Harper's illustrated Biochemistry CH 35

8/10/2019 Harper's illustrated Biochemistry CH 35

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CHAPTER 35: DNA ORGNIZATION, REPLICATION & REPAIR BIOCHEMISTRYPRELIMS SEM 2

Richelle Dianne G Ramos RPh

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

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 H4form 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


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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 outsideof its structure , available for regulatory PTMs

H3-H4tetramer 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

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 nucleusupon activation

SOME REGIONS OF CHROMATIN ARE ACTIVE AND

OTHERS ARE INACTIVE

Chromatin containing active genes

(transcriptionally or potentionally

transcriptionally active) show to differ in severa

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.


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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 heterochromatinat 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 hybridizationused 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- adeninethymine (A-T) rich region

containing repeated DNA sequences

Size range of centromere: 102 (brewers yeast)

to 10 (mammals) base pairs (bp)

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 eachchromosome, 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 dsDNAmolecule

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 1.3x108nucleotides 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


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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 ofnonprotein-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 byrecombination to occur more rapidly

Exonscoding 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

3x106bp 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

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 byaffecting 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 thatvary 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


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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 transcribedby 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 RNAtemplate 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

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, double-

stranded 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


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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

Unequal crossover affects tandem arrays of

repeated DNAs whether they are related globin

genes or more abundant repetitive DNA ex:

hemoglobins designated Lepore and anti-

Lepore

Unequal crossover through slippage in thepairing 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 polymeraseor

reverse transcriptase


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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 DNAelements 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

3poly(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,

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 containtetraploid 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 VLand VC genes for single

immunoglobulin molecule are widely separated

in the germ line DNA

Plasma cell- immunoglobulin producing cell

In differentiated plasma cell, VLand VC genes

have been moved physically closer together in

the genome and into the same transcription

unit

Rearrangement does not bring the VLand 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/ VLand 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


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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 ofE.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 oriis 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)- 80bp-

A+T-rich sequence that is easy to

unwind, origin of replication of yeast

and is bound by MCM CHON complex

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 oriby

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


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- 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 tounwinddsDNA 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, 10-

200 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 5x106bp of DNA

-

Process is completed in 3o mins,

replication rate is 3x105bp/min

- 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 sitesinitiate 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.col-

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 DNA-

RNA 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


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- 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 thecactions 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

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 G1and

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 cel

cycle

Cyclin D conc increase in late G1phase 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 G1phase, 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 G1to 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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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 damaging

agents

lesions formed

Nucleotide Excision

Repair (NER)

UV light

chemicals

Bulky adducts

pyr dimers

Mismatch Repair

(MMR)

Replication

errors

mismatch,

insertion,

deletionBasic Excision

Repair (BER)

O2radicals

hydrolysis,

alkylating

agents

Abasic sites,

single strand

breaks, 8-

oxaguanine

lesions

Homologous

Recombination (HR)

And

Xrays,

ionizing

radiation,

Double and

single strand

breaks,

Nonhomologous

End-Joining (NHEJ)

anti-tumor

drugs

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 G1and

G2check-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

Documents

Harper's illustrated Biochemistry CH 35