Download Chapter 20 DNA Metabolism Gene: A segment of DNA or RNA that

Chapter 20
DNA Metabolism
Gene: A segment of DNA or RNA that encodes
a functional biological product = Protein or RNA
E. coli gene products are sometimes named after
the gene: recA  RecA
Chromosomes: DNA macromolecules are
folded into chromosomes.
http://www.copernicusproject.ucr.edu/ssi/HighSchoolBioResources/DNA/chromosome.gif
Genome: Sum of all genes and intergenic DNA.
The Human Genome contains about 23,000
genes encoding proteins and about 10,000 genes
encoding RNA.
The human genome contains about 3 billion base
pairs. Only about 2% of the genome encodes
genes.
The E. coli genome contains about 4.6 million
base pairs and encodes 4403 genes.
Figure 20.16 shows a Map of the E. coli
circular, double-stranded chromosome and the
positions of some genes important in DNA
replication.
Exonucleases: Enzymes that degrade DNA
5'3' or 3'5' from one end.
Endonucleases: Enzymes that cleave interior
phosphodiester bonds.
DNA Polymerases are enzymes that synthesize
DNA.
Eukaryotes contain 5 DNA Polymerases.
E. coli has 3 DNA polymerases.
I
II
III
Subunits
1
4
10
Rate
Nucl/s
20
7
1000
Processivity
3-200
10,000
500,000
Processivity = the number of nucleotides added
before the enzyme dissociates.
DNA Polymerase I also has a 5'3' exonuclease
activity needed in DNA replication,
recombination, and repair.
For e.g. DNA Pol cannot seal nicks in DNA
strands.
Replisome: A complex of proteins involved in
DNA replication. It consists of:
Helicases: Use ATP to dissociate DNA strands.
Topoisomerases: Relieve topological stress
due to strand separation.
DNA-binding proteins: Keep the strands
separated.
Primases: Make short RNA primers.
DNA Ligases: Seal breaks in the DNA.
DNA Replication: The process by which a cell
copies its DNA to make a new cell.
Recall that DNA is a double helix of two,
antiparallel complementary strands in which A
H-bonds with T, and G H-bonds with C.
Recall also that DNA replication is semiconservative i.e. each strand serves as a
template for the synthesis of a new strand; half
of the old DNA is passed on to the daughter cell
and half remains with the parent cell.
There are 3 stages in DNA replication:
1. Initiation:
In order to make a copy of the DNA, the double
stranded DNA is separated at a replication
fork.
Figure 20.3 The electron micrographs show
replication forks where DNA is unwound and
replicating.
Replication is usually bidirectional for circular
DNA
Bi-directional
Synthesis
Replication Forks
ie there are two replication forks.
Replication has a definite starting point: Origin.
In E. coli it is called oriC.
About 20 DnaA-ATP proteins bind to the Origin
of replication.
The DnaA-complex denatures the Origin
forming an Open Complex.
Then DnaB aids the binding of DnaC (a
helicase) which unwinds the DNA bidirectionally.
Topoisomerase II and single-strand DNA
binding proteins cause 1000s of BP to be
unwound.
Initiation is the only point of regulation of
replication but it is not well understood.
2. Elongation:
Replication is always in the 5'  3' direction in
both strands.
The leading strand is made continuously.
The lagging strand is made in short pieces called
Okazaki fragments. The fragments are later
ligated together.
Leading Strand: DnaG (a primase) synthesizes
a 10-60 nucleotide RNA primer.
Next, DNA-Pol-III adds nucleotides
continuously.
3'
5'
3'
5'
Primase
3'
5'
RNA
3'
5'
Pol III
3'
5'
5'
3'
DNA
5'
3'
Lagging Strand: A Primosome includes DnaB,
DnaC, DnaG + 4 others.
The Primosome moves with the replication fork
in the 5'3' direction.
A primase synthesizes an RNA primer and
DNA Pol-III adds to it.
3'
Primase
5'
3'
5'
Pol III
3'
5'
5'
3'
3'
5'
DNA Pol-I removes the RNA primer, 5'3',
and replaces the RNA with DNA.
5'
3'
Pol I
3'
5'
5'
3'
Ligase
3'
5'
5'
3'
3'
5'
DNA ligase seals the nick between 2 Okazaki
fragments.
DNA synthesis is carried out by enzymes called
DNA polymerases.
DNA polymerase
DNAn + dNTP
DNAn+1 + PPi
ΔG'o = +2 kJ/mol
To make a phosphodiester bond requires the
input of +2 kJ/mol.
The energy comes from pyrophosphate
hydrolysis.
PPi  Pi + Pi
ΔG'o = -30 kJ/mol
So net the ΔG'o = -28 kJ/mol
DNA Polymerase requires a template and a
primer. i.e. it can only add to, not initiate, a
new strand.
Mechanistically, the 3'OH attacks the 5'-γPhosphate.
5
Error rates of polymerases: 1:10
Errors occur when bases tautomerize and
incorrectly H-bond.
Proofreading improves the error rates to 1:108.
Errors are detected by the 3'5' Proofreading
Exonuclease Active Site of DNA Pol.
After the DNA has been replicated, DNA Pol-I
can remove RNA or DNA paired to the template
using its 5'3' exonuclease activity; then it
extends the nontemplate DNA and moves the
nick to where it dissociates. Fig. 20.13b "Nick
Translation"
DNA or RNA
5'
Template
DNA
3'
Pol I
new DNA
5'
3'
When an error is detected the polymerase slides
back, the 3'  5' exonuclease site removes the
incorrect base, then polymerization continues.
Further error correction improves the error rates
to 1:1010 (see later).
3. Termination:
Little is known about how the 2 circular DNAs
are separated.
DNA Repair:
Mutations are permanent changes in the DNA
sequence.
There is a strong correlation between the
accumulation of mutations in cells and cancer.
Some changes cause the death of a cell.
At least 4 different repair systems are known:
Mismatch Repair , Base-Excision Repair: ,
Nucleotide-Excision Repair: , Direct Repair: