Beruflich Dokumente
Kultur Dokumente
Reference Books
•Lippincotts illustrated review of Biochemistry by Champe and Harvey.
•Medical Biochemistry by Baynes and Dominiczak.
Structure of deoxyribonuleic acid :
- Cellular nucleic acid exist of two forms. DNA and RNA .About 90% of the nucleic
within cells is RNA, and the remainder is DNA.
- DNA is the storage of genetic information within the cell.
- All the DNA in one human cell (on all 46 chromosomes) is about two meters long
,fits into a cell nucleus which is 2-3 micrometers.The DNA must still be in such a state as to
allow for enzymes to replicate the molecule or initiate the production of a protein. The 23 pairs
of human chromosomes are estimated to include about 100,000 genes. Each gene codes for
ONE protein;
NUCLEOSIDES
- Both DNA and RNA contain nucleotides with similar components. In RNA, the sugar
component is ribose, as indicated by the name "ribonucleic acid". In DNA, or
deoxyribonucleic acid, the sugar component is deoxyribose. The prefix deoxy means
that an oxygen atom is missing from one of the ribose Carbon atoms.
- When a sugar bonds together with a Nitrogen base, This structure is known as a
nucleoside.
NUCLEOTIDES
- Nucleotides are joined together in DNA and RNA by phosphate ester bonds between the
phosphate component of one nucleotide and the sugar component of the next
nucleotide.
- More and more nucleotides can be added on by the same process of forming ester bonds
until an immense chain is formed. But no matter how long a polynucleotide chain is,
one end of the nucleic acid molecule always has a free -OH group on the sugar at the
Carbon known as C3' (called the 3' end) and the other end of the molecule always has a
phosphoric acid group at C5' (the 5' end).
Types of DNA
BASE-PAIRING IN DNA :Experiments have shown that DNA samples taken from different
cells of the same species have the same proportions of the four bases. Human DNA contains
about 30% each of adenine and thymine, and 20% each of guanine and cytosine. The figure is
different for other organisms, but the amounts of A and T are always the same, as are the
amounts of C and G!
• In 1953, James Watson and Francis Crick proposed a structure for DNA. According to
the Watson-Crick model, a DNA molecule consists of two polynucleotide strands coiled
around each other in a helical "twisted ladder" structure .The sugar-phosphate
backbone is on the outside of the double helix, and the bases are on the inside, so that a
base on one strand points directly toward a base on the second strand. The sugar-
phosphate backbones as the two sides of the ladder and the bases in the middle as the
rungs of the ladder.
• The two strands of the DNA double helix run in opposite directions, one in the 5' to 3'
direction, the other in the 3' to 5' direction. The term that describes how the two strands
relate to each other is known as antiparallel.
- The G1 phase is a period of cell growth that occur prior to DNA replication.
- The phase during which DNA is synthesized or replicated is termed the S phase.
- A second growth phase termed G2 occurs after DNA replication but prior to cell
division.
- The mitosis or M phase is the period of cell division. Following mitosis ,the daughter
cells either re-enter the G1 phase or enter a quiescent phase termed G0 where growth
and replication ceases.
- The passage of cells through the cell cycle is tightly controlled by variety of protein
called cyclin_ dependent kinases.
Replication:
•Genetic material must be able to be accurately replicated and passed on from one generation
to the next. The double helix of DNA suggested the strands can separate and act as template
for the formation of a new , complementary strand.
2- Template:
DNA replication can not occur without a template .Template( nucleic acid strand whose
base sequence is copied in a polymerization reaction). A template is required to direct
addition of the appropriate complementary nucleotide to the newly synthesised DNA
strand. Each strand of parent DNA serves as a template.
3- Primer
( in nucleic acids , a short RNA or single stranded DNA segment that functions as a
growing point in polymerization. DNA synthesis can not start without a primer , which
prepares the template strand for the addition of nucleotide. Because new nucleotide are
added to the 3- of a primer, new synthesis is said to occur in 5- to 3- .
4. Enzymes :
The DNA synthesis that occurs during the process of replication is catalyzed by
enzyme called DNA-dependant DNA polymerases. The enzymes are DNA dependant
by the fact that they require a DNA template. They are called DNA polymerases.
The bacteria E-coli contains three separate DNA polymerases. These enzymes are
capable of catalyzing other reactions an important role in replication and DNA
repair.
1 11 111
5--------3-(exonuclease activity) + -ve -ve
3- ------5-( exonuclease activity) + + +
Synthesis rate( nucleotides/min) 600 30 30000
Replication + -ve +
Repair + ? -ve
- DNA polymerase
- A. Function : pol 1 function in the replication of DNA and in the repair of damaged
DNA.
- C. Other enzymatic activites : pol1 has two enzymatic acitivtes besides DNA
polymerase activity that are important to its cellular function.
1. Proofreading : Pol 1 does not typically add a nucleotide to the growing DNA
chain that can not properly base pair with template strand. If a mismatched
nucleotide is added , the enzyme halts polymerization .A 3- to 5- exonuclease
removes the mismatched nucleotide and polymerization resumes. This activity is
called proofreading.
- DNA polymerase 11
may be involved in some DNA repair processes .Is a single polypeptide of 90,000 mw.
It has proofreading activity ( 3- to 5- exonuclease activity but lacks excision – repair ( 5-
to 3- exonuclease activity )
DNA Replication
- DNA replication is semi-conservative, one strand serves as the template for the second
strand. DNA replication only occurs at a specific step in the cell cycle.
- Stage Activity
- S DNA synthesis 8 hr
- G2 Post-DNA synthesis 5 hr
- M Mitosis 1 hr
In the Beginning…
Single Strand Binding Protein : > Binds to DNA with no sequence preference
Binds tighter to single strand than double
Keeps separated strands from rejoining
DNA Polymerase :
,.;'**********';.,
Primase
• Creates a primer for DNA polymerase
• Template-dependant
• An RNA polymerase
• Active briefly at beginning of strand
synthesis
DNA Ligase
Since each new strand is complementary to its old template strand, two identical new copies of
the DNA double helix are produced during replication. In each new helix, one strand is the old
template and the other is newly synthesized, a result described by saying that the replication is
semi-conservative.
Conservative replication : would occur if , after replication and cell division , the parental
DNA strands remained together in one of the daughter cells and the newly synthesised DNA
strands went to the other daughter cell.
The process of DNA replication in all organisms is amazing. The sum of all genes in a human
cell 3 billion base pairs ,What's even more incredible is how few mistakes are made in this
process despite the immense size of human DNA! An error occurs only about once in each 10-
100 billion bases. The complete process of DNA replication in human cells takes several hours.
To replicate such huge molecules as human DNA at this speed requires not one, but many
replication forks, forming replication bubbles and producing many segments of DNA strands
that eventually meet up together and are joined to form the newly synthesized double helix.
Three fundamental processes take place in the transfer and use of genetic
information :
o Translation : is the process by which the genetic messages carried by mRNA are
decoded and used to build proteins.
Structure of RNA
• There are three major types of RNA that participate in the process of protein synthesis:
o ribosomal RNA ,
o transfer RNA and
o messenger RNA. Like DNA ,
• These three types of RNA are unbranched polymeric moleules composed of
mononucleotides joined together by phosphatebonds. Most RNA exists as single strands
.The three major types of RNA differ in size ,function and special structural
modifications.
Ribosomal RNA
• Ribosomal RNA are found in association with several proteins as components of the
ribosomes –the complex structures that serve as the site for protein synthesis. There
are three distinct size species of r RNA ( 23S , 16S, and 5S ) in prokaryotic cells. In
eukaryotic , there are four r RNA size species ( 28S , 18S , 5.8S , and 5S )
• Note S is the Svedberg unit, which is related to the molecular weight and shape of
the compound.
• R RNAs make up to 80% of the total RNA in the cell.
Transfer RNA
• The smallest RNAs of the three major species of RNA molecules ( 4S) have between
74 and 95 nucleotides residues. There is at least one specific type of t RNA molecule
for each of the twenty amino acids commonly found in proteins. tRNAs make up
about 15 % of the total RNA in the cell.
• Each tRNA serves as an adaptor molecule that carries its specific amino acid
attached to its 3” end to the site of protein synthesis. There it recognize the genetic
code word on an mRNA , which specifies the addition of its amino acid to the
growing peptide chain.
Messenger RNA
• Messenger RNA comprises only about 5% of the RNA in the cell. The messenger
RNA carries genetic information from the nuclear DNA to the cytosol , where it is
used as the template for protein synthesis.
• Special structural of eukaryotic mRNA ( but not prokaryotic) include a long
sequence of adenine nucleotides ( a poly-A tail ) on the 3” end of the RNA chain ,
plus a cap on the 5” end consisting of a moleule of 7-methylguanosine attached
through a triphosphate linkage.
DNA Repair
Substitutions
- Bases can undergo tautomerization (movement of bonds)
- Uncommon but spontaneous
- Allows for mismatched nucleotide incorporation during synthesis
DNA Modifications
Repair by Removal
- Nucleotide excision
- Distorted helix is seen
- Enzyme complex cuts bad strand on
either side
- DNA polymerase I fills in gap
- DNA ligase seals patch in place
Direct Repair
- Damage is repaired in place
o Ex: Thymine dimers and photolyase
(E. coli)
o UV damages DNA by causing cross-
linking
o Blocks replication
o Repair involves breaking covalent
bond
- Xeroderma pigentosa
o Damaged DNA repair system in skin
o Death by multiple skin cancer by age 30
- Hereditary nonpolyposis colon cancer
o Defective mismatch repair
- Cancer, is the severe medically relevant consequence of the inability to repair damaged
DNA.
- DNA damage can occur as the result of exposure to environmental stimuli such as
alkylating chemicals or ultraviolet or radioactive irradiation and free radicals
generated spontaneously in the oxidizing environment of the cell. These phenomena can
lead to the introduction of mutations in the coding capacity of the DNA.
- Mutations in DNA are of two types. Transition mutations result from the exchange of
one purine, or pyrimidine, for another purine, or pyrimidine. Transversion mutations
result from the exchange of a purine for a pyrimidine or visa versa.
- Humans defective in DNA repair, (in particular the repair of uv-induced thymine
dimers), due to autosomal recessive genetic defects suffer from the disease Xeroderma
pigmentosum (XP). There are at least nine distinct genetic defects associated with this
disease. One of these is due to a defect in the gene coding for the glycohydrolase that
cleaves the N-glycosidic bond of the thymine dimers. There are two major clinical forms
of XP, one which leads to progressive degenerative changes in the eyes and skin and the
other which also includes progressive neurological degeneration.
- Another inherited disorder affecting DNA repair in which patients suffer from sun
sensitivity, and progressive neurological degeneration without an increased incidence of
skin cancer is Cockayne Syndrome.
- Several diseases associated with defective repair of damaged DNA can be found in the
Inborn Errors .
- One of the major post-replicative reactions that modifies the DNA is methylation. The
sites of natural methylation of eukaryotic DNA is always on cytosine residues that are
present in CpG dinucleotides.
- The function of this methylation is to prevent degradation of host DNA in the presence
of enzymatic activities synthesized by bacteria called restriction endonucleases. The role
of this system in prokaryotic cells is to degrade invading viral DNAs. Since the viral
DNAs are not modified by methylation they are degraded by the host restriction
enzymes. The methylated host genome is resistant to the action of these enzymes.