Biochemical genetics
&molecular biology
Batch -2023
MBBS 1st year
AMDC,LHR
Higher Organization of DNA
CHROMATIN IS THE CHROMOSOMAL MATERIAL IN THE NUCLEI OF
CELLS OF EUKARYOTIC ORGANISMS
• DNA molecule
• Histones
• to condense the DNA
• integrally participate in gene regulation
• Nonhistone proteins
• nonhistone proteins include enzymes involved in DNA replication and repair, and
the proteins involved in RNA synthesis, processing, and transport to the cytoplasm
• RNA
Histones Are the Most Abundant Chromatin Proteins
• H1 histones
least tightly bound to chromatin
Organizational unit of soluble chromatin is
the nucleosome
• H2A
• H2B
• H3
• H4 (core Histones)
• These four core histones are subject to at least six types of covalent modification: acetylation,
methylation, phosphorylation, ADP-ribosylation, monoubiquitylation, and sumoylation. These
histone modifications play an important role in chromatin structure and function
Nucleosome Contains Histone & DNA
H3 and H4 form a tetramer containing two molecules of each (H3-H4)2
H2A and H2B form dimers (H2A-H2B)
oligomers associate to form the histone octamer of the composition (H3-H4)2
-(H2A-H2B)2
Neither the histone H1 nor the nonhistone
proteins are necessary for the reconstitution
of the nucleosome core.
Beads on a string
• The (H3-H4)2 tetramer itself can confer nucleosome-like properties
on DNA and thus has a central role in the formation of the
nucleosome.
• The addition of two H2A-H2B dimers stabilizes the primary particle
and firmly binds two additional half-turns of DNA previously bound
only loosely to the (H3-H4)2
• Thus, 1.75 superhelical turns of DNA are wrapped around the surface
of the histone octamer, protecting 146 base pairs of DNA and forming
the nucleosome core particle
Electron micrograph of nucleosomes (white, ball-shaped) attached to strands of DNA (thin,
gray line)
Histone chaperones
• Group of proteins that exhibit high-affinity histone binding
• As the nucleosome is assembled, histones are released from the
histone chaperones
• Nucleosomes appear to exhibit preference for certain regions on
specific DNA molecules
Chromatids vs chromosomes
• At metaphase, mammalian chromosomes possess a 2-fold symmetry, with the
identical duplicated sister chromatids connected at a centromere.
A human karyotype
• 3'→5'-Phosphodiester bonds
• Double helix
• Base-pairing
• Separation of the two DNA strands in the double helix
• Structural forms of the double helix
• Linear and circular DNA molecules
DNA REPLICATION
• INITIATION
• ELONGATION
• TERMINATION
Classes of Proteins Involved in Replication
• DNA polymerases
• Helicases
• Topoisomerases
• DNA primase
• Single-strand binding proteins
• DNA ligase
STEPS IN PROKARYOTIC DNA
SYNTHESIS
• Separation of the two complementary DNA strands
1)Formation of the replication fork
• DnaA protein at A+T region (autonomously replicating sequences (ARS) or replicators at origin
replication element (ORE)
• origin of replication in yeast and is bound by the MCM protein complex.
• (DnaB/DnaC) in prokaryotes while DNA helicases
• Single-stranded DNA-binding (SSB) proteins
• RNA primer
• Primase (DnaG)
• priming by a short length of RNA, about 10–200 nucleotides long
• Primosome
Use of an RNA primer to
initiate DNA synthesis
3) Direction of DNA replication
A) leading strand
B) lagging strand
2) Solving the problem of supercoils
Type I DNA topoisomerases
Type II DNA topoisomerases
Chain elongation
DNA polymerase III
Proofreading of newly synthesized DNA
. 5'→3' Exonuclease activity
• 3' → 5' Exonuclease activity
• 5'→3' Exonuclease activity
DNA ligase
EUKARYOTIC DNA REPLICATION
Steps Involved in DNA Replication in
Eukaryotes
1. Identification of the origins of replication
2. Unwinding (denaturation) of dsDNA to provide an ssDNA template
3. Formation of the replication fork; synthesis of RNA primer
4. Initiation of DNA synthesis and elongation
5. Formation of replication bubbles with ligation of the newly
synthesized DNA segments
6. Reconstitution of chromatin structure
Prokaryotes vs Eukaryotes Replication
Eukaryotic DNA polymerases
Telomeres
• Telomeres are complexes of noncoding DNA plus proteins located at
the ends of linear chromosomes
• Following removal of the RNA primer from the extreme 5'-end of the
lagging strand, there is no way to fill in the remaining gap with DNA
• Telomere shortening
• Telomerases solved the problem
What are
Telomerases????
complex contains a protein that acts as a reverse transcriptase
Reverse transcriptases, as seen with telomerase, are RNA-
directed DNA polymerases.
These viruses carry their genome in the form of ssRNA
molecules. Following infection of a host cell, the viral enzyme,
reverse transcriptase, uses the viral RNA as a template for the
5'→3' synthesis of viral DNA, which then becomes integrated
into host chromosomes
Methods of DNA REPAIR
A. Methyl-directed mismatch repair
B. Repair of damage caused by ultraviolet (UV) light (nucleotide
excision repair)
C. Correction of base alterations (base excision repair)
A. Methyl-directed
mismatch repair
1. Identification of the mismatched strand
2. Repair of damaged DNA
DNA REPAIR
damaging agents can be either chemicals
nitrous acid
ultraviolet light
Repair reduces the error rate from one in ten million bases to one in a
billion.
Types of Damage to DNA
• I. Single-base alteration
• A. Depurination
• B. Deamination of cytosine to uracil
• C. Deamination of adenine to hypoxanthine
• D. Alkylation of base
• E. Insertion or deletion of nucleotide
• F. Base-analog incorporation
• II. Two-base alteration
• A. UV light–induced thymine-thymine (pyrimidine) dimer
• B. Bifunctional alkylating agent cross-linkage
• III. Chain breaks
• A. Ionizing radiation
• B. Radioactive disintegration of backbone element
• C. Oxidative free radical formation
• IV. Cross-linkage
• A. Between bases in same or opposite strands
• B. Between DNA and protein molecules (eg, histones)
Mechanism of DNA Repair
• Mismatch repair
• Copying errors (single base or two- to five-base unpaired loops) Methyl-
directed strand cutting, exonuclease digestion, and replacement
• Base excision–repair Spontaneous, chemical, or radiation damage to a
single base Base removal by N -glycosylase, abasic sugar removal,
replacement
• Nucleotide excision–repair Spontaneous, chemical, or radiation damage to
a DNA segment Removal of an approximately 30-nucleotide oligomer and
replacement
• Double-strand break repair Ionizing radiation, chemotherapy, oxidative free
radicals Synapsis, unwinding, alignment, ligation
Nucleotide Excision-Repair
• used to replace regions of damaged DNA up to 30 bases in length
• damage include ultraviolet (UV) light
• induces the formation of cyclobutane pyrimidinepyrimidine dimers,
smoking, which causes formation of benzo[a ]pyrene-guanine adducts
• Ionizing radiation, cancer chemotherapeutic agents, and a variety of
chemicals found in the environment cause base modification
repaired by a process called nucleotide excision-repair
• A fragment of DNA 27–29 nucleotides long is excised.
• An excision nuclease (exinuclease) cuts the DNA upstream and
downstream of the defective region
• This gap is then filled in by a polymerase ( / in humans) and religated.
B. Repair of damage
caused by ultraviolet
(UV) light (nucleotide
excision repair)
Exposure of a cell to UV light can result in the covalent joining of
two adjacent pyrimidines (usually thymines), producing a dimer
Recognition and excision of dimers by UV-specific endonuclease
UV radiation and cancer
xeroderma pigmentosum (XP)
xeroderma pigmentosum (XP)
autosomal recessive genetic disease
sensitivity to sunlight (ultraviolet) with subsequent
formation of multiple skin cancers and premature
death
Cells cultured from patients with xeroderma
pigmentosum exhibit low activity for the nucleotide
excision-repair process
Seven complementation groups have been
identified using hybrid cell analyses
• At least seven gene products (XPA–XPG) are involved.
• Two of these (XPA and XPC) are involved in recognition and excision.
XPB and XPD are helicases
• Interestingly, are subunits of the transcription factor TFIIH
C. Correction of base alterations (base excision repair)
• Action of deaminating or alkylating compounds
• Bases of DNA can be altered
Removal of abnormal bases
Recognition and repair of an AP site:
Repair of double-strand breaks
High-energy radiation or oxidative free radicals can cause double- strand
breaks in DNA
Repaired by two methods
1) nonhomologous end-joining repair
2) homologous recombination repair