DNA REPLICATION (1/3) - INITIATION

During DNA replication, the 2 complementary strands making up the DNA double helix separate. Each strand then serves as a template for the production of a new strand. Since each of the resulting double-stranded DNA molecules is formed by one conserved strand, called a “parent strand”, and one new strand, termed a “nascent strand”, this process is termed semiconservative replication.
Note that DNA replication is slightly different in eukaryotic and prokaryotic cells. Except where stated otherwise, this video focuses on DNA replication in eukaryotes.
Progression though the phases of the cell cycle- Mitosis, G1, S, and G2 is regulated by checkpoints. At each checkpoint, the cell checks that all necessary steps have been completed before entering into the next phase. These checkpoints depend on cyclins and CDKs to proceed. DNA replication occurs during the S-stage of Interphase and must be completed before a cell can enter mitosis. DNA replication has 3 stages – initiation, elongation, and termination. Initiation involves the licencing of origins of replication in order to create two replication forks. Elongation involves synthesis of nascent DNA as the replication forks progress away from the origins of replication. Finally, termination is the end of DNA replication. Let’s discuss each stage in turn.
INITIATION
DNA synthesis always initiates at origins of replication –specific loci in the DNA genome with high concentrations of A and T base pairs. This is because these base pairs have only 2 hydrogen bond interstrand links, while C and G pairs have 3. Therefore, it takes less energy to separate the DNA strands at these locations.
In eukaryotes, origins of replication are defined as nucleotide sequences that bind the origin recognition complex, or ORC, which initiates replication. Eukaryotes have linear chromosomes, and they initiate replication at multiple origins. Prokaryotes, on the other hand, have much smaller genomes which are circular, and they have only one origin of replication. In eukaryotes, the regions between units of replication are called replicons.
Permission for DNA replication to begin from these loci is termed “licensing” of the origins of replication, and occurs during late M phase or early G1 phase. Licencing requires assembly of prereplication complexes at origins of replication, which involves binding of proteins to the ORC. During licensing, the ORC, in complex with Cdc6, recruits the MCM hexamer-Cdt1 complex. Then, a second MCM hexamer-Cdt1 complex is recruited. Cdc6 and Cdt1 are loading factors and replicator activator proteins. The MCM (minichromosome maintenance) complex is a replicative helicase – a molecular motor that uses ATP hydrolysis to power the unwinding of DNA. MCM complexes are “replication licensing factors” because they permit DNA replication to occur. They are composed of 6 subunits (Mcm2-7). MCM complexes are hexameric helicases which permit DNA replication by separating the two DNA strands, thus allowing access to other replication machinery. However, at the point when the MCM complexes are bound to dsDNA, the helicases are inactive.
Later on, active DNA helicase plays a crucial role in DNA replication, since it unwinds the DNA, breaking the hydrogen bonds between the two strands. The unwinding, or “melting”, of the dsDNA into 2 single strands results in replication forks that grow in both directions away from the origin, forming the replication bubble. MCM complexes are still inactive when first loaded onto the dsDNA. If they did actively split the DNA into 2 single strands, then the single strands might prematurely replicate, or be exposed to DNA damaging factors.
DNA helicase is activated only once the cell enters S phase! Active DNA helicase consists of Cdc45, one MCM complex, and GINS; as such, it's also termed the CMG complex. The MCM complex plays the principal unwinding role in the CMG complex. It’s been recently shown that Cdc45, and potentially GINS as well, block DNA from escaping through the gap between the Mcm2 and Mcm5 subunits. The MCM double hexamer surrounding double-stranded DNA transforms into one hexamer encircling the leading strand of each of the two diverging replication forks, although it is not known exactly how this happens. In prokaryotes, each helicase wraps around the lagging strand. Movement of the CMG helicase results in ssDNA. This ssDNA would naturally fold back on itself and re-anneal, forming secondary structures, which would interfere with the replication process. However, replication protein A, or RPA, binds the single stranded DNA to keep it from re-annealing.
Another problem - there is torsional resistance that builds up as helicase unwinds the DNA, forcing it to rotate and build up twists ahead. If the torsion isn’t dealt with, then it would eventually prevent the progress of the replication fork. Topoisomerases are enzymes that temporarily break DNA strands to remove positive supercoils in front of the replication fork.

Видео DNA REPLICATION (1/3) - INITIATION канала Neural Academy