The Human Chromosome,

DNA Replication and Repair

Dr. John Th’ng

GB26
jthng@auamed.net
FOI.6: Nucleus: Understand the
storage of genetic information
and how it is passed down to
successive generations and the
principles of basic techniques in
Molecular Biology
Learning objectives
FOI.6.1. Identify the packing of mammalian DNA into
chromosomes in the nucleus and describe the
importance of the histones
FOI.6.2. Describe the processes involved in the
replication of DNA in bacteria and humans, highlighting
the importance of the directionality of replication, the
role of Okazaki fragments, the key enzymes involved,
and requirement for primers
FOI.6.3. Given that DNA is exposed to damaging
agents, identify the types of damage and describe the
DNA repair mechanisms that restore the integrity of
genetic material
FOI.6.4. Describe the principles of the basic techniques
used in molecular biology and identify their applications
How is DNA organized in cells?
How is DNA organized in cells?
- bacteria vs. humans
The Prokaryote Genome

Figure 1-18. The structure of a bacterium

The Prokaryote Genome
Bacteria genome
- circular in bacteria cells
- attached to the plasma membrane
- can include a separate DNA fragment
- plasmid DNA
The Prokaryote Genome
- plasmids
- small circular DNA
- can carry antibiotic resistance genes
- can be transferred between bacteria
The Human Chromosome
The Human Chromosome

Each human chromosome

consists of a single DNA molecule
The Human Chromosome

Each human chromosome

consists of a single DNA
molecule of about 6 cm

HOW IS THE DNA PACKAGED???

The Human Chromosome

Structural organization of DNA

 Higher order organization
The Chromosome:
- Nuclear proteins: Histone and non-histone proteins
- CHROMATIN: DNA + Histones + Non-Histones
 Higher order organization
The Chromosome:
- Proteins: Histone and non-histone proteins
- CHROMATIN: DNA + Histones + Non-Histones
Histones:
- linker H1
- core H2A, H2B, H3, H4
Non-histone proteins:
- HMGs, transcription factors, etc.
 Higher order organization
Nucleosome: Basic unit of organization of DNA around a
histone octamer (two each of the core histones
H2A,H2B,H3 and H4).
 Higher order organization
Nucleosome (DNA + histones H2A,H2B,H3 and H4) bound
by a single linker histone H1.

Histone H1 binding is required for higher order folding of

chromatin (as seen in heterochromatin)
 Higher order organization
Chromatin: string of nucleosomes connected by the linker
DNA into a “beads on a string” arrangement.

Figure 4-23. Nucleosomes as seen in the electron microscope.

 Higher order organization
Chromatin: Binding of histone H1 coils the nucleosomes
into higher order

Figure 4-23. Nucleosomes as seen in the electron microscope.

 Higher order organization
Mitotic chromosome: The chromatin fibers are further folded
into the highly condensed mitotic chromosome
for cell division.

Figure 4-21. A comparison of

extended interphase chromatin
with the chromatin in a mitotic
chromosome. (A) An electron
micrograph showing chromatin
spilling out of a lysed interphase
nucleus. (B) A scanning electron
micrograph of a mitotic
chromosome. The constricted
region indicates the position of
the centromere.
 Higher order organization
Interphase chromosome: Nucleosomes arranged into
chromatin: for transcription, replication, repair,
etc.
Mitotic chromosome: Chromatin folded into highly
condensed metaphase chromosomes for cell
division
CHROMOSOME STRUCTURE
CHROMOSOME STRUCTURE
Figure 4-15. The organization of genes on a human chromosome.
(A) Chromosome 22, one of the smallest human chromosomes. Most of
the left arm of chromosome 22 consists of short repeated sequences of
DNA that are packaged in a particularly compact form of chromatin
(heterochromatin). (B) A tenfold expansion of a portion of chromosome
22, with about 40 genes indicated. Those in dark brown are known
genes and those in light brown are predicted genes. (C) An expanded
portion of (B) shows the entire length of several genes. (D) The intron-
exon arrangement of a typical gene is shown after a further tenfold
expansion. Each exon (red) codes for a portion of the protein. The
entire human genome is distributed over 22 autosomes and 2 sex
chromosomes.
 CHROMOSOME STRUCTURE
Telomere:
Morphology of chromosome - A telomere is a region of repetitive
DNA at the end of a chromosomes
- Protects the end of the chromosome
from deterioration
- Repeated sequence in humans
(TTAGGG repeats)

Centromere:
- Noncoding DNA
- Binds to mitotic spindle during mitosis
- Has satellite DNA sequences
- arrays of short repeats of DNA
- satellites, mini- and micro-satellites
 CHROMOSOME STRUCTURE
Heterochromatin:
Condensed chromatin
Morphology of chromosome Transcriptionally inactive
Eg: Barr body in female cells represents
inactive X chromosome, centromere,
telomere
Types:
Constitutive heterochromatin:
- Always condensed (inactive)
Facultative heterochromatin:
- Either condensed or dispersed
(permanent or temporarily inactive genes)

Euchromatin:
- Dispersed chromatin
- Transcriptionally active genes
 The Human Genome
Includes Nuclear Genome & Mitochondrial genome
- Nuclear Genome
- 23 pairs chromosomes (22 pairs autosome + X/Y sex)
- protein-coding DNA (~2-5% of the genome!!!)
- Mitochondria Genome
- code for genes for the electron transport chain
- generation of ATP
Learning objective
FOI.6.2. Describe the processes involved in
the replication of DNA in bacteria and
humans, highlighting the importance of the
directionality of replication, the role of
Okazaki fragments, the key enzymes
involved, and requirement for primers

DNA Replication
- how is DNA replicated?
- similar process between bacteria and
human cells
DNA Replication
 DNA Replication
- when does it happen
The Eukaryotic Cell
Cycle: DNA & Histone
G1 Synthesis
S
G2
M
 DNA Replication

Basic steps
- base pairing according to Chargaff’s rule
- polymerases can only work in 5’-3’ direction
- DNA polymerase needs a primer at 5’ end
- RNA primer synthesized by a primase
DNA Replication
- New DNA strands are synthesized by
using the parental strands as templates
in the formation of daughter strands
complementary to the parental strands
- Synthesis occurs simultaneously on
both strands of DNA
- High degree of fidelity
- Daughter strands are always
synthesized 5’ to 3’
- always in 5’ to 3’ direction
- DNA replication is bidirectional
- Semi-conservative
DNA Replication
DNA Replication
 DNA Replication
1. Bidirectional synthesis (bacteria and human)
2. Multiple origins in each human chromosome
- generate “Replication Bubbles”
- single origin in bacteria

Local opening Local opening

of double helix of double helix
 DNA Replication
1. Bidirectional synthesis (bacteria and human)
2. Multiple origins in each human chromosome
- generate “Replication Bubbles”
DNA Replication
- bidirectionality
[A-T Rich sequences]

Helicase
 DNA Replication
Requirements:
- DNA Templates
- Unwinding/separation of the strands
- unwinding by the helicases
- maintenance of single stranded status by proteins
- primer (RNA)
- Deoxyribonucleotides (dATP, dGTP,dCTP,dTTP)
- Enzymes to polymerize bases & proteins
- DNA polymerases
- polymerizes ONLY in 5’ to 3’ direction
- cannot initiate polymerization on its own
- needs short RNA primer to initiate
-Editing (proofreading & correction)
 DNA Replication
Initiation….
- Synthesis of RNA primer

Leading
strand

Lagging
strand

DNA Primase Pyrophosphorylase

UTP UMP + PPi 2Pi
 DNA Replication
Initiation….

Figure 5-8. The structure of a DNA replication fork. Because both

daughter DNA strands are polymerized in the 5 -to-3 direction, the DNA
synthesized on the lagging strand must be made initially as a series of
short DNA molecules, called Okazaki fragments.
 DNA Replication
Initiation
Antiparallel direction
- DNA polymerase can only add in the 5’ 3’ direction
- Synthesize new fragments in lagging strand
- OKAZAKI FRAGMENTS
 DNA Replication
Initiation
DNA Polymerase
dNTP dNMP + PPi
 DNA Replication
Replication
- DNA Polymerases
- synthesize DNA in the 5’-3’ direction
- never in 3’-5’ direction
- template-directed enzymes
- requires RNA primer
- common properties:
- DNA elongation
- proofreading
- checking for errors (eg. misincorporation)
- correct/repair errors
- 3’-5’ exonuclease
 DNA Replication
Termination
- DNA synthesis stops at RNA primers
- lagging strands
- removal of RNA primers
- 5’-3’ exonuclease in DNA polymerase I
in bacteria cells
- RNase H in human cells
- refilling of gaps left by RNA removal
- resealing of DNA strands by DNA ligase
 DNA Replication
Termination
 DNA Replication
Termination
DNA Replication
- overall process is similar
between bacteria and human cells
- enzymes are similar, and have
different names
 DNA Replication (Bacteria)
Helicase
- separate double stranded DNA
Primase
- synthesize RNA primer
DNA polymerase I
- has RNase activity to remove RNA primer
- DNA polymerase activity fills in resulting gap
DNA polymerase II
- DNA repair
DNA polymerase III
- take over from primase to synthesize the DNA
strands from the 3’ OH of the RNA primer
Ligase I
- join DNA fragments
 DNA Replication (Human)
Helicase
- separate double stranded DNA
Primase
- synthesize RNA primer
DNA polymerase α
- synthesis of short DNA fragment from primer
- Okazaki fragments
DNA polymerase g
- mitochondrial DNA replication
DNA polymerase δ and ε
- take over polymerase a to elongate DNA
RNAse H
- removes RNA primers
Ligase I
- join DNA fragments
DNA Replication

*
 DNA Replication
1. Bidirectional synthesis
2. Multiple origins in each human chromosome
- generate “Replication Bubbles”
- bacteria has a single origin
 DNA Replication
DNA is supercoiled in chromosomes
- separation of DNA double strands for
replication or transcription will introduce
supercoils & increase tensions on DNA
strands
 DNA Replication
DNA supercoils
- increased twisting of DNA strands caused by
unwinding of DNA for replication
- increase torsional stress
- topoisomers released by topoisomerases
 DNA Replication
DNA Topoisomerases
- relax supercoils
- unwind supercoiled DNA
- during transcription
- ahead of the replication fork
- untangle replicated DNA after replication in
preparation for mitosis
 DNA Replication
DNA supercoils
- relaxation by topoisomerases
- nick DNA strand
- unwind the supercoils
- reseal the nick
 DNA Replication
DNA supercoils
- package DNA to fit into nuclei
- topoisomerases
- relaxes superhelical coils of DNA
- during replication and transcription
- Types I and II topoisomerases
- replication, transcription
 DNA Replication
DNA supercoils
- topoisomerases
- Type I
- cleaves one strand DNA
- relaxes supercoil
- ATP not required
 DNA Replication
DNA supercoils
- topoisomerases
- Type II
- cleaves both strands DNA
 DNA Replication
DNA supercoils
- topoisomerases
- Type II
- cleaves both strands DNA
- separate interlocked DNA after replication
- separate entangled mammalian
chromosomes
- prepare for cell division at mitosis
- requires ATP
DNA Replication
- at ends of linear DNA
 DNA Replication
Telomeres
- ends of chromosomes in eukaryotes
- DNA is linear
- cannot replicate right to the end of DNA molecule
- lose repeats after each round of replication
- loss of telomeres limit replicative life-span of cells
- lead to cellular aging and senescence
- synthesized by telomerase
- contain RNA as template
- reverse transcriptase
- not expressed in most cells in the body
- only in some cells, eg. germ & stem cells, lymphocytes
- addition of telomere repeat sequences at ends
 SUMMARY
Understand the storage of genetic information,
and how it is faithfully passed down to
successive generations.
- how is DNA organized in cells
- how is DNA replicated
DNA REPAIR
Learning objective
FOI.6.3. Given that DNA is exposed to
damaging agents, identify the types of
damage and describe the DNA repair
mechanisms that restore the integrity of
genetic material

- maintain integrity of DNA sequences

• Common causes of DNA damage
• Types of DNA damages and Repair of
damages
DNA REPAIR
1. Causes of DNA damage
I. Replication errors
II. Spontaneous
III. Chemicals
- cigarette smoke
- metabolism
- foods (eg. barbeques, vegetable)
IV. Reactive oxygen species (ROS)
- external or produced in the body
V. Radiation
- UV-light
- ionizing radiation
DNA REPAIR
Repair of DNA damages
- Restore the integrity of the DNA
- thousands of random damages in human cell
each day
- fewer than one in a thousand base changes
remain as permanent mutations
- contribute to evolution
- Consequences of mutations
- nothing if amino acid is not change (eg. Wobble
position), or occur in silent regions of genes
- or conservative changes
- cell death if affect crucial genes
- growth of cancers if affect growth regulators
- responsible for senescence and aging
DNA REPAIR
- maintain integrity of DNA sequences

1. Common causes of DNA damage

2. Types of DNA damages and

Repair of damages
DNA REPAIR
Types of DNA damage:
1. Single base damages
2. Two base damages
3. DNA strand breaks
Repair pathways:
1. Base excision repair
2. Nucleotide excision repair
3. Nonhomologous end joining
4. Homologous recombination
5. Mismatch repair
DNA REPAIR
Repair of DNA damages
General mechanism
1. Recognition/identification of the damage
2. Excision of damaged nucleotide(s)
3. Replacing with correct nucleotide(s)
4. Ligation
DNA REPAIR
Types of DNA damage:
1. Single base damages
- incorrect base-pairing
- alteration of bases
- chemical changes
- eg. alkylation, oxidation
- deamination
- cytosine to uridine
- adenine to hypoxanthine
- deletion of bases
- depurination and depyrimidination
- AP sites
- insertion of extra nucleotides
DNA REPAIR
Types of DNA damage

Figure 5–38 Depurination and deamination (Albert’s et al., 6ed)

Base excision repair
- 2-step removal of damaged base initiate process
Base excision repair

Figure 5– 41 Base excision repair (Albert’s et al., 6ed)

DNA REPAIR
Types of DNA damage
2. Two-base alteration
- UV light - induced thymine-thymine dimers
- also with cytosine-pyrimidine dimers

Figure 5–39 The most common type of thymine dimer (Albert’s et al., 6ed)
DNA REPAIR
Types of DNA damage
2. Two-base alteration
- cross-linking by alkylating agent
- adjacent bases on the same DNA strand
- bases on opposite DNA strands
- cross-link DNA strands
- DNA and protein molecules
DNA REPAIR
Types of DNA damage
2. Bulky adducts
- reactions with big molecules, e.g. carcinogens
- e.g. benzopyrene, aflatoxin, etc.
- cause “kinks” in DNA structure
- e.g. pyrimidine dimers
Nucleotide excision repair

Figure 5– 41 Nucleotide excision repair (Albert’s et al., 6ed)

DNA REPAIR
Types of DNA damage
3. DNA strand breaks
- single or double-strand breaks
- caused by radiation or chemicals
- eg. radiation therapy, cosmic rays, etc.
Nonhomologous end-joining

Figure 5– 45 Nonhomologous end joining (Albert’s et al., 6ed)

- error-prone repair - result in mutations
Homologous recombination

(After DNA
replication)

Figure 5– 45 Homologous recombination (Albert’s et al., 6ed)

5. Types of damages: DNA mismatches
- Repair of misincorporated nucleotide
during DNA replication
- how do cells determine which is the
incorrectly inserted nucleotide?

- bacteria
- parental DNA is methylated
- daughter strand is unmethylated
- humans
- DNA is not methylated
- look for nick from Okazaki fragment
Mismatch repair

Figure 5– 19 Mismatch repair (Albert’s et al., 6ed)

DNA REPAIR - SUMMARY
Repair of DNA damages
Major DNA repair pathways
1. Base excision repair
- base modifications, AP sites
2.Nucleotide excision repair
- bulky adducts, pyrimidine dimers
3. Nonhomologous end-joining
- double strand breaks
4. Homologous recombination
- double strand breaks
- use sister chromatids
- only after DNA replication
5. Mismatch repair
- misincorporated base during DNA replication
 SUMMARY
DNA damages
- types and causes
DNA repair mechanisms
- enzymes for different types of damages