Genetics Lecture 4 PDF

Genetics DNA Replication LECTURE (4) DR. El-Sawy 0  Genetics DNA Replication  Pr...

Genetics DNA Replication LECTURE (4) DR. El-Sawy 0  Genetics DNA Replication  Process of formation of identical copy of DNA.  Duplication of DNA content before mitosis Def  Two DNA copies are distributed equally ( ) new daughter cells.  Semiconservative: each daughter DNA molecule contain: 1. One old strand  one parent strand is conserved. 2. One new strand  from free nucleotides in nucleus. General  Both strands serve as template “Simultaneously”. Criteria  Bidirectional.  Direction: synthesis of new DNA in direction “5′ → 3′ ”.  Done by DNA polymerases Complex. 1. DNA template: Double stranded DNA. 2. Precursors: dATP, dGTP, dCTP, dTTP. Components 3. Cofactors: Mg++, Mn++, ATP. 4. Enzymes: DNA polymerase " a starting part" 5. Proteins.  DNA helicases: Required for unwinding of dsDNA.  Single stranded DNA binding proteins (ssDB proteins). Major  DNA Primase (subunit of DNA polymerase α). enzymes &  DNA polymerases. proteins  Exonuclease.  Nick sealing enzymes: DNA ligase. DR. El-Sawy 1  Genetics DNA Replication  Can reads the template strand (3’ to 5’ direction) to synthesize a new strand in 5’ to 3’ direction.  Desoxyribonucleotides triphosphate serve as precursors (dAMP, dGMP, dCMP and dTMP). DNA Polymerase  It cannot initiate DNA synthesis, it needs an RNA primer to build new DNA strand upon it.  In eukaryotes, there are five types of DNA polymerases: α, ε, β, γ and δ. DR. El-Sawy 2  Genetics DNA Replication. Initiation = Identification of origin of replication (ori)  DNA replication start at multiple sites  origin of replication or (ori).  These sites rich in AT base pairs (consensus sequence).  DNA replication initiates at many different sites simultaneously & it's bidirectional.. Unwinding & separation of two strands  This origin of replication is unwound to generate a "replication bubble” forming V shaped two replication forks by DNA helicase enzyme (ATP-dependent).  This process starts at each replication fork & proceeds on both directions.  The two strands of DNA are kept away and separated by single strand binding proteins (ssBP). DR. El-Sawy 3  Genetics DNA Replication. Priming by primase enzyme  DNA polymerases cannot initiate DNA synthesis by themselves.  Primase utilizes DNA strands as templates and synthesizes a short stretch of RNA as a primer for DNA polymerase.  One RNA primer is formed for the leading strand.  Multiple primers for the lagging strand.. Synthesis of leading & lagging strands :  DNA polymerases  responsible for synthesis of both strands of DNA.  The presence of a large number of DNA polymerases (more than 20.000) helps to ↓↓ time needed for replication.  DNA polymerase reads template strand (3’ to 5’ direction) to synthesize a new strand in 5’ to 3’ direction.  dAMP, dGMP, dCMP & dTMP serve as precursors. Leading Strand: Lagging Strand: Replicated by DNA Replicated by DNA polymerase δ in polymerase ε in direction of opposite direction of advancing advancing replication fork. replication fork. Synthesized in a continuous Synthesized in a discontinuous manner. manner. Formed in form of Okazaki fragments. DR. El-Sawy 4  Genetics DNA Replication. Excision of RNA primers:  Removal of RNA primers by Exonulceases.. Ligation of different DNA fragments:  Final phosphodiester linkage ( ) 5'-phosphate group on DNA chain & 3‘hydroxyl group on adjacent chain is catalyzed by DNA ligase.  This reaction requires ATP. DR. El-Sawy 5  Genetics DNA Replication Proofreading of Newly Synthesized DNA Strands:  DNA polymerases (δ and ε) check the complementary base on template.  DNA polymerases remove the misplaced nucleotide (by 3'→ 5' exonuclease activity) & replace it with the correct nucleotide. Post-Replication Modification of DNA:  Occurs by methylation of 5th C atom of cytosine residues on DNA.  Changing methylation status of DNA (Hypomethylation or Hypermethylation) is reported in different tumors. Inhibitors of eukaryotic DNA replication : used as anticancer chemotherapy. Actinomycin D  inhibit initiation of replication by binding Acts on DNA: DNA template. Nucleotide Cytosine arabinoside (ara C) analogues : 5-florouracil (thymine analogue). DR. El-Sawy 6