7.1 - DNA Structure & Replication

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The role of nucleosomes in DNA packing

histones are used by the cell to package DNA into structures called nucleosomes

nucleosome: consists of a central core of eight histone proteins with DNA coiled around the proteins

the eight proteins (octamer) consist of 2 copies of 4 types of histones

H1, an additional histone protein molecule, serves to bind the DNA to the core particle

the association of histones with DNA = supercoiling

it allows a great length of DNA to be packed into a small space in the nucleus

the nucleosome is an adaptation that facilitates the packing of the large genomes that eukaryotes possess

the H1 histone binds to form a structure called the 30 nm fibre that facilitates further packing

The leading strand and the lagging strand

synthesis on the two strands occur differently

because the two strands are arranged in an anti-parallel way

on the leading strand:

the strand is made continuously following the fork as it opens

on the lagging strand:

the strand is made in fragments moving away from the fork

new fragments are created as the replication fork exposes more of the strand

they are called Okazaki fragments

The direction of replication

DNA replication begins at sites called origins of replication

the phosphate group of new DNA nucleotides is added to the 3' carbon of the deoxyribose of the nucleotide at the end of the chain

replication only occurs in the 5' to 3' direction

Non-coding regions of DNA have important functions

only some DNA sequences code for the production of polypeptides

these are called coding sequences

there are a number of non-coding sequences found in genomes,

some have functions (like the ones used to produce tRNA and rRNA)

some play a role in the regulation of gene expressions such as enhancers and silencers

most of the eukaryotic genome is non-coding

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Proteins involved

Helicase:

unwinds the DNA at the replication fork

Topoisomerase:

releases the strain the develops ahead of the helicase

Single-stranded binding proteins:

keep the strands apart, keep them from reannealing long enough for copying fo the strand

DNA primase:

creates RNA primers on the strands; one on leading, many on lagging

DNA polymerase:

it covalently links the deoxyribonucleotide monophosphate to the 3' end of the growing strand

DNA ligase:

connects the gaps between fragments

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How Replication Happens

Helicase

Helicase unwinds and separates the double-stranded DNA by breaking the hydrogen bonds between base pairs

This occurs at specific regions (origins of replication), creating a replication fork of two strands running in antiparallel directions

DNA Gyrase

DNA gyrase reduces the torsional strain created by the unwinding of DNA by helicase

It does this by relaxing positive supercoils (via negative supercoiling) that would otherwise form during the unwinding of DNA

Single-Stranded Binding (SSB) Proteins

SSB proteins bind to the DNA strands after they have been separated and prevent the strands from re-annealing

These proteins also help to prevent the single-stranded DNA from being digested by nucleases

SSB proteins will be dislodged from the strand when a new complementary strand is synthesized by DNA polymerase III

DNA Primase

DNA primase generates a short RNA primer (~10–15 nucleotides) on each of the template strands

The RNA primer provides an initiation point for DNA polymerase III, which can extend a nucleotide chain but not start one

DNA Polymerase III

Free nucleotides align opposite their complementary base partners (A = T ; G = C)

DNA pol III attaches to the 3’-end of the primer and covalently joins the free nucleotides together in a 5’ → 3’ direction

As DNA strands are antiparallel, DNA pol III moves in opposite directions on the two strands

On the leading strand, DNA pol III is moving towards the replication fork and can synthesize continuously

On the lagging strand, DNA pol III is moving away from the replication fork and synthesizes in pieces (Okazaki fragments)

DNA Polymerase I

As the lagging strand is synthesized in a series of short fragments, it has multiple RNA primers along its length

DNA polymerase I remove the RNA primers from the lagging strand and replaces them with DNA nucleotides

DNA Ligase

DNA ligase joins the Okazaki fragments together to form a continuous strand

It does this by covalently joining the sugar-phosphate backbones together with a phosphodiester bond