Eukaryotic DNA Replication

ARS

1. Highly conserved A sequence--> consensus sequence / ARS consensus sequence.; contains higher percentage of A and T bases than the rest of the chromosomal DNA 2-4. B1, B2, B3

Why so many origins?

1. If replication is stalled at a fork, their replication can be completed by an adjacent fork. 2. Allows for a specific section of the genome to replicate. (particular section at a particular time of development) 3. Areas of the genome can be densely packed and replicate more quickly if they have more origins.

Key proteins/players; Assembly of Eukaryotic Replication Fork

ARS MCM complex; helicase Origin Recognition Complex (ORC)

Origin of Replication

Autonomously Replicating Sequence (ARS) 100-200 bp in length occur at specific sites along a chromosome due to features of chromatin structure such as histone modifications, rather than a DNA sequence as in bacteria.

Polymerase switch

DNA pol alpha-primase complex dissociates, and is replaced with DNA pol epsilon for the leading strand, or DNA pol Delta for the lagging strand.

Removal of an RNA primer by flap endonuclease.

DNA pol delta elongates the left okazaki fragment and causes a short flap to occur on the right okazaki fragment. Flap endonuclease removes the flap. DNA pol delta continues to elongate and causes a second flap. Flap endonuclease removes the flap. The process continues until the entire RNA primer is removed. DNA ligase seals the two fragments together.

Why are telomeric repeat sequences needed?

DNA polymerase is unable to replicate the 3' ends of DNA strands. DNA polymerase synthesizes DNA only in a 5'-->3' direction and it cannot ink together the first two individual nucleotides. It can elongate only preexisting strands. These two features of polymerase function pose a problem at the 3' ends of linear chromosomes. 3' end of a DNA strand cannot be replicated by DNA polymerase because a primer cannot be made upstream from this point. If this problem were not solved, the chromosome would become progressively shorter with each round of DNA replication.

More complex than bacterial replication. Why?

Eukaryotic chromosomes are larger and linear. Chromatin is tightly packed by histones within nucleosomes. More complicated cell cycle regulation.

PCNA

Proliferating cell nuclear antigen. allows for processivity

DNA polymerase Epsilon

Replication of the leading strand during sphase

The replication of eukaryotic chromosomes

Replication begins from multiple origins of replication, and the replication forks move bidirectionally to replicate the DNA. Eventually, all of the replication forks will merge. The net result is two sister chromatids attached to each other at the centromere. Replication occurs during the Sphase of the cell cycle.

Why is the lagging strand looped out with respect to the DNA polymerase that synthesizes the lagging strand?

The loop allows the lagging strand polymerase to make DNA in a 5' to 3' direction yet move toward the opening of the replication fork

Eukaryotes contain several different DNA polymerases

alpha, epsilon, delta, gamma

DNA Polymerase Alpha

initiates DNA replication in conjunction with Primase

DNA replication in Eukaryotes begins with the assembly of the pre replication complex (preRC)

composed of many different proteins. Part of the preRC is a group of 6 proteins called the origin recognition complex (ORC)

Translocation of telomerase

following polymerization, telomerase then moves to the new end of this DNA strand and attaches another 6 NT to the end. DNApolalpha-primase lays down a RNA primer at the end of the chromosome that is complementary to the telomeric repeat sequence in the elongated parental strand. DNA pol delta or epsilon fills in the region, and is then sealed by ligase.

DNA pol beta

plays important role in removing incorrect bases from damaged DNA. ; lesion replicating polymerases. Attracted to damaged DNA and has special properties that enables it to synthesize a complementary strand over the abnormal region.

Telomerase

prevents chromosome shortening. Recognizes sequences at the end of eukaryotic chromosomes and synthesizes additional repeats of telomeric sequences. RNA part of telomerase contains a sequence complementary to the DNA sequence found in the telomeric repeat. This allows telomerase to bind to the 3' overhang region of the telomere. Following binding, the RNA sequence beyond the binding site functions as a template for the synthesis of a 6NT sequence at the end of the DNA strand. Telomere lengthening is catalyzed by TERT.

Flap endonuclease

removes small pieces of RNA flaps that are generated by the action of DNA polymerase delta.

DNA polymerase Gamma

replication of Mitochondrial DNA

DNA polymerases Delta

replication of the lagging strand during sphase

Functional role of the DNA polymerase alpha/primase complex

synthesize a short RNA-DNA primer of approximately 10RNA nucleotides followed by 20-30 DNA nucleotides. This short RNA-DNA strand is used by DNA polymerase Epsilon or Delta for the processive elongation of the DNA strands.

TERT

telomerase reverse transcriptase. uses RNA template of telomerase to elongate the parental DNA strand.

Telomere

telomeric sequences within the DNA and the specific proteins bound to those sequences. consists of a tandemly repeated sequence and a 3' overhang region.