CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR

BIOMEDICAL IMPORTANCE
Genetic info in DNA of chromosome can be
transmitted y exact replication or can be
exchanged by a number of processes.
Processes provide means of ensuring
adaptability and diversity for the organism
Mutations are due to change in base sequence
of DNA, may result from faulty replication,
movement or repair of DNA.
vertical transmission- Mutation in a germ cell is
transmitted to offspring
horizontal transmission- mutations of somatic
cells are passed on to successive generation but
only w/in an organism
most CAs are due to combined vertical and
horizontal transmission of induced mutations
CHROMATIN IS THE CHROMOSOMAL MATERIAL IN THE
NUCLEI OF CELLS OF EUKARYOTIC ORGANISMS
chromatin consist of very long double-stranded
DNA (dsDNA) and equal mass of histones
Histones- small basic proteins, function is to
condense the DNA, participate in gene
regulation
Non-histone proteins- acidic and larger than
histones, include enzyme involved inDNA
replication and repair, involved in RNA
synthesis, processing and transport to
cytoplasm
Nucleosome- dense spherical particle, 10 nm in
diameter,connected by DNA filaments,
composed of DNA wound around a collection of
histone molecules
Histones are the most abundant chromatin proteins
Histones- cmall family of closely related basic
CHONs
H1 histones- least tightly bound to chromatin,
easily removed w/ salt soln, more solube
Nucleosome- organizational unit of soluble
chromatin

Richelle Dianne G Ramos RPh

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Core histones- H2A, H2B, H3 and H4 : highly

conserved between species,
High conservation implies that function of
histones are identical in all eukaryotes
Carboxyl terminal 2/3 of histone molecules are
hydrophobic
Amino terminal 1/3p rich in basic amino acids
Core of histones are subject to 6 types of
covalent modification or post translational
modification (PTMs)
Acetylation- H3 and H4, associated w/
activation or inactivation of gene
transcription,
acetylation of core histones is associated
w/ chromosomal assembly during DNA
replication
Phosphorylation of H1- condensation of
chromosomes during replication cycles
ADP-ribosylation- DNA repair
Methylation- activation and repression of
gene transcription
Monoubiquitylation- gene activation,
repression and heterochromatic gene
silencing
Sumoylation- SUMO( small ubiquitin-related
modifier)- transcription repression
H3 and H4 form a tetramer containg 2
molecules of each
H2A and H2B form a dimer
Histone oligomers form histone octamer w/
(H3-H4)2-(H2A-H2B)
The nucleosome contains histone & DNA
Histone octamer is mixed w/ purified dsDNA
under physiologic conditions.
Reconstiturion of nucleosomes from DNA and
histones H2a, H# and H$ is independent of the
organismal or cellular origin components
H1 and nonhistone CHONs are NOT necessary
for nucleosome core reconstitution

CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR

In nucleosome, DNA is supercoiled in a lefthanded helix over the disk-shaped histone

octamer
Majority of histone cores interact inside the
DNA w/out protruding
Amino terminal tail of histones extend outside
of its structure , available for regulatory PTMs
H3-H4 tetramer can confer nucleosome-like
properties on DNA, has role in formation of
nucleosome
Addition of 2 H2A-H2B dimer stabilizes primary
particle, firmly binds 2 additional half turns of
DNA
1.75 superhelical turns of DNA are wrapped
around histone octamer to protect 145-150bp
of DNA, forming nucleosome core particle
Linker- separate core particles in a chromatin of
DNA
Histone chaperones- group of CHON that
exhibits high-affinity histone binding, mediate
assembly of nuclear chromatin
Phasing- basis for non-random distribution of
nucleosome
HIGHER ORDER STRUCTURES PROVIDE FOR
COMPACTION OF CHROMATIN
There are 2 higher orders of structure
10nm fibril- consist of nucleosomes
arranged w/ their edges aparated by small
distance(30bp of DNA), w/ flat faces parallel
to fibril axis
30nm chromatin fiber- form when there is
further supercoiling of 10nm fibril w/ 6 or 7
nucleosomes per turn
H1 histones- stabilize 30 nm diber
To form a mitotic chromosome, the 3onm fiber
must be compacted in length 100 fold.
Interphase chromosomes- chromatin fibers
appear to be organized by loops and domains
anchored in a scaffolding w/in the nucleus
Nuclear matrix- supporting matrix w/in the
nucleus, anchor chromatin fibers

Richelle Dianne G Ramos RPh

BIOCHEMISTRY PRELIMS

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Ach looped domain of chromatin correspond to

one or more specific function and contains
coding and noncoding regions of cognate gene
or genes
Certain gene regions are mobile w/ in the
nucleus moving to discrete loci w/in the nucleus
upon activation
SOME REGIONS OF CHROMATIN ARE ACTIVE AND
OTHERS ARE INACTIVE
Chromatin containing active genes
(transcriptionally or potentionally
transcriptionally active) show to differ in several
ways from inactive genes
Nucleosome structure appears to be altered in
highly active regions
DNA in active chromatin contains about
100,000 bases that are more sensitive to
digestion by a nuclease like DNase I
DNase I- make single-stand cuts in any segment
of DNA, digest DNA that is not protected or
bound by CHON
Sensitivity to DNase I reflects only a potential
for transcription not transcription itself
Sensitivity to DNase I can be correlated to
relative lack of 5-methyldeoxycytidine (meC)
w/in large regions of active chromatin, there
exist shorter stretches of 100-300 nucleotides
which are more sensitive to DNase I
hypersensitive sites result from structural
conformation that favours access of nuclease to
DNA
hypersensitive regions are location of
interrupted nucleosomal structure caused by
binding of nonhistone regulatory transcription
factor CHONs
if a gene is capable of being transcribed, it has a
DNase-hypersensitive site
nonhistone regulatory CHONs involved in
transcription control and maintaining access
template strand lead to formation of
hypersensitive sites.

CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR

Heterochromatin- transcriptionally INACTIVE

chromatin is densely packed during interphase.
There are 2 types
Constitutive heterochromatin- always
condensed and essentially inactive, found
near chromosomal centromere
Facultative heterochromatin at times it is
condensed, sometimes it is actively
transcribed and thus, uncondensed and
appears as euchromatin
Example:
- One of two X chromosome is
heterochromatic, but this chromosome
decondenses during gametogenesis and
becomes transcriptionally active during
embryoenesis.
Euchromatin- transcriptionally ACTIVE, stains
less densely, replicated earlier than
heterochromatin in mammalian cell cycle
Chromatin in regions of inactivity has high meC
content and histones contain relatively lower
levels of covalent modifications
Polytene chromosomes- chromosomes that
have been replicated for multiple cycles w/o
separation of daughter cells
Transcriptionally active regions of polytene
chromosomes are especially decondensed into
puff that contain enzymes responsible for
transcription and sites of RNA synthesis
Fluorescent in situ hybridization used for
mapping specific gene sequence
DNA IS ORGANIZED INTO CHROMOSOME
At metaphase, mammalian chromosome posses
a two-fold symmetry, w/ identical duplicated
sister chromatids connected at a centromere
During metaphase, chromosomes are nearly
completely transcriptionally INACTIVE
Centromere- adenine thymine (A-T) rich region
containing repeated DNA sequences
Size range of centromere: 102 (brewers yeast)
to 10 (mammals) base pairs (bp)

Richelle Dianne G Ramos RPh

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Metazoan centromeres-bound by nucleosomes

containing histone H3 variant protein CENP-A
Kinetochore- provides anchor for mitotic
spindle, essential structure for chromosomal
segregation
Telomeres- found at the end of each
chromosome, consist of TG-rich repeats.
Human telomeres have a variable number of
repeats of 5-TTAGGG-3
Telomerase- enzyme responsible for telomere
synthesis, for maintaining length of telomere,
attractive target for chemotherapy and drug
development
Telomere shortening- associated with malignant
transformation and aging
Each sister chromatid contains on dsDNA
molecule
During interphase, DNA molecule packing is
less dense than it is in the condensed
chromosome during metaphase
Human haploid genome consist of about 3x109
bp and about 1.7x 107
Each 23 chromatids in human haploid genome
contain 1.3x108 nucleotides in one dsDNA
llength of DNA must be compressed about
8000-fold to generate condensed structure of
chromosome during metaphase
in metaphase chromosome the 30nm
chromatin fibers are also folded into a series of
looped domains
proximal portions of chromosomes are
anchored to a nonhistone proteinaceous
nuclear matrix w/in the nucleus
packaging of nucleoproteins w/in chromatid is
not random
quinacrine or giemsa stain- used for observation
of patterns of chromatids
Pattern staining (banding) of entire
chromosome complement is highly
reproducible. Differs significantly between
species

CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR

Coding regions are often interrupted by intervening

sequences
Transcripts of protein coding regions of DNA
which appear in the cytoplasm as single mRNA
are usually interrupted in the eukaryotic
genome by large intervening sequences of
nonprotein-coding DNA
mRNA precursor- primary transcripts of DNA,
contain noncoding intervening sequences of
RNA that must be removed in a process by
which also joins together the appropriate
coding segments to form mature mRNA
Introns- noncoding intervening sequence,
longer than coding regions, separate functional
domains of coding information in form that
permits genetic rearrangement by
recombination to occur more rapidly
Exons coding region
Enhanced rate of genetic rearrangement allow
more rapid evolution of biologic function.
Other protein or noncoding RNAs are localized
within the intronic DNA of certain genes
MUCH OF THE MAMMALIAN GENOME APPEARS
REDUNDANT & MUCH IS NOT HIGLY TRANSCRIBED
Haploid genome of eache human cell consists of
3x106 bp of DNA subdivided into 23
chromosomes
Entire haploid contains sufficient DNA to code
for 1.5M average-sized genes
Humans have significantly fewer than 100,000
CHONs encoded by the ~1% of human genome
that is composed of exonic DA
There are 25,000 or less CHON-coding genes in
human
Most of the DNA is nonprotein-coding , its
information is never translated into an amino
acid sequence of a protein molecule
Excess DNA regulate the expression of genes by
serving as binding sites for regulatory

Richelle Dianne G Ramos RPh

BIOCHEMISTRY PRELIMS

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Some excess clearly makes up intervening

sequence of introns (24% of total human
genome) that split coding regions of genes
Small RNAs transcribed from repeats can
modulate transcription by interactic with the
transcription machinery or indirectly by
affecting the activity of the chromatin template
ENCODE Project Consortium shown that for 1%
of genome studied most of the genomic
sequence was indeed transcribed at a low rate
DNA in eukaryotic genome can be divided into
different sequence cases
Unique sequence DNA or Nonrepetitive
DNA- includes single copy of genes that
code for CHONs
Repetitive DNA- include sequences that
vary in copy number from 2 to as many as
107 copies per cell
More than half the DNA in eukaryotic DNA in
eukaryotic organisms is in Unique or Nonrepetitive
sequences
in brewers yeast about 2/3 of its 6200 genes
are expressed but only ~1/5 are required for
viability under laboratory growth conditions
in a higher eukaryote between 10,000 and
15,000 genes are actively expressed
In human DNA, at least 30% of the human geome
consists of repetitive sequences
repetitive sequence DNA can be broadly
classified as moderately repetitive or as higly
repetitive
highly repetitive sequence consist of 5-500 base pairs lengths repeated
as many times in tandem,
often clustered in centromeres and
telomeres of the chromosome
some are present in about1-10M copies per
haploid
majority are transcriptionally inactive
play a structural role in the chromosome

CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR

moderately repetitive sequence

present in numbers of less than 106 copies
per haploid genome
are not clustered but are interspersed with
unique sequences
Long interspersed repeats are transcribed
by RNA polymerase II
Contain caps indistinguishable from those
on mRNA
Depending on length, moderately repetitive
sequence are classified as long interspersed
repeat sequence (LINEs) or short
interspersed repeat sequence (SINEs)
Retroposons- arose from movement from one
location to another (transposition) by action of
reverse transcriptase that transcribes an RNA
template into DNA
Mammalian genomes contain 20,000-50,000
copies of 6-7 kbp LINEs
SINEs are shorter (70-300bp)there may be more
than 100,000 copies per genome
Alu Family- one example of SINEs in human
genome, it is present in about 500,000 copies
per haploid genome and accounts for ~10% of
the human genome
Members of Alu family are transcribed as
integral components of mRNA precursors or as
discrete RNA including 4.5S RNA and 7S RNA
which are highly conserved w/in a specie
Components of SINEs may be mobile elements,
capable of jumping into and out of various sites
w/in the genome
Alu B1 and B2 SiNE RNAs have been shown to
regulate mRNA production at the levels of
transcription and mRNA splicing
Microsatellite repeat sequence
Microsatellite sequence exist as both dispersed
and grouped tandem arrays
These sequences are found in dinucleotide
repeats of AC on one strand and TG on the
opposite strand

Richelle Dianne G Ramos RPh

BIOCHEMISTRY PRELIMS

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Number of these repeats may vary on the two

chromosomes thus providing heterozygosity in
the number of copies of a particular
microsatellite number in an individual
Polymerase chain reaction- used to detect
microsatellite sequences
PCR is used to screen families for microsatellite
polymorphism
Association of polymorphism with a gene of
affected family member , and lack of
association with the gene of unaffected may be
the first clue about the genetic basis of a
disease
Microsatellite instability- Trinucleotide
sequences that increase in number can cause
disease
Unstable p(CGG) repeat sequence is associated
with fragile X syndrome
Trinucleotide repeats that undergo dynamic
increase are associated with Hungtingtons
chorea (CAG), myotonic dystrophy (CTG),
spinobulbar muscular atrophy (CAG) and
Kennedy disease (CAG)
ONE PERCENT OF CELLULAR DNA IS IN MITOCHONDRIA
54 out of 67 polypeptides in mitochondria are
coded by nuclear genes, the rest are coded by
genes found in mitochondrial DNA (mtDNA)
Features of human mitochondrial DNA
Mitochondrial DNA is circular, doublestranded and composed of heavy and light
chains or strands
Contains 16,569 bp
Encodes 13 CHON subunits of respiratory
chain
- Encodes 7 subunits of NADH
dehydrogenase (complex I)
- Cytochrome b of complex III
- 3 subunits of cytochrome oxidase
(complex IV)
- 2 subunits of ATP synthase
Encodes 22 mt tRNA molecules

CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR

Encodes large 16S and small 12S mt

ribosomal RNAs
Has AGA and AGG, read as Arg, as stop
codon instead of UGA w/c is read as Trp
High mutation rate (5 to 10x that of nuclear
DNA)
Since all of mitochondria are contributed by the
ovum during zygote formation , it is transmitted
by maternal nonmendelian inheritance
An affected mother would pass the disease to
all of her children but only daughters would
transmit the trait
GENETIC MATERIAL CAN BE ALTERED AND
REARRANGED
An alteration in the sequence of pur and pyr
bases in a gene due to removal or insertion of
one or more bases may result in an altered
gene product
Alteration in genetic material results in a
mutation
Chromosomal recombination is one way of rearranging
genetic material
Genetic info can be exchanged between similar
or homologous chromosomes. The exchange is
called recombination
Recombination- occurs primarily during meiosis
in mammalian celland require alignment of
homologous metaphases
Alignment always occurs with great exactness
Crossing over- results in an equal and reciprocal
exchange of genetic info between homologous
chromosomes
When alignment is not exact the crossing over
or recombination event may result in an
unequal exchange of info
One chromosome may receive less material ,
and thus, a deletion
Other partner of the chromosome pair receives
more genetic material and thus an insertion or
duplication

Richelle Dianne G Ramos RPh

BIOCHEMISTRY PRELIMS

SEM 2

Unequal crossover affects tandem arrays of

repeated DNAs whether they are related globin
genes or more abundant repetitive DNA ex:
hemoglobins designated Lepore and antiLepore
Unequal crossover through slippage in the
pairing can result in expansion or contraction in
the copy number of repeat family
Unequal crossover may contribute to expansion
and fixation of variant members throughout the
repeat array
The farther apart the 2 sequences are on an
individual chromosome, the greater the
likelihood of a crossover recombination
Chromosomal integration occurs with some viruses
Bacteriophages- bacterial viruses, capable of
recombining w/ DNA of a bacterial host in a way
that the genetic info of bacteriophage is
incorporated in a linear fashion into the genetic
info of the host
Integration- the backbone of the circular
bacteriophage genome is broken as is that of
DNA molecule of the host
Bacteriophage DNA is straightened out or
linearized as it is integrated into the bacterial
DNA molecule, frequently, a closed circle as
well
If the bacteriophage contains DNA sequence
homologous to the host, a recombination event
analogous to that occurring between
homologous chromosomes can occur
Some bacteriophages synthesize proteins that
bind specific sites on bacterial chromosomes to
a nonhomologous site characteristics of the
bacteriophage DNA molecule
Integration is said to be site specific
DNA transcript of RNA viruses such as HIV that
causes AIDS is generated by the action of the
viral RNA-dependent DNA polymerase or
reverse transcriptase

BIOCHEMISTRY PRELIMS

CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR

Integration of animal virus DNA into the animal

genome is not site specific, but display site
preferences
Transposition can produce processed genes
Jumping genes- In eukaryotic cells, small DNA
elements that are not viruses can transport
themselves in and out of the host genome in
ways that affect the functions of neighbouring
DNA sequences
Jumping genes- can carry flanking regions of
DNA , proudly affect evolution
Alu family of moderately repeated DNA
sequences has same characteristics with termini
of retro viruses has the ability to move into and
out of mammalian genome
Processed genes- consist of DNA sequences
identical or nearly identical to those of the
messenger RNA for the appropriate gene
product
5-nontranslated region,coding region w/o
intron representation
3 poly(A) tail are all present contigously
this particular DNA sequence arrangement must
have resulted from reverse transcription of an
appropriately processed mRNA from w/c
introns had been removed and poly(A) tail
added (by transposition event)
processed genes have short terminal repeats at
each end as known to transposed sequences
Pseudogenes- genes that have been randomly
altered through evolution, contain nonsense
codons that prevent their ability to encode
functional and intact CHON
Gene conversion produces reaarangements
Gene conversion
occasionally pair up and eliminate
mismatched sequences,
Lead to accidental fixation of one variant or
another of repeated sequences,

Richelle Dianne G Ramos RPh

SEM 2

homogenize sequences of members of

repetitive DNA families

Sister chromatids exchange

in diploid eukaryotes such as human, after cells
progress through the S phase they contain
tetraploid content of DNA (sister chromatids)
each sister chromatids contain identical genetic
info since each is product of replication of
original parent DNA
crossing over can occur between each sister
chromatids
this sister chromatid exchange have no genetic
consequence as long as it is a result of an equal
crossover
Immunoglobulin genes rearrange
Gene rearrangement occur normally during
development and cell differentiation
In mice VL and VC genes for single
immunoglobulin molecule are widely separated
in the germ line DNA
Plasma cell- immunoglobulin producing cell
In differentiated plasma cell, VL and VC genes
have been moved physically closer together in
the genome and into the same transcription
unit
Rearrangement does not bring the VL and VC
genes into contiguity in the DNA
DNA contains interspersed or interrupted
sequence of 1200 base pairs at or near the
junction of V and C regions
Interspersed sequence is transcribed to RNA
along w/ VL and VC genes
interspersed info is removed from RNA during
nuclear processing
DNA SYNTHESIS & REPLICATION ARE RIGIDLY
CONTROLLED
primary function of DNA replication is the
provision of progeny w/ genetic info possessed
by the parent

CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR

replication must be complete and carried out in

way to maintain genetic stability
replication is complex and involves many
cellular functions and processes to ensure
fidelity of replication
about 30 CHONs are involved in replication of
E.coli
DNA polymerase I- has multiple catalytic
activities, complex structure, require 4 deoxy
ribonucleotides of A, G, C and T
Polymerization of E.coli by DNA polymerase I
has served as prototype for all DNA polymerase
Major role of polymerase is proofreading and
repair
In all cells, replication can occur only from a
single stranded DNA (ssDNA)
STEPS IN DNA REPLICATION
1. Identification of origins of replication
At the ori, there is an association of
sequence-specific dsDNA- binding
CHONs w/ a series of direct repeat DNA
sequences
- In bacteriophage , the ori is bound by
-encoded O protein to 4 adjacent sites
- In E.coli, oriC is bound by protein dna A
- A complex is form consisting 150-250
bp of DNA
- Autonomously replicating sequences
(ARS) have been identified in yeast cells
- ARS contains degenerate 11-bp called
origin replication element (ORE)
- Origin recognition complex (ORC)- set of
CHONs analogous to dnaA protein of
E.coli bound by ORE
- DNA unwinding element (DUE)- 80bpA+T-rich sequence that is easy to
unwind, origin of replication of yeast
and is bound by MCM CHON complex

Richelle Dianne G Ramos RPh

BIOCHEMISTRY PRELIMS

SEM 2

2. Unwinding of dsDNA to provide ssDNA

template
- Interaction w/ ori defines start site of
replication and provides short region of
ssDNA for initiation of nascent DNA
strand synthesis
- Requires formation of CHON-CHON and
CHON-DNA interactions
- Critical step provided by DNA helicase
- In uninfected E.coli function is provided
by complex dnaB helicase and dnaC
protein, stabilized by ssDNA binding
proteins (SSBs)
- In phage-infected E.coli the protein P
binds dnaB and P/dnaB binds to ori by
interacting w/ the O protein
- dnaB in an inactive helicase when in
P/daB/O complex
- dnaK, dnaJ GrepE- E.coli het shock
proteins, remove P protein and activate
dnaB helicase
- replication of phage is accomplished
at the expense of replication of the host
E.coli host
3. Formation of replication fork: synthesis of
RNA primer
A replication consists of 4 components
that form in the following sequence:
a. DNA helicase unwinds a short segment
of parental duplex DNA
b. A primase initiates synthesis of an RNA
molecule that is essential for priming
DNA synthesis
c. DNA polymerase initiate nascent,
daughter strand synthesis
d. SSBs bind to ssDNA and prevent
premature reannealing of ssDNA to
dsDNA
- DNA polymerase III enzyme- dnaE gene
product in Ecoli, bind to template DNA
as a part of multiprotein

BIOCHEMISTRY PRELIMS

CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR

Because DNA strands are antiparallel,

polymerase funcstions asymmetrically
- On leading (forward) strand, DNA is
synthesized in short fragments called
Okazaki fragments
- helicase acts on lagging strand to
unwinddsDNA I a 5-3 direction
- DNA polymerase cannot initiate DNA
synthesis de novo
- Primosome- mobile complex between
helicase and primase
4. Initiation of DNA synthesis and elongation
- Initiation of DNA synthesis requires
priming by a short length of RNA, 10200 nucleotides long catalyzed by dnaG
in E.coli
- In eukaryotes DNA pol asynthesizes
RNA primers
- Priming process involves nucleophilic
attack by 3-hydroxyl group of RNA
primer on the phosphate of the first
entering deoxynucleoside triphosphate
- The 3-hydroxyl group of of recently
attached deoxyribonucleoside
monophosphate is then free to carry
out nucleophilic attack on the next
entering deoxyribonucleoside
triphosphate again
- Selection of proper
deoxyribonucleotide to be attacked is
dependent upon proper base pairing
with other strand of the DNA
- Okazaki fragments- RNA initiator
component
5. Formation of replication bubbles w/
ligation of newly synthesized DNA
segments
Replication proceeds from a single ori
in the circular bacterial chromosome
composed of 5x106 bp of DNA
- Process is completed in 3o mins,
replication rate is 3x105 bp/min

Richelle Dianne G Ramos RPh

SEM 2

Replication bubbles- replication occurs

in both directions along all of the
chromosomes and both strands are
replicated simultaneously
- Initiation is regulated both spatially and
temporally, cluster adjacent sites
initiate replication synchronously
- There are more replicators and excess
ORC than needed to replicate
mammalian genome w/in S-phase
- During replication there must be
separation of 2 strands to allow each to
serve as a template by hydrogen
bonding its nucleotide bases to the
incoming deoxynucleoside triphosphate
- Separation is promoted by SSBs in E.coli
- Stabilizing CHONs bind to single strand
w/o interfering w/ the abilities of
nucleotides to serve as template
- To allow strand separation, there must
be unwinding
- Undwinding happens adjacent to
replication bubbles
- To counteract unwinding, there are
multiple swivels
- Swivel- function is provided by specific
enzymes that introduce nicks in one
strand of the unwinding doule helix
- RNase H degrades the hybridized
template RNA strand
- Reverse transcriptase-synthesize DNARNA hybrind utilizing RNA genome as
template
6. Reconstitution of chromatin structures
- nuclear organization and chromatin
structure are involved in determining
the regulation and initiation of DNA
synthesis
- rate of polymerization in eukaryotes is
slower than prokaryotes
- chromatin structure must be re-formed
after replication

CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR

newly replicated DNA is assembled into

nucleosome and the pre existing and
newly assembled histone octamers are
randomly distributed to each arm of
replication fork
reactions are facilitated through the
cactions of histone chaperone CHONs

Classes of proteins involved in replication

DNA polymerase- deoxyribonucleotide
polymerization
Helicases- unwinding of DNA
Topoisomerase- relieve torsional strain fom
helicase-induced unwinding
DNA primase- initiate RNA primers
synthesis
Single-strande binding CHONs- prevent
premature reannealing of DNA
DNA ligase- seals single strand nick between
the nascent chain and Okazaki fragments on
lagging strand
The DNA polymerasecomplex
3 important properties of DNA polymerase
1. Chain elongation- accounts for rate at w/c
polymerization occurs
2. Processivity- expression of the number of
nucleotides added to nascent chain before
polymerase disengages from template
3. Proofreading- identifies copying errors and
corrects them
DNA Polymerase III catalyzes the highest rate of
chain elongation and is most processive
Polymerase I and II- involved in proofreading
and DNA repair
Replication exhibits polarity
Enzyme capable of polymerizing DNA in 3 to 5
direction does not exist in any organism so
newly replicated DNA strands cannot grow in
the same direction simultaneous

Richelle Dianne G Ramos RPh

BIOCHEMISTRY PRELIMS

SEM 2

Same enzymes does not replicate both enzymes

at the same time
Semi discontinuous DNA synthesis
Single enzymes replicate leading strand in a
continuous manner in 5 to 3 direction
facing forward
Enzyme replicate the lagging strand
discontinuously while polymerizing
nucleotides in short spurts of 150-250
nucleotides in 5 to 3 direction facing
towards the end
DNA synthesis occurs during S phase of the cell cycle
Synthetic/ S phase- period of time where
replication occurs, temporally separated from
M phase by gap 1 (G1) and gap 2 (G2) called G
phase
Cell prepares for DNA synthesis in G1 and
prepares for mitosis in G2
Cell regulates DNA synthesis by allowing it to
occur only once per cell cycle at specific times in
cells preparing to divide by a mitosis
Cyclins- family of CHONs whose conc. increases
and decreases at specific times during cell cycle
Cyclin-dependent kinases (CDKs)-phosphorylate
substrates essential for progression through cell
cycle
Cyclin D conc increase in late G1 phase and
allow progression beyond the start in yeast, or
restriction point in mammals
CDK4 and CDK6- activated by D cyclins,
assemble as a complex in G1 phase, this
complex is an active serine-threonine CHON
kinase
Retinoblastoma (Rb)- substrate that regulates
cell cycle by binding to and inactivating a
transcription factor (E2F) necessary for
progression from G1 to S phase
Phosphorylation of R by CDK4 and CDK6 results
in release of E2F from Rb-mediated
transcription repression
Cyclin E, Cyclin A and kinase CDK2- initiate DNA
synthesis in early S phase

CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR

Cylin B and kinase CDK1- rate limiting for G2/M

phase transition
Oncovirus and oncogenes are capable of
alleviating or disrupting apparent restriction
that normally controls entry of mammalian cell
from G1 to S phase
Inappropriate production and activation in an
inappropriate time might result in abnormal or
unrestrained cell division
Bcl oncogene associated w/ B-cell lymphoma
appears to be the cyclin D1-gene
Oncoproteins target Rb transcription repressor
for inactivation
Inactivation of Rb, a tumor suppressor gene
leads to uncontrolled cell growth and tumor
formation
During S phase, nuclear DNA is completely
replicated once and only ONCE
All organism contain elaborate evolutionarily
conserved mechanism to repair damaged DNA
Repair of damaged DNA is critical for
maintaining genomic integrity and preventing
the propagation of mutation
Horizontal- DNA sequence changes in somatic
cells
Vertically-nonpaired lesions are present in
sperm or oocyte hence it can be transmitted to
progeny
5 mechanism of DNA repair
repair pathways
Nucleotide Excision
Repair (NER)
Mismatch Repair
(MMR)

damaging
agents
UV light
chemicals
Replication
errors

Basic Excision
Repair (BER)

O2 radicals
hydrolysis,
alkylating
agents

Homologous
Recombination (HR)
And

Xrays,
ionizing
radiation,

lesions formed
Bulky adducts
pyr dimers
mismatch,
insertion,
deletion
Abasic sites,
single strand
breaks, 8oxaguanine
lesions
Double and
single strand
breaks,

Richelle Dianne G Ramos RPh

BIOCHEMISTRY PRELIMS
Nonhomologous
End-Joining (NHEJ)

anti-tumor
drugs

SEM 2
intrasrand
crosslinks

DNA and chromosome integrity is monitored

throughout the cell cycle
Eukaryotic cells developed elaborate
mechanisms to monitor integrity of genetic
material
Check-point controls - The 4 specific steps at
w/c this monitoring occurs
If problems are detected , progression through
the cycle is interrupted until the damage is
repaired
Tumor suppressor p53- unstable, DNA-binding
transcription factor, plays a key role in G1 and
G2 check-point control,
P53 is subject to panoply of regulatory PTMs ,
increase levels will activate transcription
P21CIP-potent CDK-cyclin inhibitor (CK1),inhibit
action of all CDKs
If damage is too extensive to repair, affected
cells undergo apoptosis in a p53-dependent
fashion
Cells that lack function p53 fail to undergo
aopotosis
P53 is one of the most frequently mutated
genes is human cancers
80% of human cancers carry p53 loss of
function mutations