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When a eukaryotic cell divides, the process is called mitosis  The cell splits into two identical daughter cells  The DNA must be replicated so that each daughter cell has a copy 3

Basic rules of replication A.Semi-conservative B.Starts at the ‘origin’ C.Can be uni or bidirectional D.Semi-discontinuous E.Synthesis always in the 5-3’ direction F.RNA primers required 4

DNA replication: DNA replication: highly coordinated process has stages: Initiation – proteins open up the double helix and prepare it for complementary base pairing Replication stages: Initiation, elongation and termination. Elongation – proteins connects the correct sequence of nucleotides on newly formed DNA strand Termination - DNA replication ends at the telomere region of repetitive bases repeated over and over again close to the end. 5

DNA replication involves several processes: 1.first, the DNA must be unwound, separating the two strands 2. the single strands then act as templates for synthesis of the new strands, which are complimentary in sequence 3. bases are added one at a time until two new DNA strands that exactly duplicate the original DNA are produced Notes: The process is called semi-conservative replication because one strand of each daughter DNA comes from the parent DNA and one strand is new The energy for the synthesis comes from hydrolysis of phosphate groups as the phosphodiester bonds form between the bases. 6

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 The enzyme helicase unwinds several sections of parent DNA  At each open DNA section, called a replication fork, DNA polymerase catalyzes the formation of 5’-3’ester bonds of the leading strand  The lagging strand, which grows in the 3’-5’ direction, is synthesized in short sections called Okazaki fragments  The Okazaki fragments are joined by DNA ligase to give a single 3’-5’ DNA strand 8

A principal difference between prokaryotic and eukaryotic DNA replication is : Replication machinery in eukaryotes has similar function to E. coli but components have different names The absence of a nucleus in prokaryotes Replication in prokaryotes typically proceeds in both directions from one point of origin to a termination region until there are two copies of the circular chromosome. Replication of eukaryotes has many points of origin and many bubbles (places where the DNA strands are separating and replication is occurring). 9

What is the approximate size For an Okazaki Fragment? n What is the approximate size For an Okazaki Fragment? n The events occurring around a single replication fork of the E. coli chromosome. (a) Initiation. (b) Further untwisting and elongation of the new DNA strands. (c) Further untwisting and continued DNA synthesis. (d) Removal of the primer by DNA polymerase I. (e) Joining of adjacent DNA fragments by the action of DNA ligase. Green=RNA; red=new DNA. 10

 Large team of enzymes coordinates replication DNA replication begins with a partial unwinding of the double helix at an area known as the replication fork. This unwinding is accomplished by an enzyme known as DNA helicase. This unwound section appears under electron microscopes as a "bubble" and is thus known as a replication bubble. 11

Bidirectional replication of circular DNA molecules. 12

Temporal ordering of DNA replication initiation events in replication units of eukaryotic chromosomes. 13

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Initiation of DNA replication Initiation : For a cell to divide, DNA replication begins at specific locations in the genome, called "origins, contain DNA sequences recognized by replication initiator proteins (e.g., DnaA in E. coli, and the Origin Recognition Complex in yeast). These initiators recruit other proteins to separate the strands (forms a bubble) forming a replication fork. Origins tend to be "AT-rich“ 15

The mechanism of DNA replication: Elongation The correct nucleotide sequence is copied from template strand to newly synthesized strand of DNA DNA polymerase III catalyzes phosphodiester bond formation between adjacent nucleotides: Feature Fig. 6.20b 16

The mechanism of DNA replication: Elongation (cont) Leading strand has continuous synthesis (polymerase extends the leading strand in a continuous motion ) Lagging strand has discontinuous synthesis( polymerase extends the lagging strand in a discontinuous motion (due to the Okazaki fragments). Okazaki fragment – short DNA fragments on lagging strand

The mechanism of DNA replication: Elongation (cont) RNaseRNase removes the RNA fragments used DNA polymerase I replaces RNA primer with DNA sequence When this is complete, a single nick on the leading strand and several nicks on the lagging strand can be found. DNA ligase covalently joins successive Okazaki fragments together Feature Fig. 6.20b (cont) 18

 Unwind DNA  unwinds or unzip part of DNA helix by Helicase  stabilized by single-stranded binding proteins (SSBP) Replication fork Helicase Single-stranded binding proteins 19

DNA Polymerase III  Build daughter DNA strand  add new complementary bases  Energy????????? 20

energy ATP GTPTTPATP Where does energy for bonding usually come from? ADPAMPGMPTMPAMP modified nucleotide Tri-P Nucleotide comes with own energy 21

 The nucleotides arrive as nucleosides – DNA bases with P–P–P P-P-P = energy for bonding – DNA bases arrive with their own energy source for bonding – bonded by enzyme: ________________________ ATPGTPTTPCTP 22

DNA Polymerase III energy

no energy to bond  24

energy ligase 25

Limits of DNA polymerase III  can only build onto 3 end of an existing DNA strand Leading strand Lagging strand Okazaki fragments ligase DNA polymerase III  3 5 growing replication fork 26

DNA polymerase III ______________________  built by ________________  serves as starter sequence for DNA polymerase III Limits of DNA polymerase III  can only build onto 3 end of an existing DNA strand growing replication fork primase RNA 27

DNA Polymerase I  removes sections of RNA primer and replaces with DNA nucleotides But DNA polymerase I still can only build onto 3 end of an existing DNA strand growing replication fork DNA polymerase I RNA ligase 28

DNA polymerase III leading strand lagging strand leading strand lagging strand leading strand growing replication fork growing replication fork lagging strand

Scheme of the replication fork. a: template, b: leading strand, c: lagging strand, d: replication fork, e: primer, f: Okazaki fragmentsOkazaki fragmen The leading strand receives one RNA primer per active origin of replication while the lagging strand receives several; these several fragments of RNA primers found on the lagging strand of DNA. 30

Loss of bases at 5 ends in every replication  chromosomes get shorter with each replication  limit to number of cell divisions? DNA polymerase III DNA polymerases can only add to 3 end of an existing DNA strand Chromosome erosion growing replication fork DNA polymerase I 31

Repeating, non-coding sequences at the end of chromosomes = protective cap  limit to ~50 cell divisions ____________________  enzyme extends telomeres  can add DNA bases at 5 end  different level of activity in different cells  high in stem cells & cancers -- Why? telomerase Telomeres growing replication fork TTAAGGG 32

The parental strands are shown in (Red), and the newly synthesized strands are shown in (Blue). 33

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DNA polymerase III ______________________  built by ________________  serves as starter sequence for DNA polymerase III Limits of DNA polymerase III  can only build onto 3 end of an existing DNA strand growing replication fork primase RNA 37

______________________  removes sections of RNA primer and replaces with DNA nucleotides But DNA polymerase I still can only build onto 3 end of an existing DNA strand growing replication fork DNA polymerase I RNA ligase 38

Figure 3.7 Action of DNA ligase in sealing the nick between adjacent DNA fragments (e.g., Okazaki fragments) to form a longer, covalently continuous chain. 39

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Loss of bases at 5 ends in every replication  chromosomes get shorter with each replication  limit to number of cell divisions? DNA polymerase III DNA polymerases can only add to 3 end of an existing DNA strand growing replication fork DNA polymerase I 41

 What are they and Why are they important?  Telomere shortening and the end-replication problem  Telomerase  Telomere and aging 42

Ends of linear chromosomes Centromere Telomere Telomeres :  Repetitive DNA sequence (TTAGGG in vertebrates) (TTTAGGG in plants)  Specialized proteins  Non-coding sequences at the end of chromosomes.  'capped' end structure 43

Ori  DNA replication is bidirectional Polymerases move 5' to 3' Requires a labile primer 3' 5' 3' 5' 3' 5'  Each round of DNA replication leaves bp DNA unreplicated at the 3' end 44

CACACACCCACCAC Telomere Telomere functions Telomere To Centromere ChromosomeTelomere Subtelomeric Telomere repeat 5’GTGTGTGGGTGTGGTG 3’ 5’GTGTGTGGGTGTGGTGTGTGGGTGT Telomerase RNA New DNA synthesis (By Telomerase) Chromosome stability by: Telomere protection DNA damage response (TLC1) ku80 CDC13

Telomerase  Enzyme extends telomeres  can add DNA bases at 5 end  different level of activity in different cells telomerase growing replication fork TTAAGGG  Why telomerase is high in stem cells & cancers -- Why? 46

 Telomeres shorten with each cell division and therefore with age.  Short telomeres cause cell senescence and senescent cells may contribute to aging.  HYPOTHESIS: Telomere shortening causes aging and telomerase will prevent aging Let us think about it???????????????????????????? 47

 Telomere length and life time  Telomeres contribute to aging cancer  Telomerase protects against replicative senescence but not senescence induce by other causes 48

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DNA polymerase I  proofreads & corrects mistakes  repairs mismatched bases  removes abnormal bases  repairs damage throughout life  reduces error rate from 1 in 10, in 100 million bases 52

E. Coli Prokaryote Human Eukaryote Bi-directional fork rate Genome size Replication based on a single origin per chromosome Actual time to replicate the genome 180 Kbp/ min 4.5 mbp 4.5/0.180 = 25 min min 6 Kbp/ min 6,000 mbp 6,000 mbp / 46 chromosomes (130 mbp each) 130 / = 21,666 min = 361 hours = 15 days 8 hours with ~ 1000 origins (replicons per chromosome) 53

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 DNA must be packaged to protect it, but must still be accessible to allow gene expression and cellular responsiveness 55

 How do you fit all that DNA into nucleus of a eukaryotic cell?  DNA coiling & folding  double helix  nucleosomes  chromatin fiber  looped domains  chromosome from DNA double helix to condensed chromosome 56

 Main packaging proteins  5 classes: H1, H2A, H2B, H3, H4.  Rich in Lysine and Arginine  Very highly conserved in eukaryotes in both  Structure  Function 57

 “Beads on a string”  1 st level of DNA packing  histone proteins  8 protein molecules  many positively charged amino acids  arginine & lysine  DNA backbone has a negative charge  histones bind to DNA due to a positive charge 8 histone molecules 58

nucleosomes are organized in a stacked spiral structure the solenoid fibre is known as the 30 nm fibre 59

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Degree of packing of DNA regulates transcription –tightly packed= no transcription – = genes turned off darker DNA (Heterochromatin) = tightly packed lighter DNA (Euchromatin) = loosely packed 61