DNA ORGANIZATION AND REPLICATION

Transcription

1 DNA ORGANIZATION AND REPLICATION

2 THE CENTRAL DOGMA DNA Replication Transcription Translation

3 STRUCTURAL ORGANIZATION OF DNA

4 DNA is present in the nucleus as CHROMATIN. The basic unit of chromatin is NUCLEOSOME formed by the wrapping of DNA over the histone core. Histone core is formed by an octameric structure with the composition of (H3-H4)2 (H2A-H2B)2.

5 CHROMATIN Active Euchromatin Inactive Heterochromatin Constitutive Facultative

6 Genome is divided into different sequence classes Unique or nonrepetitive sequences Repetitive sequences Protein coding genes is ~30,000-40,000 Highly (5-500 bp) 1-10 million/haploid genome Moderately (10 6 /haploid genome) LINEs SINEs

7 REPLICATION: SEMICONSERVATIVE NATURE Steps in eukaryotes: 1. Identification of the origin site 2. Unwinding & formation of ssdna template 3. Formation of Replication fork and bubbles 4. Initiation of DNA synthesis and elongation 5. Ligation of newly synthesized DNA segments 6. Reconstitution of chromatin structure

8 Ori ORIGIN OF REPLICATION Ori binding protein ( ) A+T rich region AT rich region denaturation Binding of SSB ( )

9 REPLICATION FORK Leading strand Polymerase -Primase - Helicase - SSB - RNA Primer Okazaki fragment 5 5 Lagging strand 5

10 LIGATION OF NEWLY SYNTHSIZED STRANDS 1. DNA Polymerase I : 5-3 exonuclease activity that removes RNA primer. Catalyzes the addition of deoxyribonucleotides to the 3 end of the more recently made okazaki fragments. 2. DNA LIGASE: DNA binds to the nick and catalyzes the addition of a phosphodiester bond that seals the fragments.

11 TOPOISOMERASES Regulates the removal of super coils. Acts by forming a transient break in the DNA back bone and then resealing it. Topoisomerase I: Acts by making a transient single strand break in the supercoiled DNA and results in relaxation of the super coiled DNA. Topoisomerase II: Nicks both the strands and passes one DNA segment through this break before resealing.

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13 DNA GYRASE Introduces negative super coils into the relaxed DNA and neutralizes the positive coils that is formed during replication.

14 DNA POLYMERASES Chain elongation Processivity Proof reading property

15 PROOF READING PROPERTY Misreading of the template sequence results in deleterious and sometimes lethal mutations. Overcome by the 3-5 exonuclease action of the DNA POLYMERASE III enzyme. Removes the mispaired nucleotides from the 3 end of the chain.

16 DNA POLYMERASES E.Coli Mammalian Function I α Gap filling&synthesis of lagging stand II ε ß γ DNA proofreading & repair DNA repair Mt.DNA synthesis III δ Processive, leading stand synthesis, proof reading

17 PROTEIN FUNCTION DNA polymerase Primase dntp polymerization Synthesis of RNA primer Topoisomerases Regulation of supercoils Gyrases Helicases Unwinding of double helix, requires ATP. ss DNA binding protein Prevents premature reannealing of DNA, protection from nucleases. DNA ligase Seals the nick between the nascent chain and Okazaki fragments on lagging strand.

18 TELOMERASES Telomeres are repeat sequence of 6 nucleotides, GT rich, present at the end of the eukaryotic chromosome. Prevent the continuous loss of DNA at the end of chromosome during the course of replication.

19 INHIBITORS OF REPLICATION 1. Inhibitors of Topoisomersase I : Camptothecin traps the complex between the topoisomerase and DNA. 2. Inhibitors of Gyrase : Antibiotics like Ciprofloxacin, Novobiocin, Nalidixic acid block the DNA replication and multiplication of the cells. 3. Inhibitors of Topoisomerase II : Adriamycin, Etoposide, Doxorubicin

20 CELL CYCLE Incomplete replication detected S phase G2 Damaged DNA detected M Improper spindle detected G1 Restriction point Damaged DNA detected

21 CYCLINS & CDK Cyclins are a family of proteins whose concentration increases and decreases throughout cell cycle. The Cyclins turn on, at appropriate time, by different Cyclin dependent protein kinases(cdks)

22 Cyclins Kinases Function D CDK 4 Progression past CDK 6 restriction point at G1/S boundary E, A CDK 2 Initiation of DNA synthesis in early S phase B CDK 1 Transition from G2 to M phase

23 DNA DAMAGE & REPAIR MECHANISM

24 TYPES OF DNA DAMAGE Single base alteration: a)depurination b)deamination of Cytosine to Uracil c)deamination of Adenine to Hypoxanthine d)alkylation of base e)insertion/deletion of nucleotide f) Base analog incorporation

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26 Two base alteration: a) UV light induced thyminethymine dimer formation a) Bifunctional alkylating agent cross linkage

27 Chain breaks: a) Ionizing radiation b) Radioactive disintegration of backbone element c) Oxidative free radical formation

28 Cross linkage: a)between bases in same or opposite strands b)between DNA and protein molecules( e.g histones)

29 MECHANISM OF DNA REPAIR Mismatch repair Base excission repair Nucleotide excission repair Double strand break repair

30 3 CH 3 CH Single site strand cut by GATC endonuclease 3 CH 3 CH Defect removed by exonuclease 3 CH 3 CH Defect repaired by polymerase 3 CH 3 CH Religated by ligase 3 CH 3 CH M I S M A T C H R E P A I R

31 HEREDITARY NON POLYPOSIS COLON CANCER (HNPCC)

32 3 5 A T C C G A C T A C T A G G C T G A T G Heat energy BASE EXCISSION REPAIR A T C C G A U T A C T A G G C T G A T G A T C C G A C T A C T A G G C T G A T G U N- glycosylase DNA polymerase & Ligase A T C C G A * T A C T A G G C T G A T G Apurinic or Apyrimidinic endonucleases A T C C T A C T A G G C T G A T G

33 NUCLEOTIDE EXCISSION REPAIR Recognition and unwinding Oligonucleotide excision by cutting at two sites by Exinuclease Resynthesis and religation

34 XERODERMA PIGMENTOSUM (XP)

35 DOUBLE STRAND BREAK REPAIR Ku & DNA-PK Approximation Unwinding P P

36 Alignment & base pairing Ligation

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