Molecular mechanisms of DNA replication. Transcription. The painting “Dawn of the Double Helix” composes the DNA duplex as human figures. The theme in this painting is “Life forms: The basic structures that make our existence possible”. 1

Two types of nucleic acids – DNA and RNA Genome - the genetic information of an organism. The genomes of all cells are composed of DNA. Nucleic acids are biopolymers consisting of nucleotides Nucleotides have three components: (1) A weakly basic nitrogen base (2) A five-carbon sugar (3) Phosphate 2

Ribose and Deoxyribose Рибоза Дезоксірибоза Ribose is the constituent of RNA Deoxyribose is the constituent of DNA 3

Nitrogen Bases of Nucleic Acids Adenine Guanine Cytosine Thymine Uracil The nitrogen bases are derivatives of either pyrimidine or purine. 4

Nucleosides Nucleosides are composed of ribose or deoxyribose and a heterocyclic base. 5

Structure of mononucleotide Adenosine mononucleotide 6

Formation of DNA chain (5’-3’ direction) 7

Nucleotides joined by 3’-5’ phosphodi-ester linkages One end of the polynucleotide chain is said to be 5’ (no residues are attached to 5’-carbon) and other is said to be 3’. Direction from the top to bottom is called 5’→3’, from bottom to top – 3’→5. 8

Primary structure of nucleic acids 9

Two Antiparallel Strands Form a Double Helix DNA is Double-Stranded Two Antiparallel Strands Form a Double Helix Two strands run in opposite directions Bases in opposite strands pair by complementary hydrogen bonding Adenine (A) - Thymine (T) Guanine (G) - Cytosine (C) Crick Francis Watson James The double helix of DNA was discovered in 1953 by Crick F. and Watson J. Nobel prize in 1962. 10

Chemical structure of double-stranded DNA Chemical structure of double-stranded DNA. The two strands run in opposite directions. Adenine in one strand pairs with thymine in the opposite strand, and guanine pairs with cytosine 11

Comple-mentary base pairing and stacking in DNA 12

Two-stranded struc-ture of DNA 13

DNA in cells is a constituent of chromatin Chromatin – DNA plus different proteins Histones – main proteins of chromatin 14

Chromatin structure DNA is packaged by coiling of the into a solenoid (helix) structure 15

Types of RNA (1) Transfer RNA (tRNA) Carries amino acids to translation machinery Very stable molecules (2) Ribosomal RNA (rRNA) Makes up much of the ribosome Very stable, majority of cellular RNA (3) Messenger RNA (mRNA) Encodes message from DNA to ribosomes Rapidly degraded by nucleases 16

DNA Replication 17

The flow of genetic information in a typical cell The main postulate of molecular biology DNA  RNA protein 18

Replication – synthesis of DNA on the DNA template Semiconservative mechanism of DNA replication The two strands separate, and each strand is copied to generate a comple-mentary strand. Each parental strand remains associated with its newly synthesized complement, so each DNA duplex contains one parental strand and one new strand. 19

A model for DNA replication 20

Components which are necessary for replication Enzymes (most important – DNA-dependent DNA polymerase) Protein factors Parental DNA ATP, GTP, ТТP, CТP Ions Mg і Zn 21

DNA replication In eukaryotes the replication begins in many points simultaneously V-shape – replication forks - the point of the beginning of replication Helicase – enzyme untwisting the double strand 5’ 3’ Helicase Primase 5’ Okazaki fragments 3’ Primer Leading strand Lagging strand 5’ 3’ 22

Replisome - protein machinery for replication Replisome contains: primosome, DNA polymerase III, proteins Helicase is a constituent of primosome 23

Bidirectional DNA replication in E. coli New strands of DNA are synthesized at the two replication forks where replisomes are located 24

DNA polymerase DNA polymerase III – the main enzyme of replication responsible for the chain elongation Appropriate nucleotides are inserted in the correct positions according to the complementary principle DNA polymerases only synthesize new strand in the 5’-3’ direction. 25

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Напрямок синтезу 5’-3’, антипаралельно до матричного ланцюга 27

DNA polymerase synthesizes two strands simultaneously Because DNA polymerases only polymerize nucleotides 5 ’3’, both strands must be synthesized in the 5’3’ direction. Thus, the copy of the parental 3’5’ strand is synthesized continuously; this newly made strand is designated the leading strand. As the helix unwinds, the other parental strand (the 5’3’, strand) is copied in a discontinuous fashion through synthesis of a series of fragments - Okazaki fragments; the strand constructed from the Okazaki fragments is called the lagging strands 28

Синтез відстаючого ланцюга відбувається дискретно Lagging strand is copied in a discontinuous fashion (Okazaki fragments) The formation of a phosphodi-ester linkage between of adjacent Okazaki fragments is catalized by ligase 29

Okazaki Model Reiji Okazaki provided experimental evidence for discontinuous DNA synthesis 30

RNA Primer Begins Each Okazaki Fragment Primosome is a complex containing primase enzyme which synthesizes short pieces of RNA at the replication fork - primer DNA pol III uses the RNA primer to start the lagging-strand DNA synthesis Replisome - includes primosome, DNA pol III Each Okazaki fragment has the primer 31

DNA polymerase I activities Okazaki Fragments Are Joined by Action of DNA Polymerase I and DNA Ligase DNA polymerase I activities DNA pol I removes the RNA primer at the beginning of each Okazaki fragment Synthesizes DNA in place of RNA DNA ligase Catalyzes the formation of a phosphodiester linkage between of adjacent Okazaki fragments 32

Repair of Damaged DNA DNA is the only cellular macromolecule that can be repaired DNA damage includes: -base modifications -nucleotide deletions or insertions -cross-linking of DNA strands -breakage of phosphodiester backbone 33

Reparation – enzymatic deletion and synthesis of the damaged DNA fragments Recombination - exchange or transfer of pieces of DNA from one chromosome to another or within a chromosome Transposition – dislocation of gene or group of genes from one place to another 34

ТРАНСКРИПЦІЯ 35

NECESSARY COMPONENTS DNA matrix DNA-dependent RNA-polymerase АТP, GТP, CТP, UТP Мg ions 36

DIFFERENCE FROM REPLICATION Only one strand is used as a matrix Only the part of DNA is transcribed (not the entire chain) 37

RNA Polymerase There are 3 RNA-polymerases in eukaryotes (for mRNA, rRNA, tRNA) RNA pol is core of a larger transcription complex Complex assembles at one end of a gene (promoter) when transcription is initiated – transcription initiation DNA is continuously unwound as RNA pol catalyzes a processive elongation of RNA chain 38

The Chain Elongation Reaction Mechanism almost identical to that for DNA polymerase Growing RNA chain is base-paired to DNA template strand Incoming ribonucleotide triphosphates (RTPs) form correct H bonds to template New phosphodiester bond formed Direction 5’-3’ Speed - 30-85 nucleotides/sec 39

Initiation and elongation steps of transcription 40

The transcription bubble 41

RNA poly-merase reaction 42

RNA poly-merase reaction 43

Transcription Termination Only certain regions of DNA are transcribed Transcription complexes assemble at promoters and disassemble at the 3’ end of genes at specific termination sequences 44

PROCESSING Transcription occurs in the nucleus, translation in the cytoplasm Eukaryotic mRNA is processed in the nucleus In some mRNA, pieces are removed from the middle and the ends joined (splicing) 45

Specific enzymes cut out introns and join exons - splicing Introns - internal sequences that are removed from the primary RNA transcript Exons - sequences that are present in the primary transcript and the mature mRNA Specific enzymes cut out introns and join exons - splicing 46

7-methylguanosine (CAP) mRNA transcription splicing DNA exons exones PROCESSING 7-methylguanosine (CAP) Poly-A (TAIL) 5’ 3’ introns Primary transcript 47