerase prevented from diffusing away from the primer?
Sliding clamp protein encircles newly synthesised DNA and holds the DNA polymerase.
What is the difference between the lagging and the leading strand in DNA replication.
One of the template strands has a 3’ end the other a 5 prime end. The one with a 3’ is the leading strand and undergoes continuous DNA synthesis as you would expect as the new strand can be constructed in the 5’ - 3’ direction. The template strand with a 5’ end is the lagging strand because it encounters a problem. The strand is synthesized in small sections in the opposite direction to that of DNA replication. This allows for synthesis in the 5’ - 3’ direction as the 5’ end of the template strand must have a 3’ on the newly synthesised strand next to it. This is all discontinuous synthesis and the small sections made are called Okazaki fragments, all with their own RNA primer that must be removed later.
What is the trombone model of replication?
The leading strand can be synthesised continuously in a 5’-3’ direction
The lagging strand is looped out so that it passes through the polymerase active site in a 3’-5’ direction, allowing synthesis to occur in the 5’-3’ direction.
One asymmetric DNA polymerase dimer synthesises both strands.
How is DNA synthesis finished?
RNA fragments removed by RNAase enzyme
DNA polymerase fills the gaps
DNA ligase makes the final seal
What does helicase and topoisomerase do?
Helicase unwinds DNA
Topoisomerase relaxes DNA prior to unwinding by creating transient breaks which allows the DNA strands to rotate relative to each other preventing damage.
What is bidirectional DNA replication?
Best way I can explain this for me to remember. Imagine DNA as a zipper, it starts unzipping in the middle and unzips in both directions from that origin. So each side of the zipper is a strand and each of those strands undergoes DNA synthesis. If you look at one strand, one side of the origin will be a leading strand the other a lagging strand and the same for the other strand but on opposite sides.
Do eukaryotes or prokaryotes have multiple origins of DNA replication?
Eukaryotes
