Lee_Justin_Block5_MM10 (3 Phases of DNA replication (Initiation (DNA a…
3 Phases of DNA replication
DNA a binds to the 9 mer
9 mer wraps around the DNA a forming a tight knot
This wrapping motion creates a bubble in the rest of the DNA strand
Then SSB binds to the forming DNA bubble and holds the bubble in place
DNA b continues to widen and expand the DNA bubble
Elongation requires several enzymes that use the single-stranded parental DNA strands as templates for synthesizing new complementary daughter strands.
DNA replication begins at the ori and proceeds in both the 3’ and 5’ directions of the new strands.
In the 3’ direction, replication is continuous and carried out by a single DNA polymerase to produce the leading strand.
In the 5’ direction, several DNA polymerases work together to produce the lagging strand
The rate at which the leading and lagging strands are replicated can vary depending on whether or not other proteins are interacting with template DNA. If RNA polymerase is transcribing RNA, replication will be block at the point where RNA polymerase is attached to the DNA.
DNA replication in E. coli bacteria occurs so as to ensure that both the leading and lagging strands are completed simultaneously. Any blockage on one strand will cause the synthesis of the other strand to stall so that it does not complete in advance of the other strand.Stalling is regulated by a protein called Tus, which recognizes and binds to 10 different terminator sites (ter sites) on the template DNA.
A human somatic cell in G1 or G0 phases of interphase has 46 chromosomes, each comprised of a linear double-stranded DNA (dsDNA) molecule that forms a single chromatid. When a cell receives the appropriate signal to begin cell division, it crosses the G1/S checkpoint to enter the S phase of interphase where DNA replication occurs. By replication of the DNA, a sister chromatid is produced.
The dsDNA then denatures bidirectionally from the centromere toward the both ends of the molecule. Meanwhile, new complementary daughter strand are made using the parent strands as templates. This process produced a pair of sister chromatids attached to each other at the centromere to form the “X” shape of the metaphase chromosome.
DNA replication is said to be semiconservative because the each of the sister chromatids of the metaphase chromosome has one strand derived from the parent G1 chromosome and one new strand .
Replication is bidirectional
Bidirectional DNA replication has been observed with plasmid DNA from infected cells. Over time, the replication bubble expands as the growing fork unzips the DNA and new strands are copied in 3’ direction. The parent strands serve as templates for making the complementary daughter strands.
Stops and places along the DNA replication train ride
The origin of replication
DNA replication begins at the origin of replication or ori.
In E.coli bacteria, the origin of replication called oriC and includes 2 sets of consensus sequences called the 13-mer and 9-mer sequences.
The 13-mer repeat is an AT-rich region that is the DNA unwinding element or DUE. The 9-mer repeat is the DnaA box - binding site of the initiator protein DnaA.
DNA polymerase (replication challenges)
DNA polymerase synthesizes the new daughter strands. There are 3 forms of the enzymes that, due to certain limitations of the polymerization process and restrictions on DNA polymerase, require help from other proteins.
DNA pol III is unable to unwind the DNA double helix and denature the complementary template strands.
DNA pol III cannot initiate daughter strand elongation. It can only grow a DNA strand from preexisting RNA primers or DNA.
DNA pol III grows the new strands only in the 5’ to 3’ direction. As the strand denatures in the opposite direction, new strands need to be made in the 3’ to 5’ direction.
solution to challenges
Helicase unwinds and unzips the DNA double-helix and the single-stranded-binding (Ssb) protein binds to the single strands to keep them from reannealing. The unwinding of the double helix causes supercoiling. The enzyme gyrase (also called topoisomerase) stabilizes the uncoiled DNA to prevent supercoiling.
The enzyme primase adds RNA primer to the parent template strand so that DNA Pol III can polymerize the complementary daughter strand in the 3’ direction.
During replication there is a continuous leading strand that grows in the 3’ direction, and a lagging strand composed of Okazaki fragments that grow in the 5’ direction. DNA Pol I removes ribonucleotides of the RNA primer between adjacent Okazaki fragments on the lagging strand and fills the gap with deoxynucleotides. DNA ligase joins the adjacent fragments.
Leading and lagging strands
The leading strand begins at the ori with the RNA primer and elongates without breaking in the 3’ direction (downstream of the ori) until it reaches the end of the DNA molecule.
Okazaki fragments are used for replicating the rest of the DNA that is upstream of the ori.
Like the leading strand, Okazaki fragments are elongated from RNA primers in the 3’ direction.
The lagging strand is made of all the Okazaki fragments of a single daughter strand that are fused together by Polymerase I and ligase. It begins at the ori and is assembled with successive Okazaki fragments, growing in the 5’ direction rather than the polymerization direction of 5’ to 3’.
Telomeres are non-coding regions of the DNA located at the ends of eukaryotic chromosomes.
They protect the coding regions from telomere DNA deletion that occurs during replication.
Because DNA polymerase require a primer RNA to add new nucleotides, when the RNA primers on the 5’ ends of the lagging strands are removed, DNA pol I cannot replace it with DNA since it cannot initiate replication. This means that whenever the DNA replicates in preparation for cell division, a section of DNA is deleted. After a several rounds of replication, the gene coding regions of the DNA are threatened by deletion.