DNA Replication

A key Regulatory Event Each time the cell divides, its entire DNA has to be replicated The initiation of replication occurs at the origin A Replicon is a unit of DNA that is replicated. It contains one origin of replication The genome of a prokaryote constitutes one single replicon In Eukaryotes, each single chromosome is divided into a large number of replicons. A Replicon works only once per cell cycle. A signal is generated when the entire process of replicating all replicons has been completed.

The Origin of Replication An origin usually initiates bidirectional replication A replication fork is the point at which strands of parental DNA are separated for replication A replication eye is a region in which DNA has been replicated. A replication fork is initiated at the origin and then moves sequentially along DNA A replication is bidirectional when an origin creates two replication forks that move in opposite directions

In Eukaryotes Replicons are 40-100 kb in length A chromosome has many replicons Replicons are activated at specific times during S phase of the cell cycle Replicons near one another are activated at the same time. There are no termination sites. Replication forks merge into one another. The origin is constituted mainly of rich A-T sequences.

Replication The Replisome is the multiprotein structure that assembles at the replication fork to undertake synthesis of DNA It contains DNA polymerase and other enzymes. Replication happens in three steps: Initiation Elongation Termination

Basic Features of Replication Each strand of parental DNA is a template DNA polymerases (or replicases) can extend a DNA chain by adding nucleotides one at a time only to a 3’-OH end. DNA polymerases have the ability to proofread the synthesized DNA. They possess a 3’-5’exonuclease activity that excise incorrectly paired bases.

DNA is synthesized during: Semiconservative replication Repair of damaged DNA Some DNA polymerases function only for replication Other DNA polymerases function only for Repair. A damaged strand can be excised (cut out) and replaced by a new strand.

Separating the strands Helicase uses energy (one ATP for each base pair) and separates the two strands of DNA Single-strand binding protein (SSB) attaches to a single strands and prevents the DNA from renaturing.

The DNA strands are not equivalent The two DNA strands have a different modes of synthesis. The leading strand of DNA is synthesized continuously in the 5’ to 3’direction. The lagging strand of DNA must grow overall in the 3’ to 5’ direction. It needs to be synthesized discontinuously in the form of short fragments 5’-3’ These fragments are called the Okasaki fragments. They are joined later covalently by a ligase.

Priming is required to start DNA synthesis A primer is a short sequence of RNA that is paired with one strand of DNA and provides a free 3’-OH end for the DNA polymerase to start working. The Primase is an enzyme which makes short sequences of RNA.

Elongation

Initiation - Forming the Replication Eye Origin of Replication 5’ 3’ 5’ 3’ 3’ 5’ 5’ 3’

Extension - The Replication Fork 5’ 3’ Primase Single strand binding proteins Laging Strand 3’ 5’ 5’ 3’ Okazaki fragment RNA Primers DNA Polymerase Helicase Leading Strand 5’

Functions And Their Associated Enzymes Melting /Denaturing DNA Helicase SSB Proteins Topoisomerase Polymerizing DNA DNA Polymerase Providing primer Primase Joining nicks Ligase