Module IV.

Expression of the genetic information

Unit 10:
Chromosome structure, DNA replication, repair and
recombination.
The organization of prokaryotic and eukaryotic cells

Prokaryotic simplicity vs. eukaryotic complexity

 The organization of prokaryotic genome
The prokaryotic chromosome is a single molecule of double-stranded circular DNA per cell,
in a de-spiralized state (it does not fold like eukaryotic chromosomes in cell division)
although it does have supercoiling by DNA gyrase (allows enough condensation), which is
concentrated in a specific region of the cytoplasm called the nucleoid (prokaryotes do not
have a proper nucleus). They can present natural plasmids in variable number,
minicromosomes of double-stranded circular DNA with replication capacity.
 The organization of eukaryotic genome (plants and animals)
The eukaryotic cell has several chromosomes grouped in the nucleus. Each of them is a
double-stranded linear DNA in interaction with histone and non-histone proteins. At the cell
cycle interphase, DNA is presented as euchromatin, allowing its replication and expression,
and in mitosis, thanks to the folding of DNA on histone proteins, it appears as easily
separable chromosomes in daughter cells. At the level of genetic information, eukaryotic
genes have introns (non-coding for protein) and exons (they encode for protein), whereas in
prokaryotes the message is direct, without introns.
 Nucleosome
(Core: Octamer)
Structural organization of the genome
in eukaryotes: “Beads-in-a-string”

Nucleosome with
DNA (Front)

H2A H3 Electrostatic
interaction between the
H2B H4 octamer and the DNA

Nucleosome with
DNA (Side)

1,75 turns:146 bp
Structural organization of
the genome in eukaryotes:
the nucleosome and the
folding of chromatin.

(H2A-H2B)+(H3-H4)2+(H2A-H2B)
8,5 nm diameter and 6,7 nm height.
Protects 146 bp (1,75 turns).
30 pb linker with H1
Structural organization of the genome in eukaryotes:
from chromatin to chromosomes.
 Chromatin and Chromosome

Chromatin: “coloured substance”. Non-condensed DNA in interphase, bound to

histone and non-histone proteins. Heterochromatin is transcriptionally inactive:
constitutive (in centromeres and telomeres or in the chromosomal inactivation of
human X except in embryogenesis) and facultative (not always condensed, with
transcriptional activity: X in embryogenesis). Eucromatin is transcriptionally
active and replicates earlier than heterochromatin.

Chromosome: “coloured body”. DNA condensed in mitosis, bound to histone

and non-histone proteins. Mitotic chromosome presents 2 chromatids, a
centromere and defined telomeres.
 Chromatin and Chromosome
Cell cycle
Chromosomes in 25-30% of the cycle
one chromatid
state

One chromatid from Duplication of

each chromosome to the chromatid
each pole of each
chromosome

10% of the cycle

Chromosomes
in the state of
two chromatids 40% of the cycle

20-25% of the cycle

 Metaphase chromosomes and Karyotype
P arm: the symbol
"p" was chosen to
Varias Kb
designate the short
arm because "p"
stands for "petit“
(small) in French.

130 pb
Q arm: the letter "q"
was selected to signify
the long arm merely
because "q" is the
next letter in the
alphabet from P.
Varias Kb

Metaphase chromosome: 2 chromatids (1 DNA molecule of double strand each).

Kinetochore: centromere and mitotic spindle proteins.
Telomerase synthesizes and repairs telomeres.

Diploid genome (somatic cell): 2 sets of chromosomes.

Haploid genome (germ cell): 1 set of chromosomes.
Metaphase chromosomes and
Karyotype
In humans, 2n = 46 chromosomes,
22 autosomic pairs and 1 sexual pair
(XX or XY)

Chromosomal banding: Staining

with Quinacrine or Giemsa typical
and identifying each species. Highly
reproducible (evolutionary studies).
Classification of chromosomes by centromere position

Large chromosomes
Group A (chromosomes 1, 2 and 3), meta
and submetacentric.
Group B (chromosomes 4 and 5),
submetacentric.
Medium chromosomes
Group C (chromosomes 7, 8, 9, 10, 11, 12
and also the X chromosomes),
submetacentric.
Group D (chromosomes 13, 14 and 15),
acrocentric.
Small chromosomes
Group E (chromosomes 16, 17 and 18),
submetacentric.
Group F (chromosomes 19 and 20),
metacentric.
Group G (chromosomes 21 and 22),
acrocentric.

By agreement the sex chromosomes

X and Y are apart from their
corresponding groups and put together
at the end of the karyotype.
Classification of chromosomes by centromere position

Metacentric, submetacentric,
acrocentric and telocentric

Karyotype: representation ordered

by number, size, centromeric
position, etc. of the chromosomes of
a species. Metaphase chromosomes
are stained with Giemsa and
identified by their banding pattern.

In humans, 2n = 46 chromosomes,
22 autosomal pairs (autosomes) and
1 sexual pair (XX or XY)
By agreement the sex chromosomes
X and Y are apart from their
corresponding groups and put
together at the end of the karyotype.
 Gene structure and expression
Prokaryotes Eukaryotes

Three RNA
polymerases Transcription
Transcription One RNA polymerase

Processing
mRNA
Cap 5’

Translation

To the
citoplasm

Translation

Polycistronic mRNA
Monocistronic mRNA
Gene structure
and expression

Prokaryotic genes
(polycistronic messenger)

Eukaryotic genes
(Monocistronic messenger)
 Repetitive sequences in
Eukaryotes
(Repetitive DNA)

Repetitive DNA makes up to 50% of the genome. These repeats can be coding
type (minority) and non-coding type (majority).

Coding DNA: repeats of three genes encoding Ribosomal RNAs: gene for 5,8S
rRNA, gene for 18S rRNA and gene for 28S rRNA; In Homo sapiens they are located
in the short arms of the acrocentric chromosomes; each repeat is a block with these
three genes, with a size of 13 Kb; There are 50 repeats separated by 30 Kb each,
until adding 2 Mb.

Non-coding DNA: it can be intragenic (introns and other non-coding regions of the
gene) or extragenic. It is a DNA sequence that repeats itself in the genome
hundreds or thousands of times. Different types of elements, which can be found in
tandem (satellite, mini-satellite and microsatellite) or dispersed (SINE, LINE, Herv
and transposons).
 Replication

1.- High fidelity process (without mistakes, with

verification and correction mechanisms).

2.- Complete process (continuous copy from

beginning to end of the genome).

3.- Semiconservative, with leading and lagging

strands.
 Types of proteins involved in replication:

-DNA polymerases Deoxynucleotides polymerization.

-Helicases Progressive unwinding of dsDNA.

-Topoisomerases Releases the torsional stress generated by the

unwinding induced by the helicases.

-DNA primases Synthesis of RNA oligonucleotides (RNA primers).

-ssDNA binding prot. Prevent premature renaturation of ddDNA.

-DNA ligases Seals nicks in single strand (between fragments at

leading strand and between repaired Okazaki
fragments at the lagging strand).
 Cell cycle

Steps in the replication of dsDNA in eukaryotes.

1. Identification of the replication origins.
2. Uncoiling (denaturing) of the dsDNA to provide a ssDNA mold.
3. Formation of the replication fork.
4. Initiation of DNA synthesis and elongation.
5. Formation of replication and ligation bubbles of newly synthesized
DNA segments.
6. Reconstitution of the chromatin structure.
 “Bubbles” formed by
multiple points of
bidirectional replication

In eukaryotes we can find

multiple functional
replication origins at the
same linear chromosome,
thus reducing the time
needed for replication.
One chromatid Two chromatids
 Direction of synthesis and reading of the dsDNA

Synthesis of nucleic acids is always from 5’ Antiparallel arrangement, inter-strand

phosphate to 3’-OH bonds

5’ phosphate.

3’ OH.

At dsDNA, the coding strand is 5’-3’, and the Watson-Crick complementarity

template strand is 3’-5’: readind 3’-5’ the rules: A and T matches each other (2
template strand allows the DNA polimerase hydrogen bonds); G and C matches
to perform the synthesis in 5’ to 3’ sense. each other (3 hydrogen bonds)
 DNA Polymerases from E. coli

DNA Polymerase III

from E. Coli.

Elongation = polymerization rate = nucleotides / second

Processivity = number of incorporated nucleotides before separating from the template.
Prooofreading or copy error elimination activity

There is no polymerization activity (synthesis) in 3'-5' direction.

DNA polymerases can not start de novo DNA synthesis.
Observation of the Watson-Crick rules of complementarity.
 Steps in the replication of dsDNA in Prokaryotes (E. coli model):
1. Previous methylation of the GATC sitesD by Dam methylase.
2. Binding of O (dnaA) proteins to the ori sequences.
3. Unwinding (denaturation) of the region rich in A-T to provide a ssDNA mold.
4. Binding of SSB proteins to the ssDNA zone.
5. Assembly of the replication fork by helicase (dnaB + dnaC), primase and DNA
polymerase (dnaE).
6. Initiation of DNA synthesis and elongation, with union of SSB proteins to the nascent
DNA strand.
7. Formation of the bubbles of replication and ligation of the synthesized DNA segments.

Supercoiling
 Initiation
and
elongation.
Initiation and Elongation.
Uni- and bidirectional models
of elongation

Termination.
Ending of the DNA replication
Activity of DNA ligases
 Viral replication:
Retrotranscription and Replication
 Mutations: evolutionary meaning

Mutations, together with the recombination that occurs in meiosis, constitute a source
of genetic variability, and variability is the source of evolution. The paradox of genetic
material: it must be stable in order to be transmitted, but it must also be able to
change to allow evolution.

Mutation is any change or stable alteration of the structure of the hereditary material.
They can be caused by damages produced by mutagenic agents (chemical, UV, by
radiation ...) or by errors during replication, meiosis and DNA repair.

The basis of natural selection is to select those individuals whose characters allow
them to be better adapted to their environment, that is, the most favorable genetic
combinations that will be transmitted to the next generation.
 Mutations: evolutionary meaning

A mutation can involve from a small event such as the alteration of a single pair of
nucleotide bases to the gain or loss of whole chromosomes.

Classification of mutations:
1. Depending on the type of cells affected, germline or somatic mutations.
2. According to the amount of DNA affected: point or gene mutations, chromosomal
mutations (affect the structure of the chromosome and, therefore, the information
they carry) and genomic mutations (affect the number of chromosomes and usually
occur due to problems during meiosis) .
3. According to the effect they produce: lethal, silent, nonsense or recessive.
 Somatic mutations Germline mutations
They are produced in non- They are produced in
germ cells. germline cells.

They only affect the cells They affect ovules and sperm.
that descend from the one
that underwent the They affect all the cells of the
mutation. resulting individual.

They are not transmitted They can be transmitted to

to offspring. the offspring.
 Genomic mutations: euploidies and aneuploidies
1.- Euploidies: alteration of the number of complete chromosomal endowments of the cell. In
H. sapiens, each set of 23 different chromosomes is a complete chromosomal endowment.
Monoploidy: a single chromosomal set, although complete (like a gamete, but in a somatic cell).
Polyploidy: somatic cells with three or more complete chromosomal sets (typical in vegetables).
2.- Aneuploidies refer to the alteration in the number of chromosomes within the
chromosomal envdowment. They can be monosomies (missing one member of the couple:
Turner syndrome, X0, 45 chrs), trisomies (three chromosomes of the same type: Down syndrome,
3x21, 47 chrs), tetrasomies (four chromosomes instead of two), etc. In humans, monosomies and
trisomies are typical.
Both euploidy and aneuploidy are the result of defective processes of separation of the
chromosomes in meiosis, resulting in gametes with an irregular number of
chromosomes.

X chromosome 21 chromosome
monosomy trisomy
Chromosomal mutations: structural alterations
Deletion Duplication Inversion

Traslocation Insertion
 Mutations in the DNA sequence
1. Defective replication / repair or physically-induced chemically.
2. Somatic mutations (horizontal transmission) vs germinal (vertical transmission).
3. Informational consequences of the mutation:
1. Substitution mutations:
1. Silent (CCA and CCT code Pro, ATT: Ile, CTT: Leu).
2. Loss of function (alteration of the active center or due to the
appearance of STOP)
3. Attenuated-incentivated (variation of Km or VMax by alteration of the
active center, modification of the allosteric regulation, of the interaction
capacity, etc).
2. Mutations changing the open reading frame (ORF).
Mutation for physicochemical DNA damage
Informational consequences of the mutation
Mechanisms of DNA repair
 1.-Copy
mistakes
(mismatch
repairs;
additional
mechanism to
proofreading)
 2.-Base excision.

Examples:

Deamination by
hydrolysis of the
cytosine, deamination
of adenine in
hypoxanthine and
guanine in xanthine
because of the action
of nitrous acid
(industrial agent).
 3.-Nucleotide excision

(Example: Dimerization of UvrABC

thymines by UV light)

It is a more elaborate route

than proofreading to correct
pyrimidine dimers and other
UvrD (helicase II)
DNA lesions that cause and exonuclease.
structural changes.
The UvrABC endonuclease
cleaves a fragment of 12
nucleotides, which is
displaced by UvrD (helicase DNApol I and
DNA ligase
II) and exonuclease.
DNApol I and DNA ligase
replace it with new DNA.
Thymine dimers removal
4.-Repair of double-strand breaks in
DNA

-Full fidelity systems

-Systems that cause errors
Breakage repair with complete restoration of the sequence
Breakage repair without terminal homology
Repair with introduction of deletions
 Repair with
introduction of
deletions
 Recombination
It is the process by which genetic information is redistributed. It allows
"shuffling" large sets of genes, providing a source of variation and selection
for evolution faster than the mutation.

Types of recombination
1.- Homologous recombination (RecA dependent on bacteria).
2.- Specific recombination (non-homologous, independent RecA in bacteria):
2.1.- Specific site, legitimate or conservative: insertion of bacteriophages.
2.2.- Specific "illegitimate": transposable genetic elements.

Integration
Simple transposon

Viral integration
(Phage l)
5‘- GCTGGTGG -3'

Por RucV
(Nucleasa-helicasa)

Non-
recombinant
heterodúplex

Recombinant
heteroduplex

Resolution of Holliday intermediaries

Homologous
Recombination in
Prokaryotes (E. coli)
 Recombination mechanism: Holliday model
Pair of duplex Breaks in homologous
molecules strands

Exchange of homologous Displacement of

strands intersection point

Turn of
molecules

Breaks in the non-interchanged strands Breaks in the exchanged strands

(vertical line) and ligation (horizontal line) and ligation.
 Homologous Recombination in Eukaryotes
1.- Occurs during meiosis (chromatids crossover ).

2.- Chromosomes exchange DNA segments with each other.

3.- Breakage of the DNA chains takes place in both chromosomes.

4.- It is a process that happens randomly at any point of the chromosomes.

 Recombination mechanism: Holliday model
Theoretical model described in textbooks for more than 30 years.

1.- Start by single strain breaks in both chromosomes.

2.- Crosslinking of the chains: formation of the structure of Holliday

3.- Migration of the Holliday structure and formation of heteroduplex regions.

4.- Structure resolution: heteroduplex formation and recombination.

 Recombination mechanism:
1.- Updated model
2.- Recombination starts by a double-strand break in one
of the helix (chromatid of chromosome A). The other
helix (chromatid of chromosome B) is used as a template
to repair the break.
3.-
1.- Breakage of the double helix.

2.- Partial digestion of the generated 5 'ends. (Activity

5'-3 'exonuclease): the 3' ends remain in single strand
form.

4.- 3.- Mating of strands. Repair by elongation of the 3'

ends using the other chromosome as a template.

4.- Formation of an intermediate structure with two

heteroduplex regions and two Holliday structures.

5.- 5.- Rotation of Holliday structures, resolution by cutting

(formation of heteroduplex regions with or without
recombination) and subsequent ligation of two chains.