BIOCHEMISTRY
CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR
BIOMEDICAL IMPORTANCE
Genetic info in DNA of chromosome can be
PRELIMS
SEM 2
Core histones- H2A, H2B, H3 and H4 : highly conserved between species,
transmitted y exact replication or can be exchanged by a number of processes.
High conservation implies that function of histones are identical in all eukaryotes
Processes provide means of ensuring
Carboxyl terminal 2/3 of histone molecules are
adaptability and diversity for the organism Mutations are due to change in base sequence of DNA, may result from faulty replication,
hydrophobic Amino terminal 1/3p rich in basic amino acids Core of histones are subject to 6 types of
movement or repair of DNA. vertical transmission- Mutation in a germ cell is transmitted to offspring
covalent modification or post translational modification (PTMs) Acetylation- H3 and H4, associated w/
horizontal transmission- mutations of somatic
activation or inactivation of gene
cells are passed on to successive generation but only w/in an organism
transcription, core histones is associated acetylation of core w/ chromosomal assembly during DNA
most CAs are due to combined vertical and horizontal transmission of induced mutations
CHROMATIN IS THE CHROMOSOMAL MATERIAL IN THE
replication Phosphorylation of H1- condensation of Phosphorylation chromosomes during replication cycles
NUCLEI OF CELLS OF EUKARYOTIC ORGANISMS
ADP-ribosylation- DNA repair Methylation- activation and repression of gene transcription
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
MonoubiquitylationMonoubiquitylation- gene activation, repression and heterochromatic gene silencing
Non-histone proteins- acidic and larger than
Sumoylation- SUMO( small ubiquitin-related ubiquitin-related
histones, include enzyme involved inDNA replication and repair, 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
modifier)- transcription repression H3 and H4 form a tetramer containg 2 molecules of each H2A and H2B form a dimer
(H3-H4)2-(H2A-H2B) The nucleosome contains histone & DNA
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 sol’n, more solube organizational unit of o f soluble Nucleosome- organizational chromatin
Richelle Dianne G Ramos Ramos RPh
Histone oligomers form histone octamer w/
Histone octamer is mixed w/ purified dsDNA
under physiologic conditions. 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
BIOCHEMISTRY
CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR
In nucleosome, DNA is supercoiled in a left-
handed 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
PRELIMS
SEM 2
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
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
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
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
transcriptionally active) show to differ in several ways from inactive genes
bound by CHON Sensitivity to DNase I reflects only a potential for transcription not transcription itself
There are 2 higher orders of structure
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
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
conformation that favours access of nuclease to DNA
nucleosomes per turn
hypersensitive regions are location of interrupted nucleosomal structure caused by
H1 histones- stabilize 30 nm diber To form a mitotic chromosome, the 3onm fiber must be compacted in length 100 fold.
Sensitivity to DNase I can be correlated to relative lack of 5-methyldeoxycytidine (meC)
COMPACTION OF CHROMATIN
Nucleosome structure appears to be altered in
binding of nonhistone regulatory transcription factor CHONs
if a gene is capable of being transcribed, it has a
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
template strand lead to formation of
nucleus, anchor chromatin fibers
hypersensitive sites.
Richelle Dianne G Ramos RPh
DNase-hypersensitive site nonhistone regulatory CHONs involved in transcription control and maintaining access
BIOCHEMISTRY
CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR
Heterochromatin- transcriptionally INACTIVE
chromatin is densely packed during interphase. There are 2 types Constitutive heterochromatin- always
transcribed and thus, uncondensed and appears as euchromatin Example: -
becomes transcriptionally active during embryoenesis. Euchromatin- transcriptionally ACTIVE, stains less densely, replicated earlier than
Metazoan centromeres-bound by nucleosomes 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’
One of two X chromosome is heterochromatic, but this chromosome decondenses during gametogenesis and
SEM 2
containing histone H3 variant protein CENP-A
condensed and essentially inactive, found near chromosomal centromere Facultative heterochromatin – at times it is condensed, sometimes it is actively
PRELIMS
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
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
bp and about 1.7x 107 Each 23 chromatids in human haploid genome contain 1.3x108 nucleotides in one dsDNA
separation of daughter cells
less dense than it is in the condensed chromosome during metaphase
chromosomes are especially decondensed into
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
At metaphase, mammalian chromosome posses
a two-fold symmetry, w/ identical duplicated sister chromatids connected at a centromere
not random quinacrine or giemsa stain- used for observation of patterns of chromatids
During metaphase, chromosomes are nearly
completely transcriptionally INACTIVE Centromere- adenine –thymine (A-T) rich region containing repeated DNA sequences
length of DNA must be compressed about
8000-fold to generate condensed structure of
DNA IS ORGANIZED INTO CHROMOSOME
l
Transcriptionally active regions of polytene
puff that contain enzymes responsible for transcription and sites of RNA synthesis Fluorescent in situ hybridization – used for mapping specific gene sequence
Human haploid genome consist of about 3x109
Size range of centromere: 10 2 (brewer’s yeast) to 10⁶ (mammals) base pairs (bp)
Richelle Dianne G Ramos RPh
packaging of nucleoproteins w/in chromatid is
Pattern staining (banding) of entire chromosome complement is highly reproducible. Differs significantly between species
BIOCHEMISTRY
CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR
Coding regions are often interrupted by intervening
PRELIMS
SEM 2
Some excess clearly makes up intervening
sequences
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
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
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
coding segments to form mature mRNA
transcription machinery or indirectly by affecting the activity of the chromatin template ENCODE Project Consortium shown that for 1%
Introns- noncoding intervening sequence, longer than coding regions, separate functional domains of coding information in form that
Unique sequence DNA or Nonrepetitive DNA- includes single copy of genes that code for CHONs
permits genetic rearrangement by recombination to occur more rapidly
Repetitive DNA- include sequences that vary in copy number from 2 to as many as 107 copies per cell
Exons – coding region
Enhanced rate of genetic rearrangement allow more rapid evolution of biologic function.
More than half the DNA in eukaryotic DNA in
Other protein or noncoding RNAs are localized
eukaryotic organisms is in Unique or Nonrepetitive
within the intronic DNA of certain genes
sequences
in brewer’s yeast about 2/3 of its 6200 genes
MUCH OF THE MAMMALIAN GENOME APPEARS
are expressed but only ~1/5 are required for
REDUNDANT & MUCH IS NOT HIGLY TRANSCRIBED
viability under laboratory growth conditions
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
15,000 genes are actively expressed In human DNA, at least 30% of the human geome consists of repetitive sequences
that is composed of exonic DA
There are 25,000 or less CHON-coding genes in
in a higher eukaryote between 10,000 and
repetitive sequence DNA can be broadly classified as moderately repetitive or as higly repetitive
highly repetitive sequence-
human Most of the DNA is nonprotein-coding , its information is never translated into an amino
consist of 5-500 base pairs lengths repeated as many times in tandem,
often clustered in centromeres and
acid sequence of a protein molecule Excess DNA regulate the expression of genes by serving as binding sites for regulatory
telomeres of the chromosome some are present in about1-10M copies per haploid
Richelle Dianne G Ramos RPh
majority are transcriptionally inactive
play a structural role in the chromosome
BIOCHEMISTRY
CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR
moderately repetitive sequence
6
present in numbers of less than 10 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
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
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,
Number of these repeats may vary on the two 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 Hungtington’s chorea (CAG), myotonic dystrophy (CTG),
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
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
capable of jumping into and out of various sites w/in the genome
Mitochondrial DNA is circular, doublestranded and composed of heavy and light chains or strands
Alu B1 and B2 SiNE RNAs have been shown to
regulate mRNA production at the levels of
transcription and mRNA splicing
Contains 16,569 bp Encodes 13 CHON subunits of respiratory chain -
Encodes 7 subunits of NADH
-
dehydrogenase (complex I) Cytochrome b of complex III
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
SEM 2
chromosomes thus providing heterozygosity in
repeat sequence (LINEs) or short interspersed repeat sequence (SINEs) Retroposons- arose from movement from one location to another (transposition) by action of
PRELIMS
-
3 subunits of cytochrome oxidase (complex IV) 2 subunits of ATP synthase
Encodes 22 mt tRNA molecules
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CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR
Encodes large 16S and small 12S mt
SEM 2
Unequal crossover affects tandem arrays of
ribosomal RNAs
repeated DNAs whether they are related globin
Has AGA and AGG, read as Arg, as stop
genes or more abundant repetitive DNA ex: hemoglobins designated Lepore and anti-
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
Lepore
by maternal nonmendelian inheritance
PRELIMS
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
An affected mother would pass the disease to all of her children but only daughters would transmit the trait
Unequal crossover through slippage in the pairing can result in expansion or contraction in
individual chromosome, the greater the likelihood of a crossover recombination
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
Chromosomal integration occurs with some viruses
recombining w/ DNA of a bacterial host in a way
one or more bases may result in an altered gene product
Alteration in genetic material results in a
that the genetic info of bacteriophage is incorporated in a linear fashion into the genetic
mutation
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
Chromosomal recombination is one way of rearranging genetic material
Genetic info can be exchanged between similar
linearized as it is integrated into the bacterial
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
Bacteriophages- bacterial viruses, capable of
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
exchange of genetic info between homologous chromosomes
When alignment is not exact the crossing over
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
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
Some bacteriophages synthesize proteins that bind specific sites on bacterial chromosomes to
reverse transcriptase
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CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR
Integration of animal virus DNA into the animal
genome is not site specific, but display site
PRELIMS
SEM 2
homogenize sequences of members of repetitive DNA families
preferences Sister chromatids exchange 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
progress through the S phase they contain tetraploid content of DNA (sister chromatids)
messenger RNA for the appropriate gene product
5’-nontranslated region,coding region w/o
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
intron representation
in the germ line DNA
3’ poly(A) tail are all present contigously
Plasma cell- immunoglobulin producing cell
this particular DNA sequence arrangement must have resulted from reverse transcription of an
In differentiated plasma cell, VL and VC genes have been moved physically closer together in the genome and into the same transcription
each sister chromatids contain identical genetic info since each is product of replication of original parent DNA
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
in diploid eukaryotes such as human, after cells
appropriately processed mRNA from w/c introns had been removed and poly(A) tail
unit
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
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
codons that prevent their ability to encode
functional and intact CHON
along w/ VL and VC genes interspersed info is removed from RNA during nuclear processing
Gene conversion produces reaarangements
junction of V and C regions Interspersed sequence is transcribed to RNA
Gene conversion
occasionally pair up and eliminate
DNA SYNTHESIS & REPLICATION ARE RIGIDLY
mismatched sequences,
CONTROLLED
Lead to accidental fixation of one variant or another of repeated sequences,
Richelle Dianne G Ramos RPh
primary function of DNA replication is the provision of progeny w/ genetic info possessed by the parent
BIOCHEMISTRY
CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR
replication must be complete and carried out in
SEM 2
2. Unwinding of dsDNA to provide ssDNA
way to maintain genetic stability
template
replication is complex and involves many cellular functions and processes to ensure
-
Interaction w/ ori defines start site of replication and provides short region of
fidelity of replication
ssDNA for initiation of nascent DNA
about 30 CHONs are involved in replication of E.coli
strand synthesis Requires formation of CHON-CHON and
-
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
PRELIMS
Major role of polymerase is proofreading and repair
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 ʎ phage-infected E.coli the protein P
In all cells, replication can occur only from a
binds dnaB and P/dnaB binds to oriʎ by
single stranded DNA (ssDNA)
interacting w/ the O protein dnaB in an inactive helicase when in
-
P/daB/O complex
STEPS IN DNA REPLICATION
At the ori, there is an association of
dnaK, dnaJ GrepE- E.coli het shock proteins, remove P protein and activate
sequence-specific dsDNA- binding
dnaB helicase
1. Identification of origins of replication
-
-
CHONs w/ a series of direct repeat DNA sequences In bacteriophage ʎ , the ori ʎ is bound by ʎ-encoded O protein to 4 adjacent sites
-
-
replication of ʎ phage is accomplished at the expense of replication of the host E.coli host
3. Formation of replication fork: synthesis of
-
In E.coli, oriC is bound by protein dna A
RNA primer
-
A complex is form consisting 150-250
-
bp of DNA
that form in the following sequence: a. DNA helicase unwinds a short segment
-
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
-
A replication consists of 4 components
of parental duplex DNA b. A primase initiates synthesis of an RNA molecule that is essential for priming DNA synthesis
CHONs analogous to dnaA protein of
c.
E.coli bound by ORE
daughter strand synthesis d. SSBs bind to ssDNA and prevent
DNA unwinding element (DUE)- 80bp-
DNA polymerase initiate nascent,
A+T-rich sequence that is easy to
premature reannealing of ssDNA to
unwind, origin of replication of yeast and is bound by MCM CHON complex
dsDNA DNA polymerase III enzyme- dnaE gene product in Ecoli, bind to template DNA
-
as a part of multiprotein
Richelle Dianne G Ramos RPh
BIOCHEMISTRY
CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR
-
Because DNA strands are antiparallel,
-
On leading (forward) strand, DNA is synthesized in short fragments called Okazaki fragments
chromosomes and both strands are replicated simultaneously
-
DNA polymerase cannot initiate DNA
-
synthesis de novo Primosome - mobile complex between helicase and primase
-
-
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
Initiation of DNA synthesis requires priming by a short length of RNA, 10-
serve as a template by hydrogen bonding its nucleotide bases to the
200 nucleotides long catalyzed by dnaG
incoming deoxynucleoside triphosphate
in E.coli In eukaryotes DNA pol asynthesizes
-
w/o interfering w/ the abilities of
Priming process involves nucleophilic attack by 3’-hydroxyl group of RNA
nucleotides to serve as template To allow strand separation, there must
-
entering deoxynucleoside triphosphate The 3’-hydroxyl group of of recently attached deoxyribonucleoside
be unwinding -
monophosphate is then free to carry out nucleophilic attack on the next
-
Undwinding happens adjacent to replication bubbles To counteract unwinding, there are multiple swivels
-
entering deoxyribonucleoside
Swivel- function is provided by specific enzymes that introduce “nicks” in one
triphosphate again Selection of proper
strand of the unwinding doule helix RNase H degrades the hybridized
-
deoxyribonucleotide to be attacked is dependent upon proper base pairing with other strand of the DNA -
Separation is promoted by SSBs in E.coli Stabilizing CHONs bind to single strand
RNA primers
primer on the phosphate of the first -
Initiation is regulated both spatially and temporally, cluster adjacent sites initiate replication synchronously
4. Initiation of DNA synthesis and elongation
-
Replication bubbles- replication occurs in both directions along all of the
helicase acts on lagging strand to unwinddsDNA I a 5’-3’ direction
-
SEM 2
polymerase funcstions asymmetrically
-
-
PRELIMS
template RNA strand -
Okazaki fragments- RNA initiator component
Reverse transcriptase-synthesize DNARNA hybrind utilizing RNA genome as template
6. Reconstitution of chromatin structures
ligation of newly synthesized DNA
nuclear organization and chromatin structure are involved in determining
segments
the regulation and initiation of DNA
-
5. Formation of replication bubbles w/
-
-
Replication proceeds from a single ori in the circular bacterial chromosome composed of 5x106 bp of DNA
-
synthesis rate of polymerization in eukaryotes is slower than prokaryotes
Process is completed in 3o mins,
-
chromatin structure must be re-formed
replication rate is 3x10 5 bp/min
Richelle Dianne G Ramos RPh
after replication
BIOCHEMISTRY
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 -
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
replication fork
continuous manner in 5’ to 3’ direction
reactions are facilitated through the cactions of histone chaperone CHONs
facing forward
Enzyme replicate the lagging strand discontinuously while polymerizing
Classes of proteins involved in replication
nucleotides in short spurts of 150-250 nucleotides in 5’ to 3’ direction facing towards the end
DNA polymerase- deoxyribonucleotide polymerization
Helicases- unwinding of DNA
Topoisomerase- relieve torsional strain fom
replication occurs, temporally separated from
DNA primase- initiate RNA primers synthesis
M phase by gap 1 (G1) and gap 2 (G 2) called G phase
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
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
3 important properties of DNA polymerase 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
and DNA repair
Cyclin-dependent kinases (CDKs)-phosphorylate substrates essential for progression through cell
CDK4 and CDK6- activated by D cyclins, assemble as a complex in G1 phase, this complex is an active serine-threonine CHON
DNA Polymerase III catalyzes the highest rate of chain elongation and is most processive Polymerase I and II- involved in proofreading
Cell regulates DNA synthesis by allowing it to
cycle Cyclin D conc increase in late G 1 phase and allow progression beyond the start in yeast, or restriction point in mammals
corrects them
Cell prepares for DNA synthesis in G 1 and prepares for mitosis in G2
1. Chain elongation- accounts for rate at w/c
Synthetic/ S phase- period of time where
helicase-induced unwinding
The DNA polymerasecomplex
DNA synthesis occurs during S phase of the cell cycle
kinase
Retinoblastoma (Rb)- substrate that regulates cell cycle by binding to and inactivating a transcription factor (E2F) necessary for
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
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
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CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR
Cylin B and kinase CDK1- rate limiting for G 2/M
Nonhomologous End-Joining (NHEJ)
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
PRELIMS
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
