DNA_ GENES_ REPLICATION_ REPAIR

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					           3. DNA: GENES, REPLICATION, REPAIR
                                       (Jan Kovář)


GENETIC INFORMATION & GENOPHORE:
Genetic information of cell is an internal information of cell (cell is carrying this
information) which is passed from parental cell on daughter cell. It represents information
concerning the structure and functioning of cell, including the functioning of cell within the
framework of organism.

Genetic information have to be lasting and stable enough. On the other hand, it must be
capable of changing during evolution. Furthermore, it must be capable of reproducing and
must be easily available for realization (expression).

The carrier of genetic information is referred to as genophore.

The genophore of cells is DNA (deoxyribonucleic acid).
In some types of viruses, the genophore can be RNA (ribonucleic acid).


THE STRUCTURE OF DNA:
DNA is a polymer of nucleotides (polynucleotide chain). DNA molecul usually contains
two polynucleotide chains (strands) forming a double helix.

Nucleotide: nitrogen-containing base + sugar pentose + phosphate
Nucleoside: nitrogen-containing base + sugar pentose

In the case of DNA, the pentose is deoxyribose (deoxyribonluceotide).

Bases in DNA: adenine (A), guanine (G) (purine bases)
              cytosine (C), thymine (T) (pyrimidine bases)

Individual nucleotides bind each other within the framework of polynucleotide chain via OH
group of the 3' carbon of deoxyribose and phosphate of the 5' carbon of deoxyribose
(phosphodiester bond).

Polynucleotide chain contains a backbone made of repeated deoxyriboses and phosphates.
Each deoxyribose binds (at the side of chain) one of bases.
Polynucleotide chain has a polar character (3' end and 5' end).

Two polynucleotide chains in the double helix bind each other in an antiparallel way (3' end
to 5' end and 5' end to 3' end). Binding of the chains is mediated by bonds between bases.
Each base from one chain always binds specifically base from the second chain.

Complementary pairing of bases:
A (purine) binds always T (pyrimidine) and vice versa (2 hydrogen bonds).
G (purine) binds always C (pyrimidine) and vice versa (3 hydrogen bonds).


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CHROMOSOME AND ITS STRUCTURE:
DNA is present within cell in the forme of chromosomes (big amount of comprised genetic
information) or in the form of plasmids (small amount of comprised genetic information).

Procaryotic cells: 1 chromosome
Eucaryotic cells: more chromosome

Procaryotic chromosome: circular, approximately 106-107 base pairs, attached to the plasma
membrane of cell by one specific point, it contains proteins (some of them are similar to
histons of eucaryotic cells).

Eucaryotic chromosome: linear, approximately 108 base pairs (in the case of human, 5-
25x107, i.e. 1.7-8.5 cm), it is located in the nucleus of cell, it contains proteins histones (basic
proteins: they contain big amount of basic aminoacids lysine and arginine) and acidic proteins
(nonhistone proteins).

Histones: H1, H2A, H2B, H3, H4
          there are all 5 types of histones in each chromosome
          they are involved in basic structure of eucaryotic chromosomes

Acidic proteins: heteregenous group of proteins
                 they are probably involved in the three-dimensional arrangement of
                 chromosome

The basic structual unit of eucaryotic chromosome is nucleosome.
Nucleosome: core made of the octamer of histones H2A, H2B, H3, H4 (each twice), DNA is
wound around the core, the lenght of wound DNA is 146 base pairs, there are about 60 base
pairs of „free“ DNA between two nucleosomes, thus there are about 200 base pairs per one
nucleosome, individual nucleosomes are joined by histone H1.



Structural levels of eucaryotic chromosome:
 double helix of DNA (the diameter of fiber is 2 nm)
 nucleosomes with wound DNA (11 nm)
 30-nm chromatin fiber (30 nm)
 chromatin loops (300 nm)
 condensed part of chromosome (chromatid) (700 nm)
 entire metaphase chromosome (1400 nm)

Due to condensation, metaphase chromosome is 10 000x shorter than the same chromosome
in extended form.

Basic structure of metaphase chromosome: 2 chromatids, centromere, telomeres




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PLASMIDS:
Plasmids are cytoplasmic genophores which do not carry genetic information indispensable
for the functioning of cell (vital genetic information is in chromosomes).
Plasmids are usually found in procaryotic cells.

Procaryotic plasmid: circular double-stranded DNA, 1.5-200x103 base pairs.

Types of procaryotic plasmids:
 F (fertile) plasmids: necessary for the conjugation of bacteria (the exchange of genetic
   information).
 R (resistance) plasmids: they produce resistance against antibiotics and
   chemotherapeutic.
 Col plasmids: they provide the capability of producing proteins colicins which can kill
   other bacteria.


GENETIC CODE:
Genetic information in relevant DNA strand (similarly in RNA) is safed as a sequence of
individual bases, i.e. A, G, C, and T.

Genetic information encodes the sequence of aminoacids, i.e. the primary structure of cell
proteins.
It means that the sequence of bases in DNA determines the sequence of aminoacids in
relevant protein.

Genetic code is a rule by which certain sequence of bases determines relevant aminoacid.

In organism, there are 20 common aminoacids which are encoded.
One aminoacid is always determined by the sequence of 3 bases.
Thus genetic code is based on triplets.

There are 4 bases (A, G, C and T) and thus there are 64 (43) combinations of triplets, i.e.
codons.
61 triplets encode aminoacids. The codon for methionine functions as the initiation codon.
3 triplets function as stop codons.

Because there is a surplus of 61 triplets encoding only 20 aminoacids, one aminoacid can be
encoded by 1 to 6 triplets (codons):
Methionine and tryptophan are encoded only by 1 codon and, on the other hand, leucine,
serine and arginine are encoded by 6 codons.
Genetic code is redundant.

Reading frame: one of 3 possibilities how sequence of the triplets can be read.

Genetic code is universal.




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GENE:
Gene is a segment of DNA (or RNA) which encodes a single polypeptide chain (protein) or a
single RNA chain (rRNA, tRNA).

Most of genes have their own stable site within the sequence of relevant chromosome. This
site is referred to as gene locus.

 Structural genes: they encode proteins.
 Genes for RNA: they encode RNA which is not a template for translation (rRNA, tRNA).
 DNA regulatory regions: they do not encode proteins as well as RNA but they carry an
  information for binding specific molecules regulating gene expression.

Procaryotic gene: it only contains uninterrupted DNA sequence which encodes
corresponding polypeptide chain.

Eucaryotic gene: it contains coding DNA sequences (exons) which are interrupted by
noncoding sequences (introns).
Human gene comprises approximately 3x104 base pairs.

Nongene DNA:
In mammalian cells, genes, which are expressed, represent only 7-10% of entire DNA.
 Regulatory regions: they are involved in the regulation of expression.
 Repetitive sequences: highly variable (DNA fingerprinting).
 Mobile elements (transposons): they do not show a stable position within DNA
    sequence.
 Pseudogenes: Genes which lost their function due to the accumulation of mutations.


GENOME:
Genome is the complete set of DNA (set of all genophores) of the cell (organism).

Genom of procaryotic cell: chromosome + possible plasmids.

Genom of eucaryotic cell: nuclear chromosome + mitochondrial chromosome + chloroplast
chromosome (in the case of plants) + possible plasmids (only in some cases).
Human genom (haploid set) comprises approximately 30 000 genes.




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DNA REPLICATION:
DNA replication is the doubling of DNA. Two identical double helixes of DNA are derived
from one original double helix of DNA. It means entire genetic information is safed in both
new double helixes of DNA.

DNA replication has a semiconservative character. It means that a new strand is synthesized
on the basic of complementary pairing (complementary strand) according to each of both
original strands of the double helix. The original strands function as a template.

Replicon: a segment of DNA which represent a replication unit with its own replication
origin.
Procaryotic chromosome: one replicon
Eucaryotic chromosome: many (hundreds to thousands) replicons

Replication origin: a specific sequence of DNA (rich in A-T base pairs) where the
replication starts after unwinding the double helix. It happens after binding initiator proteins.

Replication fork: From the replication origin, the replication continues in both opposite
directions. It results in two replications forks (the shape of letter Y) which are moving apart.
Procaryotic chromosome: replication fork moves at 1000 bp/s.
Eucaryotic chromosome: replication fork moves at 100 bp/s.


DNA POLYMERASE:
DNA polymerase catalyzes the formation of phosphodiester bond between two nucleotides
(between the 3' end of deoxyribose of one nucleotide and the 5' end of deoxyribose of other
nucleotide via relevant phosphate). In this way it catalyzes the formation of new DNA strand.

First, a nucleotide added to new growing DNA strand pairs complementary by its base with
the base of relevant nucleotide of the template. Afterwards, the nucleotide forms
phosphodiester bond with the previous nucleotide of growing strand.
Nucleotide enters the reaction as nucleoside triphosphate (ATP, GTP, CTP, TTP).
Energy released by freeing pyrophosphate (PPi) is used for polymerization reaction.

DNA polymerase has 2 important and limiting properties:
 It can synthesize new DNA strand only in 5'→3' direction. It means according to the
  template in 3'→5' direction.
 It is unable to start the synthesis of new DNA strand. It can only extend existing strand of
  nucleic acid.

There are several types of DNA polymerases in eucaryotic cells:
 DNA polymerase α
 DNA polymerase δ
and other types (DNA polymerase β, DNA polymerase γ).




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THE MECHANISM OF DNA REPLICATION:
After the binding to replication origin, enzyme helicase unwinds the double helix of DNA
(energy from ATP is used here).

Single-stranded DNA is stabilized by binding molecules of single-strand binding protein.

The replication is started by enzyme primase which produces a short strand of RNA referred
to as primer.
primer in procaryotic cell: 5 bp
primer in eucaryotic cell: 10 bp
Primer provides DNA polymerase with 3' end. DNA polymerase continues the synthesis of
new DNA strand according to the template.

Protein known as sliding clamp keeps DNA polymerase attached to the template strand and it
allows DNA polymerase to slide along the template strand.

The synthesis of new strand in 5'→3' direction on 3'→5' template runs continuously (DNA
polymerase δ): the leading strand.

The synthesis of new strand in 3'→5' direction on the 5'→3' template runs discontinuously
(DNA polymerase α): the lagging strand.
DNA polymerase „skips“ here forward along the template and then it synthesizes backward in
proper direction 5'→3'.
The synthesis of new strand is performed piece after piece. These pieces are reffered to as
Okazaki fragments. Each fragment starts with its own primer.
Afterwards, RNA primers are removed and missing DNA is synthesized by relevant DNA
polymerase. Finally, individual fragments are joined by enzyme DNA ligase.
Okazaki fragments of procaryotic cell: about 1000 nucleotides
Okazakiho fragments of eucaryotic cell: about 200 nucleotides

Telomerase: it solves a problem of synthesizing the lagging strand at the end of chromosome
(telomere).


PROOFREADING:

DNA polymerase synthesizes a new DNA strand in 5'→3' direction but simultaneously it has
proofreading activity in 3'→5' direction on the new DNA strand.

Before binding a new nucleotide, DNA polymerase verifies whether previously bound
nucleotide has the base complementary to template. If yes, DNA polymerase continues by
binding a new nucleotide. If no, DNA polymerase removes previous wrong nucleotide and,
instead of this nucleotide, relevant nucleotide is bound.

The proof reading activity of DNA polymerase can explain why DNA polymerase has only
5'→3' polymerase activity. Proofreading in 5'→3' direction (in the case of hypothetical
polymerization in 3'→5' direction) is not possible from the chemical point of view.




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DNA REPAIR:
Mismatch repair mechanism corrects wrongly paired bases of newly synthesized DNA
strand. Thus it corrects mistakes of replication machinery.
Proteins, involved in mismatch repair, recognize pairing which is not complementary
(mismatch) due to the deformation of DNA double helix.
Afterwards, they remove wrong segment of new DNA strand and synthesize this segment
again.

Replication machinery makes 1 error/107 nucleotides.
Mismatch repair mechanism corrects 99% of the errors of replication machinery.
Therefore, overall accuracy of DNA reproduction within cell and thus the accuracy of genetic
information transfer from one DNA molecul to other DNA molecule is 1 error/109
nucleotides.

The repair of accidentally demaged DNA (thymine dimers, depurination, deamination):
1. Recognition of the demage on DNA strand and excision of the damaged DNA by specific
   nucleases.
2. Synthesis of the removed DNA according to complementary strand by repair DNA
   polymerases.
3. Rejoining newly synthesized DNA segment with repaired DNA strand by DNA ligase
   (ligation).

The stability of DNA and thus also the stability of genetic information depends on
mechanisms of DNA repair.


LITERATURE:

 Alberts B. et al.: Essential Cell Biology. Garland Science, New York and London, pp.
  169-187 & 195-215, 2004

 Alberts B. et al.: Molecular Biology of the Cell. Garland Science, New York, pp. 195-245
  & 266-304, 2008




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