DNA replication

case is equal to reasonThe view that a glance passing from age to age from generation to generation can not be completely false - the purest illusion ... After all, with the exception of a few philosophical minds, no one will ever think of testing whether what everyone is saying is true.

Pierre Beyle

Genetic information transfer

t RNA r RNAm RNA

protein

tRNA mRNA rRNA

protein

replication

TRANSCRIPTION(direct and inverse of viruses)

translation

DNA

The central dogma of molecular biology:the transfer of genetic information is carried out onlyfrom the nucleic acid (DNA and RNA). The recipientof the information can be another nucleic acid (DNAor RNA) and a protein.

It is a sequence of deoxyribonucleotides interconnected by 3 ', 5'-phosphodiester bonds.The free 3 '-end contains a free hydroxyl group and is called the OH-terminus,The 5 '-end contains a phosphate group and is called the P-terminus.The direction of the chain in the primary structure is 5 '3'.The primary structure determines the uniqueness of the structure and the functional individuality of the DNA. In this case, the backbone of the DNA chain is always constant throughout and represents the alternation of the groups: pentose-phosphate-pentose. Variable groups are nitrogen bases.

PRIMARY DNA STRUCTURE

Nucleotid

PO

OOH

HOO

Nitrogen base(A or G or T or C or Y)

Pentose(ribose or

deoxyribose)

Phosphat

1’

3’

5’

Thymine

Thymine nucleotide

OH OH

Principles of DNA structure1. Irregularity. There is a regular sugar-phosphate backbone to which nitrogenous bases are attached. Their alternation is irregular.2. Antiparallelism. The DNA consists of two polynucleotide chains oriented antiparallel. The 3`-end of one is located opposite the 5`-end of the other.3. Complementarity. Each nitrogenous base of one chain corresponds to a strictly defined nitrogen base of another chain. Compliance is given by chemistry. Purine and pyrimidine form hydrogen bonds in a pair. In pair A-T, there are two hydrogen bonds, in pair G-C-three.4. The presence of a regular secondary structure. Two complementary, antiparallel polynucleotide strands form right spirals with a common axis.

Minor DNA basesThe composition of tRNA nucleotides includes minor bases - an average of 10-12 bases per molecule.They are represented by methylated bases, isomers and analogues of pyrimidines.Minor bases perform the following functions:1) are necessary for the formation of the secondary structure of ND(for example, the formation of loops in tRNA),2) make tRNA resistant to the action of nucleases of the cytoplasm and support a certain tertiary structure of the molecule, because they can not participate in the formation of complementary pairs,3) prevent spiraling of certain areas a polynucleotide sequence of tRNA,4) perform a protective function (for example, methylated sites in mRNA),5) methylated sites can act as markers, on which special regulatory proteins recognize damage to DNA, areas of the onset of matrix synthesis,6) methylation is also used for recognition again synthesized DNA and parental (during cell division and DNA synthesis).

Minor bases

N

HN

O

O

ribosa

N

HN

O

O

ribosa

Dihydrouridine

CH3

N

HN

O

O

H

ribosa

N

HN

S

O

ribosa

Ribothymidine Pseudouridine Thyouridinethyopirimidine

N

NHN+

N

OCH3

NH2

7-Methylguanine

N

N

O

NH2

H3CNHHN

O

O

5-Methylcitosine Pseudouracile

ribosa ribosa ribosa

N

NHN

N

O

ribosa

N

NN

N

ribosa

NHH3C

N

NHN

N

O

ribosa

NH CH3

Inosine N-Methyladenosine N-Methylguanosine

1. Nitrogenousgrounds:- Adenin;- Guanine;- Cytosine- Timin(Uracil)

2. Carbohydrate:Deoxyribose(Ribose) 3. The

remainder of phosphoric acid (PA)

The structure of DNA (RNA)

DNA polymerMonomers - nucleotidesNucleotide is a chemical compound of the remains of three substances:

REPLICATION IS REQUIRED FOR THE FOLLOWING TERMS

A sufficient number of deoxyribonucleotides

(dATP, dTTP, dGTP, dCTP)

Weaving of the double helix DNA

Primer formation (primers of RNA)

Availability of the necessary enzymes

Four types of polymerasesDNA-dependent DNA polymeraseRNA-dependent DNA polymerase (revertase)DNA-dependent RNA polymerasesRNA-dependent RNA polymerases

Polimerases build up DNA or RNA from the 5` end to the 3` end, since they need a free OH group on the ribose of the 3 'terminal nucleotide.For the same reason, continuous copying of the parallel circuit in the opposite direction is not possible.

procariotic eucariotic

In cells of eukaryotes there are at leastat least six different DNA-dependentDNA polymerases:α, β, δ, ε, γ, ζ.Four of them - α, β, δ, ε directly participate in replication chromosomal DNA

DNA polymerase IDNA polymerase IIDNA polymerase III

DNA-polimerases

NUCLEASE ACTIVITYhydrolysis of phosphodiester bondsDNA polymerase β removes the RNA primer (acts as RNase).DNA polymerases δ and ε can correct synthesis errors.The DNA polymerase ε recognizesRNA primer and beginssynthesize DNA

POLYMERASE ACTIVITY

Education 5 → 3 phosphodiester bondsbetween deoxyribonucleotides.

DNA-polimerases α, β, δ, εhave 2 types of activity

DNA polymerase inserts deoxyribonucleoside triphosphates (dATP, dGTP, dCTP, dTTP) into the DNA chain under construction according to the complementarity rules, unambiguously determining the position of the corresponding nucleotide of the matrix. When nucleotides are inserted, pyrophosphate is released, which is cleaved by inorganic pyrophosphatase, which makes the polymerization reaction practically irreversible.

DNA ligases are enzymes that crosslink DNA strands. When replicating ligases, chains of Okazaki fragments are sewn. DNA ligases are involved in replication, stitching Okazaki fragments, in damage repair and in DNA recombination.

Enzymes and proteins of DNA replication

DNA primase is an enzyme that synthesizes an RNA primer-RNA primer for the subsequent synthesis of Ozakaki fragments, as well as the synthesis of RNA primers during the synthesis of the replicative form of bacteriophage DNA, as well as the synthesis of RNA primers during the synthesis of the replicative form of bacteriophage DNA .In eukaryotes, DNA primase is a subunit of αDNA polymerase. DNA primase. differs from conventional RNA polymerases in that it is able to use both ribo and deoxyribonucleotides as a substrate. In addition, the DNA primase forms a primusome.RNA primase is a DNA-dependent RNA polymerase from E. coli that catalyzes the polymerization of RNA primers, which are necessary for DNA replication for the synthesis of a delayed chain. Along with other proteins, primase RNA is part of the primoxoma.

Enzymes and proteins of DNA synthesis

Enzymes and proteins of DNA replication

Primers are short, 20-30 bases in length, single-stranded DNA (deoxyoligonucleotides) complementary to the 3-ends of chains of the copied DNA template, due to which a DNA fragment is cut that is millions of times copied by the enzyme Taq-DNA polymerase joining the 3-termini of the primers and finishing them up to a given length of several hundred or thousand base pairs.

DNA polymerase

β-ring

γ-complex

New DNA chain

complementary bases

τ-protein

helicase

DNA

Structure of DNA polymerase

DNA helicase – The enzymes that untwist the double-stranded DNA helix with the energy of hydrolysis of triphosphates are necessary for replication, repair, recombination and transcription. Helicase is present in all organisms.DNA helicases are divided into several families:Superfamily I: UvrD (E. coli, DNA repair), Rep (E. coli, DNA replication), PcrA(Bacillus stearothermophilus, unknown), Dda (bacteriophage T4, initiation of repair).Superfamily II: RecQ (E. coli, DNA reperation), BLM (human, DNA reperation), WRN (human, DNA reperation), NS3 [1] (Hepatitis C virus, replication). TRCF (Mfd) (E. coli, transcription factor of adhesion).Superfamily III: LTag (Simian Virus 40, replication), E1 (human papillomavirus, replication).DnaB-like family: DnaB (E. coli, replication), gp41 (bacteriophage T4, DNA replication), T7gp4 (bacteriophage T7, DNA replication).Rho-like family: Rho (E. coli, transcription termination factor).

Enzymes and proteins of DNA replication

Helicaza (continued)To begin the operation of helicase, a single-stranded region of DNA is required, i.e. Helicase can not begin melting of native DNA without defects.

SSB proteins (single strand bind) They do not denature DNA, but only fix a single-chain condition.They have an increased affinity for single-stranded DNA. The protein does not bind to double-stranded DNA, which does not have molten sections.Proteins bind to double-stranded DNA if there are disturbances in the secondary structure.Selectively stimulate the DNA polymerase.

Enzymes and proteins of DNA replication

Топоизомеразы – ферменты, изменяющие топологию ДНК, т.е. катализирующие переходы в молекулах ДНК, связанные с изменением степени сверхспирализации. Топоизомеразы меняют число зацеплений одной цепи за другую. Делятся на два класса: Тип I (релаксазы) – уменьшают число зацеплений. Тип II (гиразы) – увеличивают число зацеплений.

Enzymes and proteins of DNA replication

TOPOSYOMERASE(DNA topoisomerase),

enzymes that catalyze the transformation of one topological DNA isomer into another by forming or removing nodes and engaging, reducing or increasing the degree of super-spiralization in the molecule.

Structure of the 92K fragment oftopoisomerase II Saccharomices cerevisiae,catalytic DNA binding domain (PDB accession code 1BGW.

DNA replicationThe process of replication is divided into three stages:

initiation, elongation and termination.In order for DNA polymerase molecules to begin DNA

synthesis, they need primer, a short oligodeoxyribonucleotide or oligoribonucleotide, complementary to the corresponding site of the DNA matrix, which has a free 3'-OH group at the end.

InitiationReplication begins at the specific "origin of replication" (THP - ori) -regions of DNA with a specific sequence of nucleotides - 300 nucleotide pairs - to which proteins are attached that unravel the double helix and initiate replication.

DNA helicases unleash a double helix of DNA using ATP energy. The segment of the beginning of the discordance of the chains is called the replicative fork because of the characteristic Y-shape.DNA topoisomerases remove topological stress (supercoiling) when DNA is untwisted. For this, the enzyme first breaks the DNA strand, then covalently joins the ruptured end. This connection has a significant energy, so the reaction is reversible and does not require additional energy costs. There are 2 types of topoisomerases: topoisomerase I (introduces single-strand breaks) and topoisomerase II (introduces double-strand breaks in DNA).SSB proteins (from the English single-strand DNA-binding proteins) bind to single-stranded regions and stabilize the unraveled duplex, preventing the formation of hairpins.

DNA replicationReplicon – a DNA molecule capable of

autonomous replication. The replicon contains all the necessary genes and regulatory sequences that provide an adjustable doubling of its DNA. The points of origin of replication (origin) are specific DNA sites in which replication begins (in E. coli - oriC).

Replicative fork - the copying process continues through the formation of replicative plugs in one or both directions until the DNA is fully doubled.

The points of origin of replication can be one or many, replicative forks can move in one or two directions.

Since the DNA polymerase is unable to build up the 5 end, the synthesis of the DNA strand, the one that grows in the "convenient" direction for the enzyme, goes continuously (from the 5 end to 3 end), this thread is called the leading or leading thread. Synthesis of the filament from the 3 end to 5 end is carried out by short fragments (fragments of Okazaki),which are then sewn together, and such a thread is called retarded, in general the replication of this thread is slower. The structure that is formed during replication is called a replicative plug.This replication mechanism is called semicontinuous.

DNA replication

5΄→3΄ (5΄- PPP, 3΄ - ОН).The direction of synthesis coincides with the direction of motion of the replicative plugfor only one (leading) chain.For the other (lagging behind) - against the movement of the replicative fork.

Direction of movement of the replicative plug

MODELS OF REPLICATION DNA

DNA Replication Modelsa - semiconservativeb - conservativec - dispersionParent chains are depicted in the form of red ribbons, newly synthesized are shown inblue (On Russel, 1998, p. 345

Parentchains

Firstgeneration

Secondgeneration

The speed of genome replication depends on the number of points of initiation.In E. coli, the copy speed in each replicating plug is ~ 1500 p / s; Therefore, a complete genome of length 4 × 106 bp replicates approximately40 min. If the chromosome replicates faster, it means that the frequency of initiation acts at the same origin of replication increases with the previous copy speed.

DNA replicationReplication by type Θ

Replication fork

Репликация ДНК у прокариот и эукариот

Each eukaryotic chromosome is a polyreppleton

DNA eucariotes

DNA parents

Early stage

Late stage

Child Duplexes

The stages of replication

Formation of a replicative fork and

RNA primer(DNA-chelicase,primase (DNApolymerase α)

The formation of a hybridforms of DNA-RNA andfragments of Okaucasia

DNA polymerases δ & ε

HydrolysisRNA primer

helpDNA polymerases β

(ribonuclease)

Formation of DNA instead of RNA primer(DNA polymerase β)

Sewing fragmentsOkazaki (DNA ligase)

The rate of replication in prokaryotes is 500 nucleotides / second

Features of replication in eukaryotes1. Replication occurs in the S-period of the mitotic cell cycle.2. There are many replicons in one DNA molecule, i.e. there are several replication start points.3. DNP-polymerase:α-DNA polymerase. The main enzyme is replication. It also has the activity of primase. Synthesizes the fragments of Okaucasi.β - DNA polymerase - repair enzyme (eliminates DNA damage).γ - DNA polymerase provides the synthesis of mitochondrial DNAδ - DNA polymerase is involved in the synthesis of the host chain.4. The length of the Okaaki fragments is 100-200 nucleotides.5. The rate of replication is 50 nucleotides / second.6. There is a telomerase enzyme that lengthens the 3'-end of DNA before replication; each time after replication, the length of the 3 'end of the linear DNA molecule is reduced by the size of the primer. Disturbances in elongation of telomeres are associated with carcinogenesis and aging.